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Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα
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  • Chiara Borsari
    Chiara Borsari
    Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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    Orcidhttps://orcid.org/0000-0002-4688-8362
  • Erhan Keles
    Erhan Keles
    Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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  • Jacob A. McPhail
    Jacob A. McPhail
    Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada
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  • Alexander Schaefer
    Alexander Schaefer
    Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
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  • Rohitha Sriramaratnam
    Rohitha Sriramaratnam
    Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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  • Wojciech Goch
    Wojciech Goch
    Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland
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  • Thorsten Schaefer
    Thorsten Schaefer
    Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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  • Martina De Pascale
    Martina De Pascale
    Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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    Orcidhttps://orcid.org/0000-0002-7546-2766
  • Wojciech Bal
    Wojciech Bal
    Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
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    Orcidhttps://orcid.org/0000-0003-3780-083X
  • Matthias Gstaiger
    Matthias Gstaiger
    Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
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    John E. Burke
    Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada
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  • Matthias P. Wymann*
    Matthias P. Wymann
    Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
    *Email: [email protected]. Tel: +41 61 207 5046. Fax: +41 61 207 3566.
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2022, 144, 14, 6326–6342
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https://doi.org/10.1021/jacs.1c13568
Published March 30, 2022

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Abstract

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Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5′-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinact and reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.

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Introduction

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Eight covalent kinase inhibitors, afatinib, (1) ibrutinib, (2) osimertinib, (3) neratinib, (4) dacomitinib, (5) acalabrutinib, (6) zanubrutinib, (7) and mobocertinib, (8,9) have been approved by the Food and Drug Administration (FDA) as anticancer agents to date. This renewed the interest in targeted covalent inhibitors (TCIs). Advantages of irreversible inhibitors include increased ligand efficiency, specificity, and prolonged duration of action. (10) Besides therapeutic approaches, TCIs find application as chemical probes to explore the role of kinases in the onset of human disease. (11) Currently, only a limited number of signaling proteins’ cysteines have been successfully targeted. (12,13) TCIs are usually generated using a reversible-inhibitor scaffold with a high affinity for the primary target, which is then extended by an electrophilic, reactive functional group. Often dubbed warhead, these electrophiles engage in a Michael addition to form a covalent link with the target cysteine. This strategy has been successfully exploited to target protein kinases, including Bruton’s tyrosine kinase (BTK) (2,14) and epidermal growth factor receptor (EGFR) (15) (Figure 1a,c). The incorporation of an electrophile on PCI-29732 and gefitinib scaffolds yielded the cysteine-targeting covalent inhibitors acalabrutinib and ibrutinib for BTK (Figure 1b), and dacomitinib and afatinib for EGFR (Figure 1d). Typically, highly reactive warheads in covalent drugs lead to toxicity and off-target reactions with cellular nucleophiles such as thiols, including glutathione (GSH). Sadly, even for marketed drugs like ibrutinib, clear structure–activity relationship (SAR) studies leading to compound selection are obscured. (2) The intrinsic reactivity with thiols has not been determined for recent covalent inhibitors, (16−19) even though a variety of warheads have been explored. (7,20) Besides warhead reactivity, the optimization of warhead proximity is often not considered in the generation of covalent probes. (21) Most of the explored targeted cysteines are in close proximity to the reversible scaffold, (22) and structural analysis of the linker has not been examined or disclosed. Targeting of remote cysteines has been rarely approached, but a few examples are present in the literature, including anaplastic lymphoma kinase (Alk), (23) cyclin-dependent kinases 12 and 13 (CDK12 and CDK13), (24,25) and CDK7 (26) covalent inhibitors.

Figure 1

Figure 1. (a, c) Design strategy for the development of irreversible inhibitors targeting (a) BTK and (c) EGFR. X-ray crystallographic structure of (a) BTK (colored wheat) in complex with PCI-29732 (teal; PDB ID 3GEN) (78) and (c) EGFR (cyan) in complex with gefitinib (brown, PDB ID 4I22). (79) (b, d) Chemical structure of the reversible-inhibitor scaffolds and covalent derivatives are depicted. The incorporation of the warhead on PCI-29732 led to acabrutinib and ibrutinib, and gefitinib served as a scaffold for dacomitinib and afatinib. (e) PQR514 (46) (magenta) bound to phosphoinositide 3-kinase α (PI3Kα) (gray; based on PDB ID 6OAC (59)). Cys862 positions from eight superimposed PI3Kα-inhibitor complexes are shown (see Table S1 for details). The position of the cysteine thiols (yellow) is conserved across the different complexes and deviates less than 2.6 Å. (f) Schematic design of the covalent PI3Kαi library. The distances between reversible inhibitor skeletons and the targeted nucleophiles are shown as dashed green lines (∼4 Å for BTK and EGFR vs ∼11 Å for PI3Kα).

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CNX-1351 is the only known covalent inhibitor of phosphoinositide 3-kinase α (PI3Kα) and targets its remote Cys862, (27) which is not conserved in other PI3Ks. CNX-1351 shows a relatively poor in vitro and cellular potency, and its chemical features and warhead do not make it suitable for a lead optimization process.
Thus, a rational path to the identification of highly selective, covalent druglike inhibitors is currently elusive. A systematic study of reactivity, proximity, and the integration of chemical and biochemical measurements, paired with kinetic and structural modeling, is required for the design of high-quality covalent chemical probes.
Herein, we have selected PI3Kα to outline a roadmap to target solvent-exposed cysteines at >10 Å from a reversible binding core module. The PI3K signaling pathway plays a key role in human carcinogenesis, and the PI3K family is divided into three classes according to sequence homology and substrate specificity. (28,29) The class I PI3K family consists of four isoforms (PI3Kα, β, δ, and γ). The genetic locus encoding PI3Kα, PIK3CA, is frequently mutated and implicated in tumor progression. (30,31) Structurally diverse, reversible pan-class I PI3K inhibitors (PI3Ki) are currently evaluated in clinical trials, either as monotherapy, or as part of combination regimens. (32−34) Alpelisib (BYL719) (35) has been approved in combination with Fulvestrant for the treatment of HR+/HER2– advanced breast cancer bearing activating mutations in PIK3CA. (36−39) BYL719 shows biochemical selectivity for PI3Kα, (35) but is not sufficiently isoform-selective to serve as a chemical probe to dissect specifically PI3Kα-dependent biological processes. However, BYL719’s preference for PI3Kα enlarges its therapeutic window in PIK3CA-mutant tumors (40) and makes it suitable to treat PIK3CA-related overgrowth syndrome (PROS). (41) Targeting the PI3Kα-specific Cys862 with covalent inhibitors provides a strategy to achieve exquisite PI3K isoform selectivity and to generate tools to elucidate the role of PI3Kα in cancer and metabolism. Here, a rational path to covalent inhibitors targeting distal cysteines is depicted, which is driven by a stepwise adaptation of warhead reactivity and proximity, supervised by a rigorous determination of chemical and enzymatic reactivities validating the proximity to the targeted nucleophile. The approach is generally suited to target unexplored Cys residues in biologically active enzymes.

Results and Discussion

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Design of Covalent PI3Kα Inhibitors

Starting from the clinical candidate PQR309, (42−45) we have previously identified the preclinical candidate PQR514 with improved, low nanomolar affinity for PI3Kα (Ki for PI3Kα = 2.2 nM (46)). The PQR514 core was a suitable starting point to serve as PI3Kα-binding module. Cys862 in PI3Kα is located at ∼11 Å from the solvent-exposed morpholine oxygen atom of PQR514 (Figure 1e). An overlay of eight PI3Kα-inhibitor crystal structures showed that the position and orientation of the Cys862 side chain were maintained across all structures (Figure 1e and Table S1), thus providing a fair estimate of the target space. Subsequently, a variety of warheads and linkers were studied to assess the required warhead reactivity and the spatial trajectory of the Michael acceptor (for a schematic setup, see Figure 1f).

Tuning of Warhead Reactivity

The control of warhead reactivity is a pivotal parameter in the development of covalent chemical probes and drug candidates. It has been reported that the rate of thiol-Michael addition to α,β-unsaturated carbonyls decreases from enones to acrylamides and that electron-donating groups and alkyl moieties reduce the rate of thiol addition due to electronic and steric effects. (47) Even though chemical and computational methods have been used to assess the half-life of warheads, (48) their intrinsic reactivity, as well as its correlation with the rate of covalent bond formation, is often neglected in SAR elucidation.
Up-to-date, several approaches have been applied to predict warhead reactivity, including NMR chemical shifts, (49) pKa assessments, (50) and quantum mechanics/molecular mechanics (QM/MM). (51)1H and 13C NMR have been previously used to predict the reactivity of structurally similar α,β-unsaturated carbonyls. (52) Experimentally, the half-life to adduct formation with GSH has served as a surrogate for measuring the relative reactivity of cysteine-targeting warheads, (53,54) and absolute rate constants (kGSH or kchem) have been systematically established for a collection of N-aryl (55) and N-phenyl (56) acrylamides. Here, we designed and synthesized PI3Kis bearing nine different warheads, including enones and acrylamides (1–9, Figure 2a).

Figure 2

Figure 2. (a) Chemical structure of a set of nine warhead-containing compounds (1–9). (b) General reaction of warhead-containing compounds with βME. (c) High-performance liquid chromatography (HPLC) reaction monitoring of inhibitor (3) and its βME adduct (3-βME) formation. (d) Time-dependent inhibitor consumption curves used to calculate kchem. Values are mean ± standard deviation (SD) (n = 3). Error bars are not shown when smaller than symbols. (e) Chemical shift of the α-proton in 1H NMR spectra of compounds bearing an unsubstituted double bond (7) and β-methyl (8)- and β,β-dimethyl (9)-substituted warheads. (f) Intrinsic reactivity of the inhibitors (kchem) plotted against the chemical shift of the α-protons of the indicated compounds. Raw data and details for kchem calculations are reported in Table S2 and in the Materials and Methods section in the Supporting Information. The second-order rate constant kchem could not be measured for compounds 6 and 9 due to lack of reactivity with 12 M βME, and also not for 1 due to its extremely fast reaction with 1 mM βME. kchem for compound 8 was lower than 1 × 10–6 M–1·s–1 and was thus considered nonreactive. These three values are therefore “out-off scale” (denoted with red arrows) for kchem. (g) Representation of energy of the lowest-unoccupied molecular orbital (LUMO) electron density (top) and LUMO map (bottom) for compound 1. Electron deficiency ranges from blue (high) to red (low). Given that the LUMO designates space available for a pair of electrons, color tints toward blue in the LUMO map indicate where a nucleophilic attack would likely occur. Calculations were performed using Spartan 18, Wavefunction, Inc. (h) ELUMO values plotted against the intrinsic reactivity of the inhibitors (kchem). Raw data and details for kchem calculations are reported in Table S2 and in the Materials and Methods section in the Supporting Information. As in (f), values for kchem denoted with red arrows are “out-off scale”. (i) Chemical shift of α-protons of indicated compounds plotted against ELUMO values. (j) Model for cellular on- and off-target reactions of covalent inhibitors. In the on-target reaction (green), enzyme E and inhibitor I form first a reversible E∼I complex (equilibrium dependent on Ki), which is then converted to a covalent EI complex (controlled by kinact). Competing off-target reactions (red) with cellular sulfhydryls (S) consume the inhibitor and form a stable SI adduct. (k) On- and off-target covalent reactions of inhibitors as depicted in (j) were modeled using KinTek Global Kinetic Explorer: concentrations were set to 7 mM for intracellular sulfhydryls (for intracellular reduced glutathione (GSH) concentrations, see ref (80)); for the targeted enzyme (PI3Kα) to 10 nM; and for indicated covalent inhibitors to 100 nM. Experimental values for kinact and kchem used for the modeling are listed in Table S2 and Figure S1. Details for the determination of kinact are given in Figure 3 and associated materials.

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We experimentally determined the rate constants of adduct formation of this diverse set of Michael acceptors with β-mercaptoethanol (βME; Figure 2b): the nine warhead-containing compounds (1 mM) were incubated with βME at a selected concentration from 1 mM to 12 M at 37 °C in phosphate-buffered saline (PBS, pH 7.4)/dimethyl sulfoxide (DMSO) mixtures (v/v 1:4). The temporal progress of the reactions was monitored by HLPC (Figure 2c) and βME adducts were confirmed by mass spectrometry (MS) (see the MALDI-MS Spectra of β-Mercaptoethanol Adducts section in the Supporting Information). The resulting time-dependent inhibitor consumption/adduct formation curves (Figure 2d) were fitted using pseudo-first-order reaction kinetics (yielding k′), and were used to calculate the second-order reaction rate constant (kchem, Table S2). Second-order rate constants could not be reliably calculated for compounds 6 and 9 due to lack of reactivity with 12 M βME, and also not for 1 because of the extremely fast reaction with 1 mM βME. The presented set of Michael acceptors showed a broad range of reactivities spanning over 5 log units for kchem.
As a predictive analytical parameter for kchem,1H NMR chemical shifts of the alkene α-proton of the warheads were explored: relative to the unsubstituted double bond in 1, 4, 7, the signal of the alkene α-proton was shifted upfield in β-methyl (2, 5, 8)- and β,β-dimethyl (3, 6, 9)-substituted warheads (see, for example, compounds 7–9, Figure 2e). When Log(kchem) values were, however, plotted against chemical shifts of the α position in 1H NMR, no reliable correlation could be established (Figure 2f).
Subsequently, LUMO maps (Figure 2g) with the correspondent energies of lowest-unoccupied molecular orbitals (ELUMO) were calculated. Low-energy LUMO makes chemical moieties reactive toward nucleophiles. As LUMO energies have been used to assess the electrophilicity of molecules, they could be predictive of kchem values. A moderate correlation between ELUMO energies and experimentally determined Log(kchem) values was found (R2 = 0.68, Figure 2h). Similarly, no fair correlation was obtained when ELUMO values were plotted against the chemical shift of the α-proton (R2 = 0.34, Figure 2i).
Altogether, these results highlight that a measurement of kchem is essential for a systematic classification of warhead reactivity. To estimate on-target and off-target reactivity, the reaction of the warhead with Cys862 of PI3Kα or cellular thiols was modeled using KinTek software using the two competitive on-target and off-target reactions depicted in Figure 2j. The on-target model involves the inhibitor dissociation constant Ki for the formation of the reversible enzyme–inhibitor complex (E∼I) and the rate of the covalent reaction with the protein (kinact; for the measurement of Ki and kinact, see next section) to form the irreversible inhibitor complex (EI).
The results of this modeling (Figures 2k and S1) illustrate that (i) highly reactive electrophiles (2) react very rapidly with the target, but are mostly consumed by off-target reactions; (ii) moderate electrophiles (3, 4, 7) selectively engage the target and show negligible side reactions; and (iii) minimally reactive molecules (5) engage neither the desired target nor undesired cellular thiols.
This defines a range for kchem from ∼5 × 10–5 to 5 × 10–3 M–1·s–1 for warheads with an acceptable off-target activity. On-target reactivity is, however, promoted by reversible affinity and warhead proximity to the targeted nucleophilic amino acid side chain.
While not suitable for drug development, we show below that highly electrophilic warheads (see 2) can be exploited in the early stage of hit identification to assess the spatial proximity of the warhead to the target cysteine.

Scan of Protein Target Site and Assessment of Proximity

The β-methyl-substituted enone (2) and its corresponding moderately electrophilic derivative, β,β-dimethyl enone (3), were selected to explore the target space. A collection of compounds with pairwise matched linkers but either fast (2, 10–12) or moderately (3, 13–15a) reactive Michael acceptors were synthesized (Figure 3a).

Figure 3

Figure 3. (a) Chemical structure of a collection of nine compounds with pairwise matched linkers and either fast (2, 10–12) or moderately (3, 13–15a) reactive Michael acceptors, and of 15b and 15c. (b) The reactive volume (Vr) of the warhead was estimated by a sphere around the sulfur atom of Cys862, with a radius equal to the distance (d) between the Cys862 sulfur and the β-carbon of the Michael acceptor (Vr = 4/3·π·d3); see Table S4 for values. The modeled locations of the β-carbon of the Michael acceptor are indicated by spheres colored as in (a). (c) Correlation between kinact and the local warhead concentration (see Table S4 for calculation). (d, e) Time-dependent IC50 determinations derived from time- and concentration-dependent time-resolved fluorescence resonance energy transfer (TR-FRET) ratios curves depicted in Figure S2 (IC50s for all compounds with error bars, see Figure S13). Strong (2, 10–12 (d)) and weak (3, 13–15a (e)) electrophiles were investigated. (f) Compound-specific intrinsic reactivity (kchem) determined as in Figure 2; (g) dissociation constants (Ki) calculated from concentration and time-dependent TR-FRET experiments as shown in Figure S2 (h) rate constants for covalent binding to PI3Kα (kinact); (i) second-order rate constant used to characterize covalent binding of irreversible inhibitors to the target protein (kinact/Ki). The calculation of kinetic parameters was carried out through global fitting for numerical integration to a kinetic model using KinTek Global Kinetic Explorer modeling software. (73−76,81,82) All values are shown as mean ± SD (n = 3). Error bars are not shown when smaller than the symbols. Orange, strong electrophiles; green, weak electrophiles. The distance between the Michael acceptor (β-carbon) and Cys862 thiol was calculated using PyMOL 2.3.5 Schrödinger, Inc. (see Figure S3 and Table S3). (j) TR-FRET experiments for the reversible analogue of compound 2 (2r). (k) Covalent docking of compounds 2, 10–12 using CovDock Schrödinger. Representation of the shift of the morpholine oxygen (spheres, in hinge region defined by Val851) and of the NH2 (spheres, in binding affinity region defined by Asp810) with respect to PQR530 scaffold (magenta) is displayed. (l) CovDock Schrödinger modeling has been used to investigate the displacement of the PI3K core module of the indicated compounds when covalently bound to Cys862 of PI3Kα. The distances of (i) the morpholine oxygen atom (O17) and the N nitrogen atom of Val851 backbone amide (O17-V851, red) in the hinge region, and (ii) the amino group (N26) of the aminopyrimidine and the closest oxygen atom in COO– side chain of Asp810 (N26-D810, light blue) in the affinity region were determined from CovDock modeling coordinates from 50 predicted poses, and average and SD were calculated. As a control for a structural distortion of the PI3K-binding module, the distances between the O17 and N26 atoms (O17-N26, black) were determined. Numbering of inhibitor atoms can be found in (a) and (k). Distances are reported in angstrom. Modeling was performed using PDB ID 6OAC (59) as a starting point. All compounds are depicted in Figure S6.

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When bound to the adenosine 5′-triphosphate (ATP)-binding site of PI3Kα, the linker length of the covalent inhibitor defines the proximity of the warhead to Cys862. As explained below in detail, the warhead will thus reside within a reactive volume (Vr), which defines the local concentration of the sulfhydryl group of Cys862 and the β-carbon of the Michael acceptor of the warhead. Based on molecular modeling, Vr can be approximated by a sphere with a radius equal to the distance of the warhead and Cys862 (Figure 3b and Table S4). An excellent correlation was obtained between the predicted local concentration (estimated by 1/(NA·Vr); NA, Avogadro’s number) of the warhead and the rate constant for the irreversible reaction with Cys862 (kinact; R2 = 0.999, Figure 3c). The irreversible reaction rate kinact was determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) tracer displacement assay. Fitting time- and concentration-dependent TR-FRET ratio curves (Figure S2) using KinTek kinetic modeling software yielded kinact, the calculated Ki, and a kinact/Ki ratio typically used to characterize the covalent binding of irreversible inhibitors to the target protein (Table S3). The time- and concentration-dependent TR-FRET data can be represented as time-dependent IC50 curves (exposure times of 10, 15, 30, 60, and 90 min), where a shift in IC50 visualizes a covalent, time-dependent reaction of inhibitors with PI3Kα (Figure 3d,e). For compounds bearing a fast-reacting warhead (2, 10–12) a significant IC50 shift was observed even when their Michael acceptor was predicted to be located at >3.5 Å from Cys862 thiol (10: 5.1 Å and 11: 3.8 Å; Figures 3d and S3). Compounds 2 and 12, with longer linear linkers (n = 3 and 4), displayed an increased efficiency in covalent bond formation with PI3Kα, and compound 12 showed the highest rate of covalent inactivation (kinact/Ki = 1.27 × 10–3 nM–1·s–1, Table S3). Reversible analogues did not display a time-dependent IC50 shift (Figure 3j for 2r). Similarly, β,β-dimethyl enone-containing compounds 13 and 14 showed no detectable IC50 shift (Figure 3e) and very low kinact/Ki ratios (13: 3.25 × 10–6 and 14: 3.21 × 10–6 nM–1·s–1) compatible with negligible covalent target engagement. Compound 15a, placing the warhead <2 Å from Cys862, showed an increased kinact/Ki ratio (5.82 × 10–5 nM–1·s–1, Table S3). The fact that 15a displayed a 22-fold lower kinact/Ki ratio compared to 12 with an identical linker length demonstrates that the faster warhead in 12 is much better suited to monitor the proximity to the target.
The increase in linker length did not influence the intrinsic reactivity of the inhibitors for both fast and moderately reactive Michael acceptors (Figure 3f). The reversible binding (see Ki, Figure 3g) was moderately attenuated by extended hydrophobic linkers in a solvent-exposed region of the ATP-binding site. These two findings clearly illustrate that the increased efficiency in covalent bond formation for 12 is related to the optimized proximity of the warhead to Cys862. The comparison of molecules with equal linker length and fast vs moderate reactivity warheads shows clearly that fast warheads produce a significant increase in kinact (Figure 3h) and kinact/Ki (Figure 3i) producing a proximity “hit alert” that is not detected with moderately reactive compounds. Fast-reactive warheads therefore provide an exquisite opportunity to rapidly (i) approach a nucleophilic target in biologically relevant proteins and (ii) prove the feasibility to convert a reversible scaffold into a covalent molecule.
To complement the experimental approaches, modeling of covalent docking was performed using CovDock Schrödinger. (57,58) As CovDock affinity values are only reliable when intrinsic warhead reactivities are equal, (57) they cannot be exploited to compare our different hit sets including compounds with enones and acrylamides (2, 3, and 10–24). Therefore, as an output parameter, we assessed the displacement of the PI3K-binding core module from the hinge (V851) and the binding affinity region (D810) after covalent binding to Cys862. Fifty poses per ligand were generated, and the distance between (i) the morpholine oxygen atom (O17) and the backbone amide nitrogen atom of Val851 (O17-V851, Figure 3a,k), and (ii) the amino group (N26) of the aminopyrimidine and the closest oxygen in the side chain carboxylate of Asp810 (N26-D810, Figure 3a,k) were calculated. Considering the compounds 2 and 10–12, the morpholine oxygen (O17) of 12 was juxtaposed with that of PQR530 (PDB ID 6OAC) (59) (Figure 3k), highlighting that the reversible core module was well accommodated in the ATP-site upon the modeling of the covalent reaction with Cys862. On the contrary, the morpholine oxygen in compound 10 was displaced by ∼2 Å (Figure 3k) to allow the warhead to react with Cys862. A similar trend was observed determining the N26-D810 distance, where N26 of compounds 10 and 11 was displaced by ∼1.8 and 2.5 Å, respectively, compared to N26 in 12 (Figure 3l). Analysis of the whole panel of compounds, shown in Figure S6, pointed out that CovDock can be exploited to narrow down the hit list in covalent inhibitor design, and allows the exclusion of molecules with a bad match in linker length.
A further demonstration that the scan of the target site is more likely to succeed using reactive warheads was demonstrated with acrylamide compounds 15b and 15c, which showed a drop of kinact to 0.96 × 10–4 and 1.27 × 10–4 s–1, respectively, compared to a kinact of 9.03 × 10–4 s–1 for 15a.

Linker Optimization Using Druglike Warheads

As shown above, unsubstituted acrylamides (4 and 7) can be exploited in covalent chemical probes with minimal off-target reactions with unspecific nucleophiles inside cells (Figure 2i). To correlate the spatial trajectory to Cys862 with the above warhead series (see Figure S14), nine molecules (16–24, Figure 4a) bearing an acrylamide warhead and different linker modules were synthesized. A piperidine ring was introduced in these molecules as the attachment point for the warhead. Piperidine has been proposed as a privileged scaffold in drug discovery and is present in many marketed anticancer agents. (60) The piperidine ring was connected to the PI3Kα-binding module through a carbon-chain (16–18) or an amide-containing (19–24) linker. Two possibilities have been explored for amide-linker compounds: secondary (compounds 19–21) and tertiary (22–24) amides were introduced (Figure 4a). The presence of an additional methyl slightly affected the physicochemical properties (clog P, see Table S5) and inhibitor–protein affinity (see next section).

Figure 4

Figure 4. (a) Library of acrylamide-containing compounds (16–24). (b) Intrinsic reactivity (kchem) of compounds 16–24, ibrutinib and CNX-1351. Experiments performed with HPLC (1 mM inhibitor and 600 mM βME, n = 3). All values are reported as mean ± SD. Error bars are not shown when smaller than the symbols. (c) Efficiency in covalent bond formation (kinact/Ki) plotted against distance from Cys862. Zero on the x-axis corresponds to Cys862 positioning; zero to left: shorter linkers; zero to right: longer linkers. Kinetic parameters are reported in Table S5. The distances between the Michael acceptors (β-carbons) and Cys862 thiol were calculated using Maestro 11.1 and PyMOL 2.3.5 Schrödinger LLC (see Figure S5). (d) Plot of kchem/kinact ratios vs the reactive volume Vr of compounds 12, 2, 11, 10 (red circles), 15a, 3, 14, 13 (green squares), 19, 22, 18, 17, 16 (blue circles), and 20, 23, 21, 24 (orange diamonds). The top x-axis shows the Vr·NA term utilized as in eq S14b to determine the scaling factor σ for each compound group as shown in Figure S7 and Table S6. The compound group-specific σ factors were used to normalize the kchem/kinact ratios by 1/σ. The (blue) regression line includes all compounds listed above except for 20, 23, 21, 24, with a linker reaching beyond the target Cys862. (e) Normalized kcat reaction rates were calculated according to eq 15 with a spherical Vr approximation using the modeled distance to the target d. For Ki, the indicated values were used. (f) Normalized kcat reaction rates were calculated according to eq 17a as a function of d and the inhibitor concentration [I]. For both graphs in (e) and (f), a mean kchem averaged over the values from compounds 19, 22, 18, 17, and 16 was used (kchem (19, 22, 18, 17, 16) = 3.38 × 10–4 M–1·s–1). (g) LC-SRM quantification of Cys862-modification by 19, 22, and 22r. (h, i) X-ray structure of (h) 19 (deep teal) bound to PI3Kα (PDB ID 7R9V, resolution 2.69 Å) and (i) 22 (deep purple) bound to PI3Kα (PDB ID 7R9Y, resolution 2.85 Å). H-bonds are depicted as dashed black lines. As for the CHF2-group, a H-bond either with (i) Lys802 or with (ii) the triazine core (intramolecular interaction) is possible based on the proton positioning. (j, k) Electron density maps of the inhibitor (2Fo–Fc map contoured at 1σ, blue mesh) fit the structure of (j) 19 and (k) 22, and the electron density clearly shows that the acrylamide forms a covalent bond with the thiol group of Cys862.

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Intrinsic reaction rates (kchem) were measured by adduct formation with βME, to validate that chemical modifications of the linker did not affect the electrophilicity of the acrylamide warhead. All compounds displayed similar intrinsic reactivities, matching the values of CNX-1351 and ibrutinib (see the Supporting Information for chemical structures) within the same order of magnitude (Figure 4b). TR-FRET measurements of kinact, Ki, and calculation of kinact/Ki, as reported in Table S5, document that the efficiency of covalent bond formation was dramatically increased passing from compound 16 to 19 and 22 (see Figure S4 for time- and concentration-dependent TR-FRET ratio curves used for parameter fitting, and Figure S5 for distance measurements). After reaching a maximum efficiency in covalent bond formation (19 and 22kinact/Ki = 4.14 × 10–4 and 2.10 × 10–4 nM–1·s–1, respectively; Table 1), a drop in kinact/Ki values was observed further elongating the linker beyond Cys862 (compounds 20, 21, 23 and 24, Figure 4c). Our data show that the excellent reactivity of 19 and 22 with PI3Kα is related to the optimal positioning of the warhead close to Cys862 and is not influenced by intrinsic warhead reactivity (Figure 4b,c). Compounds 19 and 22 displayed kinact/Ki values >10–20 times higher than that of CNX-1351 (41.4, 21.0 vs 1.74 × 10–5 M–1·s–1).
Table 1. Key Parameters for Acrylamide Derivatives (16–24), CNX-1351, Ibrutinib
compoundkchem × 104 (M–1·s–1)kinact PI3Kα × 104 (s–1)aKi PI3Kα (nM)akinact/Ki × 105 (nM–1·s–1)aIC50 pPKB (S473) SKOV3c
164.100.5810.80.5451
172.490.9810.70.9347
183.072.928.563.4348
193.7012.02.9341.482
204.522.5214.01.80664
214.371.9510.61.87686
223.5410.85.1421.086
233.883.7814.52.61250
243.864.0612.23.36260
19r 0b2.550b200
22r 0b6.150b227
CNX-13512.226.5738.01.74165
ibrutinib10.4    
a

Mean (and SD) were calculated from three independent measurements.

b

Values modeled with KinTek Global Kinetic Explorer are lower than 10–11 (nM–1·s–1), no covalent reaction was detected. The full dataset and SD are reported in Table S5.

c

Mean (and SD) were calculated from three independent measurements.

Stochastic Approach to Calculation of Covalent Reaction Rates as f(kchem, kinact, Ki, and Vr)

To better understand how the various kinetic parameters influence the overall covalent reaction rates, we developed a novel stochastic approach to describe the relation of kchem, kinact, Ki, and the distance of the warhead to the target cysteine (d), which defines the reactive volume Vr. Our proposed model unites the macroscopic and microscopic parameters of the covalent reaction and takes into account the probabilistic nature of the chemical reactions. Using Chemical Master Equations (CME), we derived a linear relation of the kchem/kinact ratio to the reactive volume Vr (eq 14a), for all equations and their full derivation, see the Supporting Information, SI.
(14a)
Here, σ is a scaling factor, which allows for the compensation for nonintrinsic warhead reactivity due to the steric effects and the warhead’s chemical environment. The application of the model to three groups of covalent PI3Kα inhibitors (2 and 10–12; 3 and 13–15a; 16–19 and 22) showed that kchem/kinact ratios plotted vs Vr fit a simple linear relationship (R2 = 0.98, 0.98 and 0.99, respectively; see Figure S7 and Table S6), and thus validate the proposed eq S14b.
Moreover, the scaling factor allows the association of the three investigated compound groups in unified kchem/kinact ratio to reactive volume Vr plots as depicted in Figure 4d. The excellent linear fit obtained in Figure 4d (R2 for 2, 10–12, 3, 13–15a; 16–19, and 22 was 0.988) characterizes σ as a useful predictor of changing compound properties, but also visualizes deviations in the targeting approach: compounds 20, 21, 23, and 24, with linker reaching beyond the targeted Cys862, appear below the regression line when fitted using the σ derived from compounds 16–19 and 22 based on the similarity of warhead structure and environment. This illustrates that the kinact values of 20, 21, 23, and 24 are larger than predicted based on the spherical approximation of Vr. The reason for this is that molecules with linkers reaching beyond the target Cys can bend and fold back to form Cys adducts while still bound to the ATP-binding site. Molecules with a short linker must detach from the ATP-binding site to reach the target Cys (Figure 3k,l).
Also, the resulting interactions between warhead and target cysteine may be altered by the steric constraints. Overall, the results indicate that molecules with longer linkers have an increased probability to bind to Cys862 as predicted from a distance from the warhead to the Cys when measured in a stretched conformation, which in turn yields higher kinact values and consequently lower kchem/kinact ratios. This shows some limitations of the current Vr estimation when outside of the validated distance from the target Cys (see Figure 4d). Future improvements and inclusion of steric factor calculations in the σ·Vr term will extend its validity across diverse structurally restricted linkers.
The stochastic approach also allows the integration of equations established by Copeland (61) and Strelow (62) to calculate kcat and kobs values and to predict overall rates as a function of kchem, kinact, Ki, and Vr
(15)
(17a)
These equations can be utilized to generally define the dependencies of reaction rates as a function of Ki and the inhibitor concentration, [I]: as shown in Figure 4e for the dependency of kcat on the distance to target Cys (d) and Ki, the reversible affinity to the target (Ki) is important, but an improvement in Ki by a factor of ∼10 can be matched by a closer approach to the target Cys by ca. 3Å. This illustrates that the distance of the warhead to the target cysteine dominates kcat. For kobs, the overall rate of the covalent reaction, values converge rapidly to a maximal value if [I] > Ki, but can still be dramatically accelerated by shortening the distance to the targeted nucleophile (Figure 4f).

Confirmation of Covalent Cys862 Engagement

The covalent bonds between compounds 19 and 22 and the targeted Cys862 in PI3Kα were confirmed by mass spectrometry and X-ray crystallography. Bottom-up LC-MS/MS-based proteomics demonstrated covalent modification of Cys862 by compounds 19 and 22. A reversible analogue of compound 22 (22r) was synthesized (Scheme S3) and used as a negative control. A selective covalent modification of Cys862-containing tryptic peptide (NSHTIMQIQCK) was observed for 19 and 22, but not for 22r. MS1 extracted-ion chromatogram (XIC) traces are reported in Figure S8. Afterward, the presence of modified forms of NSHTIMQIQCK was established by peptide-spectrum matching. All MS2 spectra were searched against the whole set of human entries of the Uniprot database (20 308 total protein entries). Only peptide spectrum matches with a high confidence (>0.9) were considered (see proteomics data in the Supporting Information). Among these, the only compound-modified peptide was NSHTIMQIQCK carrying 19 and 22 (Table S7). The fragment spectra were assigned with a very high confidence of >99.9% (Figure S9). LC-SRM was used to quantify the conversion rates of Cys862 by 19, 22, and 22r. The reactive compounds displayed a conversion rate >90% (19: 94% and 22: 92%), while no conversion was observed for the reversible analogue 22r (Figure 4g).
X-ray crystallographic studies with compounds 19 and 22 mapped continuous electron densities between the side-chain thiol group of Cys862 and the inhibitors’ acrylamide moiety, indicating the formation of a covalent bond between PI3Kα and the inhibitor (Figure 4h,i). Due to linker flexibility, a gap in the electron density map was observed for both 19 and 22 (Figure 4j,k). The core molecule maintained the major interactions observed for the PI3Kα-PQR530 complex (PDB ID: 6OAC, resolution 3.15 Å), (59) including the H-bond between the morpholine oxygen and the backbone amide of Val851, as well as between the NH2-group of the aminopyrimidine and Asp805/810. The CHF2-group could either interact (i) with the triazine core in an intramolecular manner or (ii) with Lys802 (distances <2.5 Å). The linker connecting the core module with the warhead is located in the solvent-exposed region and the carbonyl group linked to the piperazine establishes a pivotal interaction with Gln859 (distance <3.0 Å) for both 19 and 22 structures (Figure 4h,i). Met858 and Thr856 interact differentially with 19 and 22 linkers due to the higher rigidity of the tertiary amide, leading to an optimal stabilization of 19 (see Figure 4c for kinact/Ki values). Altogether these results confirm that 19 and 22 irreversibly inhibit PI3Kα by a covalent modification of Cys862.

NanoBRET Assay Detects Drug-Target Engagement in Cells

A bioluminescence resonance energy transfer (BRET) inhibitor displacement assay was used to dynamically quantify target engagement in living cells. The used NanoBRET approach exploits light emission from a small, bright, and stable Nanoluciferase (NanoLuc) (63,64) fused to PI3Ks, and an energy transfer to a fluorescent (a pyrrolylBODIPY tracer; Energy Transfer Probe 3 in ref (62)) tracer binding to the ATP-binding pocket of PI3Ks. We have examined the irreversible covalent behavior of 19 and 22 in HEK293 cells expressing NanoLuc-PI3K fusion proteins (PI3Kα, a PI3Kα Cys862 to Ser mutant, PI3Kβ, and PI3Kδ). The reversible analogues of 19 and 22 (19r and 22r) were included as negative controls (Figure 5), and CNX-1351 as a positive control (Figure S10). After exposure to 19 or 22, subsequent inhibitor washout, and tracer addition, the cells expressing wild-type PI3Kα did not show a BRET signal and tracer binding for >3 h (Figure 5a). In contrast, the PI3Kα C862S mutant displayed comparable dissociation rates for 19, 22, and their corresponding reversible analogues 19r and 22r (Figure 5b). Tracer binding and BRET signal increase were achieved also after drug washout in PI3Kβ and PI3Kδ (Figure 5c,d). The NanoBRET assays demonstrated therefore that compounds 19 and 22 selectively and irreversibly react with Cys862 in wild-type PI3Kα, but not with PI3Kα C862S, PI3Kβ, and PI3Kδ.

Figure 5

Figure 5. (a–d) Residence time experiment using BRET in intact HEK293 cells expressing NanoLuc fused to (a) PI3Kα, (b) PI3Kα Cys862Ser, (c) PI3Kβ, and (d) PI3Kδ. The cells were incubated with inhibitors (3 μM) for 2 h. Subsequently, free inhibitor was washed out twice using Opti-MEM medium (for 10 min at 37 °C and 5% CO2), before the cell-permeable BRET tracer was added (0.2 μM final concentration). A tracer bearing a pyrrolylBODIPY fluorescent moiety (83) (excitation at 460 nm and emission at 618 nm) was used to determine the on-target residence time of the inhibitors after drug washout. (64) Displacement of the inhibitors by the BRET tracer from the ATP-binding pocket in the indicated PI3K isoforms was monitored by the recovery of the BRET signal between the NanoLuc-fused PI3K (donor) and the BRET tracer (acceptor). A prolonged residence time of inhibitors (after drug washout) diminishes the BRET signal and points to a covalent interaction. Data shown are mean ± standard error of the mean (SEM) (n = 3). Results for CNX-1351 are shown in Figure S10.

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Validation of Extended Duration of Action in Cellular Washout Studies

The library of acrylamide-containing compounds (16–24) was evaluated in SKOV3 ovarian cancer cells with a constitutively active PI3Kα due to a His1047 to Arg mutation in PIK3CA (65) and enhanced expression of ERBB2 (HER2). (66) PI3K output was monitored by protein kinase B (PKB/Akt) phosphorylation on Ser473. Compounds 19 and 22 displayed a good cellular activity (IC50 for PKB/Akt phosphorylation was 82 and 86 nM, respectively), being twice as potent in SKOV3 cells compared to CNX-1351 (IC50 of 165 nM, Tables 1 and S5). Despite the poor efficiency in covalent bond formation, compounds 16–18 showed high cellular potency (IC50 of 51, 47, 48 nM, Table 1) due to the increased clog P compared to 19 and 22 (clog P: 16: 1.72; 17: 1.89; 18: 2.28; 19: 0.65; 22: 0.89, Table S5).
To gain further insight into long-term inhibition of PI3K signaling, cell lines with mutated, constitutively activated PI3Kα (MCF7 breast carcinoma, PIK3CA Glu545Lys; SKOV3 ovarian carcinoma, His1047Arg; and T47D breast carcinoma, His1047Arg) (65) were exposed to compounds 19, 22, and their reversible analogues (19r and 22r) lacking the Michael acceptor (see Scheme S3), as well as BYL719. BYL719 (35) was used as a reversible inhibitor benchmark. The cells were incubated with inhibitor (3 μM for 2 h) before free compound was washed out. Subsequently, PI3K signaling output was measured by in-cell western (ICW) detection of phosphorylated PKB/Akt. A complete and prolonged inhibition (>6 h) of PKB/Akt phosphorylation was observed in MCF7 (Figure 6a,b) and T47D (Figure 6c,d) cells after washout of 19 and 22, demonstrating that the irreversible elimination of PI3Kα activity was sufficient to fully downregulate all PI3K signaling. This suggests that mutated PI3Kα is the prevalent driver of PI3K signaling in these cells. In contrast, ca. 25% of PKB/Akt phosphorylation persisted in SKOV3 cells after the washout of 19 or 22 (Figure 6e,f), which is best explained by the enforced signaling of over-expressed HER2 to other class IA PI3K. In line with this, further addition of the PI3Kβ-selective inhibitor TGX221 (67) after the transient exposure to either 19 or 22 yielded a complete inhibition of PI3K signaling also in SKOV3 cells (Figure 6e,f).

Figure 6

Figure 6. (a–f) Cellular washout experiments in cancer cells with a mutated and constitutively activated PI3Kα. (a, b) MCF7 (PIK3CA E545K), (c, d) T47D (PIK3CA H1047R), and (e, f) SKOV3 (PIK3CA H1047R; HER2+) cancer cell lines were treated with 3 μM of the indicated compounds for 2 h, followed by drug washout (twice with fully supplemented Dulbecco’s modified Eagle’s medium (DMEM) for 10 min each). The cells were then incubated at 37 °C and 5% CO2 for the indicated times before they were fixed, and the phosphorylation status of PKB/Akt was determined by in-cell western; t = 0 values indicate reference measurements in the presence of inhibitor (i.e., immediately after the 2 h treatment window and without removal of drug). Where indicated, TGX221 (3 μM) was added 1 h before the fixation of cells, to investigate a potential cell-line-dependent further contribution of PI3Kβ to PKB/Akt phosphorylation. Data shown are mean ± SEM (n = 4; two independent measurements). Error bars are not shown when smaller than symbols. (g, h) Plot of clog D vs biological activity ((g) pKi; (h) pIC50) of compounds 19, 22, and CNX-1351. Lipophilic ligand efficiency (LipE) values higher than 5 are considered to be the threshold for lead compounds and high-quality chemical probes. LipE calculations are shown in Table S9. (i) On-target covalent modification modeled after 40 min using KinTek Global Kinetic Explorer. See Figure S1c,d for additional details. (j) Metabolic stability of 19, 22, and CNX-1351 using rat liver microsomes fortified with phase I metabolism cofactor NADPH. The time-dependent degradation of test items (starting concentration 1 μM) in the presence of rat liver microsomes (0.5 mg microsomal protein/mL) was measured (mean ± SD; n = 2). Error bars are not shown when smaller than symbols. Corresponding raw data are reported in Table S10.

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In the presence of all inhibitors (before washout, t = 0), including the reversible 19r, 22r, and BYL719, PI3K output was completely blocked. This illustrates that BYL719 also inhibited the TGX221-sensitive PI3Kβ signaling in SKOV3 cells and acted as pan-PI3Ki. Altogether, an incubation/washout cycle with 19 and 22 reveals reliably which part of a PI3K output is strictly PI3Kα-dependent, and qualifies 19 and 22 as specific chemical probes to investigate selectively PI3Kα signaling.

Lipophilicity and Lipophilic Ligand Efficiency (LipE)

Lipophilicity plays a central role in compounds’ pharmacokinetics and safety; thus, the concept of lipophilic ligand efficiency (LipE) has been developed to normalize potency relative to lipophilicity. The potential of achieving good in vivo performance increases when chemical modifications improved potency and simultaneously lower lipophilicity. (68) Proposed acceptable values of LipE for drug candidates are ≥5. (69) We calculated LipE values for our lead compounds (19 and 22) as well as CNX-1351 to evaluate the effect of the structural changes on both potency and lipophilicity. LipE profiles of CNX-1351 did not reach the standard threshold value of 5 [LipE (Ki) = 2.78; (IC50) = 2.13, Figure 6g,h; see Table S9 for calculations]. On the contrary, compounds 19 and 22 significantly increased potency and decreased lipophilicity, resulting in optimal LipE values [LipE (Ki) = 19, 7.89; 22, 7.40; LipE (IC50) = 19, 6.44; 22, 6.18, Figure 6g,h]. Employing LipE allowed to move into the desirable property space and to select high-quality chemical probes. Based on KinTek modeling using experimentally determined Ki and kinact, 19 and 22 are predicted to reach covalent modification of PI3Kα faster than with CNX-1351 (Figure 6i). KinTek modeling also predicts that all three compounds display negligible off-target reactivity (Figure S1d).

Assessment of Metabolic Stability

The metabolic stability of 19, 22, and CNX-1351 was evaluated in vitro in rat liver microsomes fortified with NADPH as a phase I metabolism cofactor. Compounds were tested at 1 μM concentration with liver microsomes and the percentage of remaining compounds after 60 min of incubation was detected. Verapamil was run in parallel as a high-clearance control compound. Compounds 19 and 22 were stable over the tested time frame with 97% of 19 and 87% of 22 remaining. CNX-1351 was rapidly metabolized with only 2% remaining after 60 min of incubation (Figure 6j and Table S10). Compounds 19 and 22, containing a moderately reactive, druglike warhead, as well as a metabolically favorable linker, outperformed the rapidly metabolized CNX-1351.

Profiling of Protein and Lipid Kinase Selectivity

Although off-target reactions with free thiols are negligible for 19 due to its low inherent reactivity, reversible binding to off-target proteins could generate the proximity to bring about off-target reactions. As 19 contains an ATP-site binding module, protein kinases are a possible target. The cysteinome of protein kinases has been elegantly reviewed by Chaikuad et al. (70) and provides a projection of the cysteinome to protein kinase A (PKA). Modeling of 19 into this template’s ATP-binding reveals only one close target cysteine at a distance of ca. 8 Å, which belongs to the c-Jun amino-terminal kinase (JNK1, 2, 3) family (Figure 7a, Cys154 in JNK3, labeled as F3), and previously targeted by the selective, covalent JNK-IN-8 inhibitor. (71)

Figure 7

Figure 7. (a) Analysis of putative off-target reactions of covalent PI3Kα inhibitors with protein kinases. Coordinates for a cysteinome protein kinase template were kindly provided by Chaikuad and Knapp. Their template was generated based on the PDB ID 1ATP of the catalytic subunit of cAMP-dependent protein kinase (PKA), onto which cysteines throughout the kinome have been projected as reported in ref (70). These coordinates were aligned with PI3K crystallographic data from PDB IDs 5OQ4 (PQR309/PI3Kγ complex), 1E8X (ATP/PI3Kγ complex), and structures of compound 19 bound to PI3Kα provided here, to dock 19 into the PKA template’s ATP-binding site. In the depicted compound 19 (dark blue), the sulfur atom of Cys862 (oversize yellow sphere) is located at a distance of 8.0 Å from Cys F3 (Cys annotation as in ref (70)), which corresponds to a Cys in JNK1, 2, and 3. Closer is only Cys F4 at a distance of 4.0 Å, which is the Cys862 of PI3Kα projected onto the PKA template. The rest of the projected protein kinase Cys residues are ≫10 Å away from the warhead of 19 and cannot be targeted without displacement of the ATP-binding module. (b) Validation of off-target binding of 19 at 1 μM to protein kinases in KINOMEScan experiments (comparisons with results from PQR514, PQR309, BYL719, GDC-0980, and PKI-587, and lipid kinase interactions are shown in Figure S11 and quantitative data are listed in Table S13). The three red spots in the kinase tree represent CSF1R (autoinhibited; juxtamembrane domains of some protein tyrosine kinase receptors stabilize the kinase domain in an inactive state), JAK1 (JH2 domain-pseudokinase), and KIT (autoinhibited) with remaining binding of 20, 32, and 35%, respectively. CSF1R, JAK1, and KIT (and mutant) catalytic domains (in active conformations) showed no relevant interactions with 19.

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To explore the selectivity of compound 19 for PI3K, a DiscoverX scanMAX kinase assay panel including a wide range of protein and lipid kinases was used. Compound 19 displayed negligible binding to protein kinases including JNK1-3 at a concentration of 1 μM (Figure 7b, full dataset and comparisons with PQR514, PQR309, BYL719, GDC-0980, and PKI-587 in Figure S11 and Table S13). The only minor hits involved CSF1R (autoinhibited), JAK1 (JH2 domain-pseudokinase), KIT (autoinhibited), with a remaining tracer binding of 20, 32, and 35%, respectively. At the same time, CSF1R, JAK1, and KIT (and mutant) catalytic domains showed no relevant interactions with 19, as also apparent from excellent selectivity scores (Table S12). This selectivity is based in part on the PI3K-binding motif derived from PQR514, a compound that shows a Kd of 2 nM for PI3Kα in a KdELECT assay (Table S11), which drops to 0.1 nM by a factor of 20 for 19.
Compound 19 is thus a potent covalent PI3Kα inhibitor and acts at higher concentrations as pan-PI3K. This profile allows the transient, reversible inhibition of class I PI3Ks (PI3Kβ, PI3Kδ, and PI3Kγ; Table S11), while PI3Kα inactivation persists after the washout of free inhibitor and completely abrogates PI3K signaling in cells with mutated, constitutively activated PI3Kα (Figure 6).
Our novel covalent tools have been fully characterized and met all of the criteria proposed in the validation process of a covalent tool compound, (72) including (i) irreversible ligand and control compounds; (ii) time-dependent in vitro activity; (iii) retained cell-based activity upon washout; (iv) confirmed target labeling using mass spectrometry; (v) confirmed residue-specific labeling using X-ray crystallography and/or MS; (vi) cell-based target validation (resistance mutation, C862S); and (vii) selectivity profiling (KinomeScan, TreeSpot representation).

Conclusions

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Selective irreversible inhibition relies on warhead reactivity and proximity. Here, we delineate a path to an efficient design to proximity-optimized druglike covalent inhibitors targeting remote cysteines. As moderate electrophiles with a distant target nucleophile do not produce hits in assays for covalency, we use fast-reactive warheads as tools in an early stage of the screening process to explore a bigger reactive space. The proximity/reactivity (kinact) information acquired as a function of linker length served then to design molecules with an expected placement of the warhead in close proximity to the target cysteine. At that stage, acrylamide-containing warheads could be introduced, avoiding metabolic instability and off-target thiol reactivity. The optimization of linkers allowed a close positioning of the electrophile relative to Cys862 in PI3Kα. A thorough assessment of intrinsic chemical reactivity (kchem) and rate of covalent bond formation (kinact) with PI3Kα provided a guided path to proximity-driven reactivity. Following this recipe, a two-order of magnitude increase in kinact was achieved, while the electrophilicity of the warhead remained unchanged. Finally, we have developed irreversible inhibitors of PI3Kα (19 and 22) with rapid and covalent bond formation and higher cellular potency compared to CNX-1351. (27) X-ray crystallography confirmed the covalent bond formation between the cysteine thiol and the acrylamide. This specificity for Cys862 in PI3Kα was confirmed by NanoBRET assays. Compounds 19 and 22 showed excellent stability in rat liver microsomes compared to CNX-1351, highlighting the use of the described approach to design metabolically stable warheads and linkers, and to properly direct the electrophiles to remote cysteines. Considering the importance of PI3Kα as a target in oncology and vascular malformations, irreversible PI3Kα inhibitors could provide highly specific, long-acting, on-target therapeutics with unprecedented pharmacological properties.
The presented workflow, including a stepwise optimization of proximity and metabolic stability, can serve as a template to target distal cysteines across the kinome, accelerating the characterization of the role of different kinases in human disease.

Methods

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Compounds Synthesis and Characterization

All final compounds were synthesized in a purity ≥95% (see the Supporting Information for synthetic procedures).1H,19F, and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer. NMR spectra were obtained in deuterated solvents, namely, CDCl3 or (CD3)2SO. The chemical shift (δ values) are reported in ppm and corrected to the signal of the deuterated solvents [7.26 ppm (1H NMR) and 77.16 ppm (13C NMR) for CDCl3; and 2.50 ppm (1H NMR) and 39.52 ppm (13C NMR) for (CD3)2SO].19F NMR spectra are calibrated relative to CFCl3 (δ = 0 ppm) as external standard (see the Supporting Information).

Determination of kchem Using β-Mercaptoethanol (βME)

The tested compounds (1 mM) were reacted with indicated concentrations of βME (Tables S2, S3, and S5) at 37 °C in a 4:1 DMSO/PBS (pH 7.4) mixture. The loss of inhibitor and formation of inhibitor-βME adduct was monitored by HPLC (Ultimate 3000SD System, ThermoFisher) at 20 min to 1 h intervals depending on reaction rates. Inhibitor-βME adduct was identified by MALDI-ToF MS (Voyager-DeTM Pro measured in m/z; see the MALDI-MS Spectra of β-Mercaptoethanol Adducts section in the Supporting Information). The experiments were performed in triplicate. For further details, see the Supporting Information.

Structure Modeling of PI3K and mTOR Kinase Complexes

The coordinates of PQR530 in PI3Kα (PDB ID 6OAC) (59) were used as starting points to dock molecules into the ATP-binding sites. Ligands in crystal structures were manually docked using Maestro 11.1 and energy minimization was subsequently carried out. Further measurements and figures were generated in Maestro 11.1 and Chimera UCSF.

Determination of Inhibitor Binding by TR-FRET

TR-FRET was performed by a LanthaScreen Technology (Life Technologies) assay as described in ref (42). In brief, displacement of AlexaFluor647-labeled Kinase Tracer 314 (#PV6087, Kd of 2.2 nM for PI3Kα) from a kinase/antibody/tracer (KAT) complex induced by the addition of increasing inhibitor concentrations was measured. The KAT was premixed in PI3Kα assay buffer composed of 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.01% (v/v) Brij-35 from recombinant N-terminally (His)6-tagged PI3Kα [(His)6-p110α/p85α complex, 5 nM, #PV4789], biotinylated anti-(His)6-tag antibody (2 nM, #PV6089), and LanthaScreen Eu-steptavidin (2 nM, #PV5899) as well as the AlexaFluor647-labeled Kinase Tracer 314 (20 nM). Rapid dispensing of inhibitors was performed on an I.DOT One dispenser (Dispendix). For experimental details and data analysis, refer to the Supporting Information.

Determination of Inhibitor Dissociation and Kinetic Constants

Dissociation constants (Ki) and the rate of covalent bond formation (kinact) for PI3Kα were determined by LanthaScreen Technology as above, and fluorescence changes were recorded as a function of time and inhibitor concentration. Recordings were started immediately after compound addition and continued at 60 s intervals for 1.5 h. FRET emission ratios were calculated by dividing blank-corrected FRET acceptor signals (AlexaFluor647-labeled Tracer 314, 665 nm) by donor signals (Eu-labeled steptavidin, 620 nm). Kinetic parameters (Ki and kinact) were obtained through the global fitting of time- and concentration-dependent FRET ratios using KinTek Global Kinetic Explorer software, (73,74) with adaptions to fit enzymatic covalent reaction data as previously described. (75,76) For details on the experimental setup and data analysis, see the Supporting Information.

KinTek Modeling of On-/Off-Target Reactions

The reaction progression curves of on-/off-target covalent binding for each inhibitor were modeled using KinTek Kinetic software. (73,74) The underlying enzymatic reaction mechanism is detailed in the Supporting Information.

Stochastic Approach to Calculation of Covalent Reaction Rates

The details of covalent binding reaction models and the derivation of stochastic and macroscopic equations are explained in detail in the Supporting Information.

Protein Mass Spectrometry

Details on samples preparation are reported in the Supporting Information. LC-MS/MS data were acquired in data-dependent mode on a system consisting of a Proxeon Ultra easy LC and an Orbitrap Elite (Thermo). Peptides were identified using the Trans-Proteomic-Pipeline (TPP) v5.1 with the search engine Myrimatch and the parameters: precursor tolerance: 10 ppm; fragment tolerance: 0.5 Da; dynamic modifications: iodoacetamide (C), oxidation (M); 19 (C), 22 (C) and 22r (C), enzyme: trypsin; missed cleavages: 2. Peptide probabilities were assigned with PeptideProphet and iProphet and the dataset was filtered with a peptide probability (iprobability) of 0.9. MS1 XIC traces of the Cys862-containing peptides with different covalent modifications were extracted using Skyline v4.2. The conversion rate of Cys862 through the compounds was quantified using LC-SRM.

Purification of PI3Kα Protein for Structural Studies

PI3Kα (ΔABD-LBS p110α 105–1048) was expressed by recombinant baculovirus in Sf9 cells, purified, and initially crystallized as previously published. (59,77) Additional details are provided in the Supporting Information.

Crystallography

For co-crystallization of 19 and 22, protein at 5.8 mg/mL was initially incubated for 20 h on ice with a 2-fold molar excess of inhibitor. Crystals collected for diffraction were obtained from 1 μL hanging drops containing 0.5 μL of inhibitor-bound protein mixed with 0.4 μL reservoir (6–10% poly(ethylene glycol) (PEG)6000, 0.6 M sodium formate, 0.1 M N-cyclohexyl-2-aminoethanesulfonic acid (CHES) pH 9.5, 5 mM tris(2-carboxyethyl)phosphine (TCEP) pH 7.5) and 0.1 μL of 1/1000 diluted micro-seeds crushed from a drop of the original apo crystals. Diffraction data for PI3K crystals were collected at beamline 08ID-1 of the Canadian Light Source (19 and 22). Data were collected at 12.670 keV (CLS) for 19 and 22. For details on the experimental setup, data analysis, and full data collection details, see the Supporting Information.

NanoBRET Target Engagement Assay

To investigate drug-target engagement on a cellular level, HEK293 cells were transiently transfected (using jetPEI from Polyplus-transfection, #101B-010N) with expression plasmids for nano-luciferase (NanoLuc) fused to the N-termini of the catalytic PI3K subunits p110α, p110α Cys862Ser, p110β, and p110δ (Promega #CS1810C52, #CS1810C250, #CS1810C390, and #CS1810C246) in combination with a vector for the regulatory p85α subunit. Transfected cells were seeded to 96-well assay plates (Falcon, #359296) in Opti-MEM I medium without phenol red (Gibco, #11058021) and exposed to inhibitors (3 μM) for 2 h (37 °C, 5% CO2). Inhibitors were then removed, and cells were washed twice with medium (10 min intervals, each). Finally, energy transfer probe (Tracer 03, Promega, # N260B), NanoGlo Substrate (Promega, #N157C), and extracellular NanoLuc inhibitor (Promega, #N235B) were added as detailed in the Supporting Information, before donor and acceptor luminescences were recorded on a Berthold Mithras (2) LB943 luminometer equipped with 460 ± 35 nm BP (donor) and 600 nm LP (acceptor) filters (integration time set to 0.5 s). To generate raw BRET ratio values, the acceptor emission intensity (618 nm) was divided by the donor emission intensity (460 nm). Background correction was performed by subtracting the BRET ratio in the absence of tracer (average of no tracer control) from the BRET ratio of each sample.

Determination of Cellular PKB/Akt Phosphorylation

For in-cell western (ICW) assays, 1.2 × 104 SKOV3 cells in DMEM (Sigma, D-5671) supplemented with 10% heat-inactivated fetal calf serum (FCS), l-glutamine (2 mM), and 1% penicillin–streptomycin were seeded into 96-well plates (Cell Carrier, PerkinElmer, #6005550) and pre-incubated for 18 h (37 °C, 5% CO2). Inhibitors (2 mM stock solutions in DMSO) were added using an I.DOT One dispenser (Dispendix) to generate a concentration range of 0.8 nM to 3 μM (11 intervals) in 0.05% DMSO per plate. Samples were incubated for 1 h (37 °C, 5% CO2), fixed (4% paraformaldehyde [PFA] in PBS for 30 min at RT), blocked and permeabilized (1% bovine serum albumin [BSA], 0.1% Triton X-100, 5% goat serum in PBS, 30 min at RT), and co-stained with rabbit monoclonal anti-phospho-Ser473 PKB/Akt (Cell Signaling Technology, #4058) and mouse anti-α-tubulin (Sigma, # T9026) antibodies. The following day, primary antibodies were removed by washes with PBS (1% BSA, 0.1% Triton X-100), and secondary antibodies (IRDye680-conjugated goat anti-mouse, and IRDye800-conjugated goat anti-rabbit antibodies) were added for 90 min (LICOR #926-68070 and #926-32211, shaking, RT, in the dark). After removal of secondary antibodies, fluorescence was finally recorded on an Odyssey CLx infrared imaging scanner (LICOR) and the ratio of pPKB (800 nm) over α-tubulin (680 nm) was determined, as further described in ref (42) and the Supporting Information.

In-Cell Western (ICW) Washout Experiments

For ICW assays with timed removal of inhibitor (so-called “washout” experiments), 1.2 × 104 SKOV3, 1.6 × 104 MCF7, or 1.4 × 104 T47D cells (propagated as indicated above) were seeded per well of a 96-well assay plate (Cell Carrier; PerkinElmer, #6005550) and incubated for 40 h (37 °C, 5% CO2). Inhibitor stock solutions (2 mM in DMSO) were diluted to 60 μM working solutions in DMEM medium (with 10% FCS, see above) and added to a final concentration of 3 μM (max. 0.15% DMSO) per well. After inhibitor addition, the cells were incubated for 2 h, then washed twice with medium to remove free inhibitors, and further incubated for the indicated times (37 °C, 5% CO2). Values at t = 0 represent the measurement in the presence of inhibitors (no washout). Where indicated, TGX221 was added (3 μM, 1 h prior to fixation) to probe for cell-line-dependent, PI3Kβ-derived contributions to pPKB/Akt formation. Finally, the cells were fixed, blocked, permeabilized, and stained for fluorescence detection as described above.

Metabolic Stability in Rat Liver Microsomes

The assay was performed using Sprague Dawley rat liver microsomes at 0.5 mg/mL. Details on the experimental setup are reported in the Supporting Information. The experiment was initiated by the addition of 70 μL of a 10 mM NADPH solution in phosphate buffer (100 mM, pH 7.4, supplemented with 2 mM MgCl2) to the prewarmed (37 °C) microsomes/buffer/test item mix. Samples (70 μL) were removed from the incubations after 0, 10, 30, and 60 min and transferred to the quenching plate for sample preparation containing acetonitrile (ACN) supplemented with the internal standards. The experimental test item incubations were run in duplicate (n = 2). Verapamil was used as a high-clearance positive control (n = 2) to demonstrate the microsomal CYP enzyme activity. For quantitative analysis of test and reference items, LC-MS systems were used (see the Supporting Information).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.1c13568.

  • Comparison of PI3Kα X-ray crystallographic complexes (Table S1); reaction rate constants (Table S2); KinTek modeling (Figure S1); time- and concentration-dependent TR-FRET data and time-dependent IC50 shift calculations (Figures S2 and S4); summary data (Tables S3 and S5); modeling into PI3Kα (Figures S3 and S5); estimation of reactive volume (Vr) and local concentration of warhead (Table S4); CovDock Schrödinger modeling (Figure S6); kchem/kinact ratio vs reactive volume relations (Figure S7); calculation of σ factors (Table S6); protein mass spectrometry (Figure S8); summary of compound-modified peptides (Table S7); MS/MS spectra (Figure S9); data collection and refinement statistics (molecular replacement, Table S8); NanoBRET experiments with CNX-1351 (Figure S10); lipophilic efficiency (Table S9); metabolic stability (Table S10); affinities to PI3K and PI3K-related kinases (PIKK, Table S11); TREEspot data visualization of KINOMEScan interactions (Figure S11); selectivity profile (Table S12); protein kinase interactions (KINOMEscan data, Table S13); time- and concentration-dependent TR-FRET determination of IC50s (Figures S12 and S13); comparison of chemical structures (Figure S14); synthetic Schemes S1–S3; detailed experimental procedures; synthesis and characterization of intermediates and final compounds;1H NMR,13C{1H} NMR, MALDI-MS, and high-resolution mass spectrometry (HRMS) spectra; HPLC chromatograms; MALDI-MS spectra of β-mercaptoethanol adducts; chemical structures of final compounds, intermediates, and reference compounds; KinTek global kinetic explorer data fitting of TR-FRET traces; and chemical structures of final compounds, intermediates, and reference compounds (PDF)

  • Proteomics data set file: peptide probabilities assigned with PeptideProphet and iProphet, MS1 XIC traces of the Cys862-containing peptides with different covalent modifications extracted using Skyline v4.2 (XLSX)

Accession Codes

The coordinates of compounds 19 and 22 covalently bound to the PI3Kα catalytic subunit p110α have been deposited at www.pdb.org and www.rcsb.org with PDB ID code 7R9V and 7R9Y, respectively.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Chiara Borsari - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland;  Orcidhttps://orcid.org/0000-0002-4688-8362
    • Erhan Keles - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
    • Jacob A. McPhail - Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada
    • Alexander Schaefer - Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
    • Rohitha Sriramaratnam - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
    • Wojciech Goch - Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland
    • Thorsten Schaefer - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
    • Martina De Pascale - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland;  Orcidhttps://orcid.org/0000-0002-7546-2766
    • Wojciech Bal - Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland;  Orcidhttps://orcid.org/0000-0003-3780-083X
    • Matthias Gstaiger - Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
    • John E. Burke - Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada;  Orcidhttps://orcid.org/0000-0001-7904-9859
  • Author Contributions

    C.B. and E.K. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Funding

    This work was supported by the Stiftung für Krebsbekämpfung grant 341; the Swiss National Science Foundation grants 310030_153211, 316030_133860, and 200021_204602; a Swiss Cancer Research KFS-5442-08-2021 grant (to M.P.W.); and the Peter and Traudl Engelhorn Foundation (to R.S.). J.E.B. was supported by a Michael Smith Foundation for Health Research (MSFHR) Scholar award (17686) and an operating grant from the Cancer Research Society (CRS-24368). M.G. was supported by EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank P. Hebeisen, D. Rageot, A. Melone, and T. Bohnacker for advice and discussions; A. Ontani, A. Christy, J. Klein, P. Knecht, L. Wisson, and R. Triaud for discussions and contribution to synthetic efforts and kinetic experiments; and M. Pfeffer and the mass spectrometry and elemental analysis team at the University of Basel for HRMS data. They also thank A. Chaikuad and S. Knapp for kindly providing the pdb coordinates of their cysteinome template kinase used to compose Figure 7a.

References

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This article references 83 other publications.

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    8
    Properties of FDA-approved small molecule protein kinase inhibitors: A 2022 update
    Roskoski Jr., Robert
    Pharmacological Research (2022), 175 (), 106037CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
    A review. Owing to the dysregulation of protein kinase activity in many diseases including cancer, this enzyme family has become one of the most important drug targets in the 21st century. There are 68 FDA-approved therapeutic agents that target about two dozen different protein kinases and six of these drugs were approved in 2021. Of the approved drugs, twelve target protein-serine/threonine protein kinases, four are directed against dual specificity protein kinases (MEK1/2), thirteen block nonreceptor protein-tyrosine kinases, and 39 target receptor protein-tyrosine kinases. The data indicate that 58 of these drugs are prescribed for the treatment of neoplasms (49 against solid tumors including breast, lung, and colon, five against nonsolid tumors such as leukemias, and four against both solid and nonsolid tumors: acalabrutinib, ibrutinib, imatinib, and midostaurin). Three drugs (baricitinib, tofacitinib, upadacitinib) are used for the treatment of inflammatory diseases including rheumatoid arthritis. Of the 68 approved drugs, eighteen are used in the treatment of multiple diseases. The following six drugs received FDA approval in 2021 for the treatment of these specified diseases: belumosudil (graft vs. host disease), infigratinib (cholangiocarcinomas), mobocertinib and tepotinib (specific forms of non-small cell lung cancer), tivozanib (renal cell carcinoma), and trilaciclib (to decrease chemotherapy-induced myelosuppression). All of the FDA-approved drugs are orally effective with the exception of netarsudil, temsirolimus, and the newly approved trilaciclib. This review summarizes the physicochem. properties of all 68 FDA-approved small mol. protein kinase inhibitors including lipophilic efficiency and ligand efficiency.
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  9. 9
    Riely, G. J.; Neal, J. W.; Camidge, D. R.; Spira, A. I.; Piotrowska, Z.; Costa, D. B.; Tsao, A. S.; Patel, J. D.; Gadgeel, S. M.; Bazhenova, L.; Zhu, V. W.; West, H. L.; Mekhail, T.; Gentzler, R. D.; Nguyen, D.; Vincent, S.; Zhang, S.; Lin, J.; Bunn, V.; Jin, S.; Li, S.; Jänne, P. A. Activity and Safety of Mobocertinib (TAK-788) in Previously Treated Non-Small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations from a Phase I/II Trial. Cancer Discovery 2021, 11, 1688– 1699,  DOI: 10.1158/2159-8290.Cd-20-1598
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    9
    Activity and safety of mobocertinib (TAK-788) in previousiytreated non-small cell lung cancer with EGFR exon 20 insertion mutations from a phase I/ll trial
    Riely, Gregory J.; Neal, Joel W.; Ross Camidge, D.; Spira, Alexander I.; Piotrowska, Zofia; Costa, Daniel B.; Tsao, Anne S.; Patel, Jyoti D.; Gadgeel, Shirish M.; Bazhenova, Lyudmila; Zhu, Viola W.; West, Howard L.; Mekhail, Tarek; Gentzler, Ryan D.; Nguyen, Danny; Vincent, Sylvie; Zhang, Steven; Lin, Jianchang; Bunn, Veronica; Jin, Shu; Li, Shuanglian; Janne, Pasi A.
    Cancer Discovery (2021), 11 (7), 1688-1699CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)
    Mobocertinib, an oral epidermal growth factor receptor (EGFR) inhibitor targeting EGFR gene mutations, including exon 20 insertions (EGFRex20ins), in nonsmall cell lung cancer, was evaluated in a phase I/ll dose-escalation/expansion trial (ClinicalTrials. gov NCT02716116). Dose escalation identified 160 mg/d as the recommended phase 2 dose and max. tolerated dose. Among 136 patients treated with 160 mg/d, the most common any-grade treatment-related adverse events (TRAE; >25%) were diarrhea (83%), nausea (43%), rash (33%), and vomiting (26%), with diarrhea (21%) the only grade ≥3 TRAE >5%. Among 28 EGFRex20ins patients treated at 160 mg/d, the investigator-assessed confirmed response rate was 43% (12/28; 95% confidence interval, 24%-63%) with median duration of response of 14 mo (5.0-not reached) and median progression-free survival of 7.3 mo (4.4-15.6). Mobocertinib demonstrated antitumor activity in patients with diverse EGFRex20ins variants with a safety profile consistent with other EGFR inhibitors. SIGNIFICANCE: No oral EGFR-targeted therapies are currently approved for patients with EGFRex- 20ins NSCLC. Mobocertinib demonstrated antitumor activity with manageable toxicity in patients with advanced EGFRex20ins NSCLC in this study, supporting addnl. development of mobocertinib in this patient population.
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  10. 10
    Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307– 317,  DOI: 10.1038/nrd3410
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    10
    The resurgence of covalent drugs
    Singh, Juswinder; Petter, Russell C.; Baillie, Thomas A.; Whitty, Adrian
    Nature Reviews Drug Discovery (2011), 10 (4), 307-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacol. advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.
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  11. 11
    London, N.; Miller, R. M.; Krishnan, S.; Uchida, K.; Irwin, J. J.; Eidam, O.; Gibold, L.; Cimermančič, P.; Bonnet, R.; Shoichet, B. K.; Taunton, J. Covalent docking of large libraries for the discovery of chemical probes. Nat. Chem. Biol. 2014, 10, 1066– 1072,  DOI: 10.1038/nchembio.1666
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    11
    Covalent docking of large libraries for the discovery of chemical probes
    London, Nir; Miller, Rand M.; Krishnan, Shyam; Uchida, Kenji; Irwin, John J.; Eidam, Oliv; Gibold, Lucie; Cimermancic, Peter; Bonnet, Richard; Shoichet, Brian K.; Taunton, Jack
    Nature Chemical Biology (2014), 10 (12), 1066-1072CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Chem. probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biol. studies. Despite these advantages, protein-reactive compds. are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small mols. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chem. probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).
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  12. 12
    Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60, 2879– 2889,  DOI: 10.1021/acs.jmedchem.6b01815
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    12
    Determining Cysteines Available for Covalent Inhibition Across the Human Kinome
    Zhao, Zheng; Liu, Qingsong; Bliven, Spencer; Xie, Lei; Bourne, Philip E.
    Journal of Medicinal Chemistry (2017), 60 (7), 2879-2889CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Covalently bound protein kinase inhibitors have been frequently designed to target non-catalytic Cys residues at the ATP binding site. Thus, it is important to know if a given Cys residue can form a covalent bond. Here, we combined a function-site interaction fingerprint method and DFT calcns. to det. the potential of Cys residues to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site Cys dataset was assembled. The orientation of the Cys SH group indicated which Cys residues could potentially form covalent bonds. These covalent inhibitor accessible Cys residues were located within 5 kinase regions (P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop and DFG-3 close to the DFG peptide). In an independent test set, these Cys residues covered 95% of covalent kinase inhibitors. Thus, this study provides new insights into Cys reactivity and preference which is important for the prospective development of covalent kinase inhibitors.
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  13. 13
    Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 2013, 20, 146– 159,  DOI: 10.1016/j.chembiol.2012.12.006
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    13
    Developing Irreversible Inhibitors of the Protein Kinase Cysteinome
    Liu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.
    Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 146-159CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    A review. Protein kinases are a large family of approx. 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clin. success of a no. of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large no. of kinases may represent therapeutically relevant targets. To date, the majority of clin. and preclin. kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a no. of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacol. specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biol. activity profiles. In addn., we provided an informatics assessment of the potential "kinase cysteinome" and discuss strategies for the efficient development of new covalent inhibitors.
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  14. 14
    Barf, T.; Covey, T.; Izumi, R.; van de Kar, B.; Gulrajani, M.; van Lith, B.; van Hoek, M.; de Zwart, E.; Mittag, D.; Demont, D.; Verkaik, S.; Krantz, F.; Pearson, P. G.; Ulrich, R.; Kaptein, A. Acalabrutinib (ACP-196): A Covalent Bruton Tyrosine Kinase Inhibitor with a Differentiated Selectivity and In Vivo Potency Profile. J. Pharmacol. Exp. Ther. 2017, 363, 240– 252,  DOI: 10.1124/jpet.117.242909
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    14
    Acalabrutinib (ACP-196): a covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile
    Barf, Tjeerd; Covey, Todd; Izumi, Raquel; van de Kar, Bas; Gulrajani, Michael; van Lith, Bart; van Hoek, Maaike; de Zwart, Edwin; Mittag, Diana; Demont, Dennis; Verkaik, Saskia; Krantz, Fanny; Pearson, Paul G.; Ulrich, Roger; Kaptein, Allard
    Journal of Pharmacology and Experimental Therapeutics (2017), 363 (2), 240-252CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)
    Several small-mol. Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacol. characterization of BTK inhibitor acalabrutinib [compd. 1, ACP-196 (4-[8- amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin1-yl]-N-(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochem. and cellular selectivity than ibrutinib and spebrutinib (compds. 2 and 3, resp.). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Detn. of the inhibitory potential of anti-IgM-induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclin. and clin. studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approx. 99% median target coverage at 3 and 12 h and around 90% at 24 h in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacol. differentiators vs. ibrutinib and spebrutinib and is currently being evaluated in clin. trials.
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  15. 15
    Cheng, H.; Planken, S. Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer. ACS Med. Chem. Lett. 2018, 9, 861– 863,  DOI: 10.1021/acsmedchemlett.8b00311
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    15
    Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer
    Cheng, Hengmiao; Planken, Simon
    ACS Medicinal Chemistry Letters (2018), 9 (9), 861-863CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are leading causes for lung cancer. Extensive drug discovery efforts targeting EGFR have led to the discovery and FDA approval of both reversible and covalent inhibitors. Second and third generation covalent inhibitors for EGFR have also been described, with the latter targeting specific emerging mutations. After decades of extensive effort, KRAS is widely regarded as an intractable therapeutic target; however, recent publications suggest covalent inhibition is a promising strategy to deliver inhibitors of the KRASG12C mutation.
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  16. 16
    Tomassi, S.; Lategahn, J.; Engel, J.; Keul, M.; Tumbrink, H. L.; Ketzer, J.; Mühlenberg, T.; Baumann, M.; Schultz-Fademrecht, C.; Bauer, S.; Rauh, D. Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor. J. Med. Chem. 2017, 60, 2361– 2372,  DOI: 10.1021/acs.jmedchem.6b01626
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    16
    Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor
    Tomassi, Stefano; Lategahn, Jonas; Engel, Julian; Keul, Marina; Tumbrink, Hannah L.; Ketzer, Julia; Muehlenberg, Thomas; Baumann, Matthias; Schultz-Fademrecht, Carsten; Bauer, Sebastian; Rauh, Daniel
    Journal of Medicinal Chemistry (2017), 60 (6), 2361-2372CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chem. entities that efficiently inhibit drug-resistant EGFR. Herein, the authors report the optimization process for a hit compd. that has emerged from a phenotypic screen resulting in indazole-based compds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug resistant EGFR-L858R/T790M and covalently alkylate Cys 797. Western blot anal., as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates the authors' approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.
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  17. 17
    Planken, S.; Behenna, D. C.; Nair, S. K.; Johnson, T. O.; Nagata, A.; Almaden, C.; Bailey, S.; Ballard, T. E.; Bernier, L.; Cheng, H.; Cho-Schultz, S.; Dalvie, D.; Deal, J. G.; Dinh, D. M.; Edwards, M. P.; Ferre, R. A.; Gajiwala, K. S.; Hemkens, M.; Kania, R. S.; Kath, J. C.; Matthews, J.; Murray, B. W.; Niessen, S.; Orr, S. T.; Pairish, M.; Sach, N. W.; Shen, H.; Shi, M.; Solowiej, J.; Tran, K.; Tseng, E.; Vicini, P.; Wang, Y.; Weinrich, S. L.; Zhou, R.; Zientek, M.; Liu, L.; Luo, Y.; Xin, S.; Zhang, C.; Lafontaine, J. Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR. J. Med. Chem. 2017, 60, 3002– 3019,  DOI: 10.1021/acs.jmedchem.6b01894
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    17
    Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR
    Planken, Simon; Behenna, Douglas C.; Nair, Sajiv K.; Johnson, Theodore O.; Nagata, Asako; Almaden, Chau; Bailey, Simon; Ballard, T. Eric; Bernier, Louise; Cheng, Hengmiao; Cho-Schultz, Sujin; Dalvie, Deepak; Deal, Judith G.; Dinh, Dac M.; Edwards, Martin P.; Ferre, Rose Ann; Gajiwala, Ketan S.; Hemkens, Michelle; Kania, Robert S.; Kath, John C.; Matthews, Jean; Murray, Brion W.; Niessen, Sherry; Orr, Suvi T. M.; Pairish, Mason; Sach, Neal W.; Shen, Hong; Shi, Manli; Solowiej, James; Tran, Khanh; Tseng, Elaine; Vicini, Paolo; Wang, Yuli; Weinrich, Scott L.; Zhou, Ru; Zientek, Michael; Liu, Longqing; Luo, Yiqin; Xin, Shuibo; Zhang, Chengyi; Lafontaine, Jennifer
    Journal of Medicinal Chemistry (2017), 60 (7), 3002-3019CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approx. 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed. Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clin. candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compd. 21 is currently being evaluated in phase-I clin. trials of mutant EGFR driven NSCLC.
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  18. 18
    Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent inhibitors of interleukin-2 inducible T cell kinase (itk) with nanomolar potency in a whole-blood assay. J. Med. Chem. 2012, 55, 10047– 10063,  DOI: 10.1021/jm301190s
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    18
    Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay
    Zapf, Christoph W.; Gerstenberger, Brian S.; Xing, Li; Limburg, David C.; Anderson, David R.; Caspers, Nicole; Han, Seungil; Aulabaugh, Ann; Kurumbail, Ravi; Shakya, Subarna; Li, Xin; Spaulding, Vikki; Czerwinski, Robert M.; Seth, Nilufer; Medley, Quintus G.
    Journal of Medicinal Chemistry (2012), 55 (22), 10047-10063CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concns. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogs are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approx. 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
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  19. 19
    Ward, R. A.; Colclough, N.; Challinor, M.; Debreczeni, J. E.; Eckersley, K.; Fairley, G.; Feron, L.; Flemington, V.; Graham, M. A.; Greenwood, R.; Hopcroft, P.; Howard, T. D.; James, M.; Jones, C. D.; Jones, C. R.; Renshaw, J.; Roberts, K.; Snow, L.; Tonge, M.; Yeung, K. Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2. J. Med. Chem. 2015, 58, 4790– 4801,  DOI: 10.1021/acs.jmedchem.5b00466
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    19
    Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2
    Ward, Richard A.; Colclough, Nicola; Challinor, Mairi; Debreczeni, Judit E.; Eckersley, Kay; Fairley, Gary; Feron, Lyman; Flemington, Vikki; Graham, Mark A.; Greenwood, Ryan; Hopcroft, Philip; Howard, Tina D.; James, Michael; Jones, Clifford D.; Jones, Christopher R.; Renshaw, Jonathan; Roberts, Karen; Snow, Lindsay; Tonge, Michael; Yeung, Kay
    Journal of Medicinal Chemistry (2015), 58 (11), 4790-4801CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The RAS/RAF/MEK/ERK signaling pathway has been targeted with a no. of small mol. inhibitors in oncol. clin. development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small mol. inhibitors. There are a no. of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogs) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chem. series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compds. for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.
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  20. 20
    Angst, D.; Gessier, F.; Janser, P.; Vulpetti, A.; Wälchli, R.; Beerli, C.; Littlewood-Evans, A.; Dawson, J.; Nuesslein-Hildesheim, B.; Wieczorek, G.; Gutmann, S.; Scheufler, C.; Hinniger, A.; Zimmerlin, A.; Funhoff, E. G.; Pulz, R.; Cenni, B. Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase. J. Med. Chem. 2020, 63, 5102– 5118,  DOI: 10.1021/acs.jmedchem.9b01916
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    20
    Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase
    Angst, Daniela; Gessier, Francois; Janser, Philipp; Vulpetti, Anna; Walchli, Rudolf; Beerli, Christian; Littlewood-Evans, Amanda; Dawson, Janet; Nuesslein-Hildesheim, Barbara; Wieczorek, Grazyna; Gutmann, Sascha; Scheufler, Clemens; Hinniger, Alexandra; Zimmerlin, Alfred; Funhoff, Enrico G.; Pulz, Robert; Cenni, Bruno
    Journal of Medicinal Chemistry (2020), 63 (10), 5102-5118CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncol. indications based on their suboptimal kinase selectivity. We describe the discovery and preclin. profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clin. studies for chronic spontaneous urticaria and Sjoegren's syndrome.
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  21. 21
    Gurbani, D.; Du, G.; Henning, N. J.; Rao, S.; Bera, A. K.; Zhang, T.; Gray, N. S.; Westover, K. D. Structure and Characterization of a Covalent Inhibitor of Src Kinase. Front. Mol. Biosci. 2020, 7, 81  DOI: 10.3389/fmolb.2020.00081
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    Structure and characterization of a covalent inhibitor of Src kinase
    Gurbani, Deepak; Du, Guangyan; Henning, Nathaniel J.; Rao, Suman; Bera, Asim K.; Zhang, Tinghu; Gray, Nathanael S.; Westover, Kenneth D.
    Frontiers in Molecular Biosciences (2020), 7 (), 00081CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
    Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clin., possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochem. and biophys. characterization of this compd. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic anal. shows a slow inactivation rate compared to other clin. approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.
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  22. 22
    Abdeldayem, A.; Raouf, Y. S.; Constantinescu, S. N.; Moriggl, R.; Gunning, P. T. Advances in covalent kinase inhibitors. Chem. Soc. Rev. 2020, 49, 2617– 2687,  DOI: 10.1039/c9cs00720b
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    Advances in covalent kinase inhibitors
    Abdeldayem, Ayah; Raouf, Yasir S.; Constantinescu, Stefan N.; Moriggl, Richard; Gunning, Patrick T.
    Chemical Society Reviews (2020), 49 (9), 2617-2687CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Over the past decade, covalent kinase inhibitors (CKI) have seen a resurgence in drug discovery. Covalency affords a unique set of advantages as well as challenges relative to their non-covalent counterpart. After reversible protein target recognition and binding, covalent inhibitors irreversibly modify a proximal nucleophilic residue on the protein via reaction with an electrophile. To date, the acrylamide group remains the predominantly employed electrophile in CKI development, with its incorporation in the majority of clin. candidates and FDA approved covalent therapies. Nonetheless, in recent years considerable efforts have ensued to characterize alternative electrophiles that exhibit irreversible or reversibly covalent binding mechanisms towards cysteine thiols and other amino acids. This review article provides a comprehensive overview of CKIs reported in the literature over a decade period, 2007-2018. Emphasis is placed on the rationale behind warhead choice, optimization approach, and inhibitor design. Current FDA approved CKIs are also highlighted, in addn. to a detailed anal. of the common trends and themes obsd. within the listed data set.
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  23. 23
    Yan, G.; Zhong, X.; Pu, C.; Yue, L.; Shan, H.; Lan, S.; Zhou, M.; Hou, X.; Yang, J.; Li, D.; Fan, S.; Li, R. Targeting Cysteine Located Outside the Active Site: An Effective Strategy for Covalent ALKi Design. J. Med. Chem. 2021, 64, 1558– 1569,  DOI: 10.1021/acs.jmedchem.0c01707
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    Targeting Cysteine Located Outside the Active Site: An Effective Strategy for Covalent ALKi Design
    Yan, Guoyi; Zhong, Xinxin; Pu, Chunlan; Yue, Lin; Shan, Huifang; Lan, Suke; Zhou, Meng; Hou, Xueyan; Yang, Jie; Li, Deyu; Fan, Shilong; Li, Rui
    Journal of Medicinal Chemistry (2021), 64 (3), 1558-1569CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Potent inhibitors of ALK are highly desired because of the occurrence of drug resistance. We herein firstly report the development of a rationally designed inhibitor, Con B-1, which can covalently bind to Cys1259, a cysteine located outside the ALK active site by linking a warhead with Ceritinib through a 2,2'-Oxybis(ethylamine) linker. The in vitro and in vivo assays showed ConB-1 is a potent selective ALKi with low toxicity to normal cells. In addn., the mol. showed significant improvement of anticancer activities and potential antidrug resistant activity compared with Ceritinib, demonstrating the covalent inhibitor of ALK can be a promising drug candidate for the treatment of NSCLC. This work may provide a novel perspective on the design of covalent inhibitors.
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  24. 24
    Jiang, B.; Jiang, J.; Kaltheuner, I. H.; Iniguez, A. B.; Anand, K.; Ferguson, F. M.; Ficarro, S. B.; Seong, B. K. A.; Greifenberg, A. K.; Dust, S.; Kwiatkowski, N. P.; Marto, J. A.; Stegmaier, K.; Zhang, T.; Geyer, M.; Gray, N. S. Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma. Eur. J. Med. Chem. 2021, 221, 113481  DOI: 10.1016/j.ejmech.2021.113481
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    24
    Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma
    Jiang, Baishan; Jiang, Jie; Kaltheuner, Ines H.; Iniguez, Amanda Balboni; Anand, Kanchan; Ferguson, Fleur M.; Ficarro, Scott B.; Seong, Bo Kyung Alex; Greifenberg, Ann Katrin; Dust, Sofia; Kwiatkowski, Nicholas P.; Marto, Jarrod A.; Stegmaier, Kimberly; Zhang, Tinghu; Geyer, Matthias; Gray, Nathanael S.
    European Journal of Medicinal Chemistry (2021), 221 (), 113481CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compds. inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chem. campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, i.p. administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.
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    Zhang, T.; Kwiatkowski, N.; Olson, C. M.; Dixon-Clarke, S. E.; Abraham, B. J.; Greifenberg, A. K.; Ficarro, S. B.; Elkins, J. M.; Liang, Y.; Hannett, N. M.; Manz, T.; Hao, M.; Bartkowiak, B.; Greenleaf, A. L.; Marto, J. A.; Geyer, M.; Bullock, A. N.; Young, R. A.; Gray, N. S. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 2016, 12, 876– 884,  DOI: 10.1038/nchembio.2166
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    Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors
    Zhang, Tinghu; Kwiatkowski, Nicholas; Olson, Calla M.; Dixon-Clarke, Sarah E.; Abraham, Brian J.; Greifenberg, Ann K.; Ficarro, Scott B.; Elkins, Jonathan M.; Liang, Yanke; Hannett, Nancy M.; Manz, Theresa; Hao, Mingfeng; Bartkowiak, Bartlomiej; Greenleaf, Arno L.; Marto, Jarrod A.; Geyer, Matthias; Bullock, Alex N.; Young, Richard A.; Gray, Nathanael S.
    Nature Chemical Biology (2016), 12 (10), 876-884CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play crit. roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystn. of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-assocd. transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small mols. capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.
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    Kwiatkowski, N.; Zhang, T.; Rahl, P. B.; Abraham, B. J.; Reddy, J.; Ficarro, S. B.; Dastur, A.; Amzallag, A.; Ramaswamy, S.; Tesar, B.; Jenkins, C. E.; Hannett, N. M.; McMillin, D.; Sanda, T.; Sim, T.; Kim, N. D.; Look, T.; Mitsiades, C. S.; Weng, A. P.; Brown, J. R.; Benes, C. H.; Marto, J. A.; Young, R. A.; Gray, N. S. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 2014, 511, 616– 620,  DOI: 10.1038/nature13393
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    Targeting transcription regulation in cancer with a covalent CDK7 inhibitor
    Kwiatkowski, Nicholas; Zhang, Tinghu; Rahl, Peter B.; Abraham, Brian J.; Reddy, Jessica; Ficarro, Scott B.; Dastur, Anahita; Amzallag, Arnaud; Ramaswamy, Sridhar; Tesar, Bethany; Jenkins, Catherine E.; Hannett, Nancy M.; McMillin, Douglas; Sanda, Takaomi; Sim, Taebo; Kim, Nam Doo; Look, Thomas; Mitsiades, Constantine S.; Weng, Andrew P.; Brown, Jennifer R.; Benes, Cyril H.; Marto, Jarrod A.; Young, Richard A.; Gray, Nathanael S.
    Nature (London, United Kingdom) (2014), 511 (7511), 616-620CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacol. inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here the authors present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide anal. in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumor cells. Pharmacol. modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types that are dependent on transcription for maintenance of the oncogenic state.
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    Nacht, M.; Qiao, L.; Sheets, M. P.; St Martin, T.; Labenski, M.; Mazdiyasni, H.; Karp, R.; Zhu, Z.; Chaturvedi, P.; Bhavsar, D.; Niu, D.; Westlin, W.; Petter, R. C.; Medikonda, A. P.; Singh, J. Discovery of a Potent and Isoform-Selective Targeted Covalent Inhibitor of the Lipid Kinase PI3Kα. J. Med. Chem. 2013, 56, 712– 721,  DOI: 10.1021/jm3008745
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    Discovery of a Potent and Isoform-Selective Targeted Covalent Inhibitor of the Lipid Kinase PI3Kα
    Nacht, Mariana; Qiao, Lixin; Sheets, Michael P.; St. Martin, Thia; Labenski, Matthew; Mazdiyasni, Hormoz; Karp, Russell; Zhu, Zhendong; Chaturvedi, Prasoon; Bhavsar, Deepa; Niu, Deqiang; Westlin, William; Petter, Russell C.; Medikonda, Aravind Prasad; Singh, Juswinder
    Journal of Medicinal Chemistry (2013), 56 (3), 712-721CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallog., that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC50 < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI50 < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biol. impact of selectively targeting PI3Kα.
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    Wymann, M. P.; Schneiter, R. Lipid signalling in disease. Nat. Rev. Mol. Cell Biol. 2008, 9, 162– 176,  DOI: 10.1038/nrm2335
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    Lipid signalling in disease
    Wymann, Matthias P.; Schneiter, Roger
    Nature Reviews Molecular Cell Biology (2008), 9 (2), 162-176CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. Signaling lipids such as eicosanoids, phosphoinositides, sphingolipids and fatty acids control important cellular processes, including cell proliferation, apoptosis, metab. and migration. Extracellular signals from cytokines, growth factors and nutrients control the activity of a key set of lipid-modifying enzymes: phospholipases, prostaglandin synthase, 5-lipoxygenase, phosphoinositide 3-kinase, sphingosine kinase and sphingomyelinase. These enzymes and their downstream targets constitute a complex lipid signaling network with multiple nodes of interaction and cross-regulation. Imbalances in this network contribute to the pathogenesis of human disease. Although the function of a particular signaling lipid is traditionally studied in isolation, this review attempts a more integrated overview of the key role of these signaling lipids in inflammation, cancer and metabolic disease, and discusses emerging strategies for therapeutic intervention.
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    Fruman, D. A.; Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discovery 2014, 13, 140– 156,  DOI: 10.1038/nrd4204
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    PI3K and cancer: lessons, challenges and opportunities
    Fruman, David A.; Rommel, Christian
    Nature Reviews Drug Discovery (2014), 13 (2), 140-156CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. The central role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biol. has prompted a sizeable effort to target PI3K and/or downstream kinases such as AKT and mammalian target of rapamycin (mTOR) in cancer. However, emerging clin. data show limited single-agent activity of inhibitors targeting PI3K, AKT or mTOR at tolerated doses. One exception is the response to PI3Kδ inhibitors in chronic lymphocytic leukemia, where a combination of cell-intrinsic and -extrinsic activities drive efficacy. Here, we review key challenges and opportunities for the clin. development of inhibitors targeting the PI3K-AKT-mTOR pathway. Through a greater focus on patient selection, increased understanding of immune modulation and strategic application of rational combinations, it should be possible to realize the potential of this promising class of targeted anticancer agents.
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    Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.; Gazdar, A.; Powell, S. M.; Riggins, G. J.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.; Velculescu, V. E. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004, 304, 554,  DOI: 10.1126/science.1096502
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    Brevia: High frequency of mutations of the PIK3Ca gene in human cancers
    Samuels, Yardena; Wang, Zhenghe; Bardelli, Alberto; Silliman, Natalie; Ptak, Janine; Szabo, Steve; Yan, Hai; Gazdar, Adi; Powell, Steven M.; Riggins, Gregory J.; Willson, James K. V.; Markowitz, Sanford; Kinzler, Kenneth W.; Vogelstein, Bert; Velculescu, Victor E.
    Science (Washington, DC, United States) (2004), 304 (5670), 554CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    There is no expanded citation for this reference.
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    Vanhaesebroeck, B.; Perry, M. W. D.; Brown, J. R.; André, F.; Okkenhaug, K. PI3K inhibitors are finally coming of age. Nat. Rev. Drug Discovery 2021, 20, 741– 769,  DOI: 10.1038/s41573-021-00209-1
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    Phosphoinositide 3-kinase inhibitors are finally coming of age
    Vanhaesebroeck, Bart; Perry, Matthew W. D.; Brown, Jennifer R.; Andre, Fabrice; Okkenhaug, Klaus
    Nature Reviews Drug Discovery (2021), 20 (10), 741-769CODEN: NRDDAG; ISSN:1474-1776. (Nature Portfolio)
    A review. Overactive phosphoinositide 3-kinase (PI3K) in cancer and immune dysregulation has spurred extensive efforts to develop therapeutic PI3K inhibitors. Although progress has been hampered by issues such as poor drug tolerance and drug resistance, several PI3K inhibitors have now received regulatory approval-the PI3Kα isoform-selective inhibitor alpelisib for the treatment of breast cancer and inhibitors mainly aimed at the leukocyte-enriched PI3Kδ in B cell malignancies. In addn. to targeting cancer cell-intrinsic PI3K activity, emerging evidence highlights the potential of PI3K inhibitors in cancer immunotherapy. This Review summarizes key discoveries that aid the clin. translation of PI3Kα and PI3Kδ inhibitors, highlighting lessons learnt and future opportunities.
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    Yang, J.; Nie, J.; Ma, X.; Wei, Y.; Peng, Y.; Wei, X. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol. Cancer 2019, 18, 26  DOI: 10.1186/s12943-019-0954-x
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    Targeting PI3K in cancer: mechanisms and advances in clinical trials
    Yang Jing; Nie Ji; Ma Xuelei; Wei Yuquan; Peng Yong; Wei Xiawei
    Molecular cancer (2019), 18 (1), 26 ISSN:.
    Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is one of the most important intracellular pathways, which can be considered as a master regulator for cancer. Enormous efforts have been dedicated to the development of drugs targeting PI3K signaling, many of which are currently employed in clinical trials evaluation, and it is becoming increasingly clear that PI3K inhibitors are effective in inhibiting tumor progression. PI3K inhibitors are subdivided into dual PI3K/mTOR inhibitors, pan-PI3K inhibitors and isoform-specific inhibitors. In this review, we performed a critical review to summarize the role of the PI3K pathway in tumor development, recent PI3K inhibitors development based on clinical trials, and the mechanisms of resistance to PI3K inhibition.
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    Massacesi, C.; Di Tomaso, E.; Urban, P.; Germa, C.; Quadt, C.; Trandafir, L.; Aimone, P.; Fretault, N.; Dharan, B.; Tavorath, R.; Hirawat, S. PI3K inhibitors as new cancer therapeutics: implications for clinical trial design. OncoTargets Ther. 2016, 9, 203– 210,  DOI: 10.2147/OTT.S89967
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    PI3K inhibitors as new cancer therapeutics: implications for clinical trial design
    Massacesi, Cristian; Di Tomaso, Emmanuelle; Urban, Patrick; Germa, Caroline; Quadt, Cornelia; Trandafir, Lucia; Aimone, Paola; Fretault, Nathalie; Dharan, Bharani; Tavorath, Ranjana; Hirawat, Samit
    OncoTargets and Therapy (2016), 9 (), 203-210CODEN: OTNHAZ; ISSN:1178-6930. (Dove Medical Press Ltd.)
    The PI3K-AKT-mTOR pathway is frequently activated in cancer. PI3K inhibitors, including the pan-PI3K inhibitor buparlisib (BKM120) and the PI3Ka-selective inhibitor alpelisib (BYL719), currently in clin. development by Novartis Oncol., may therefore be effective as anticancer agents. Early clin. studies with PI3K inhibitors have demonstrated preliminary antitumor activity and acceptable safety profiles. However, a no. of unanswered questions regarding PI3K inhibition in cancer remain, including: what is the best approach for different tumor types, and which biomarkers will accurately identify the patient populations most likely to benefit from specific PI3K inhibitors. This review summarizes the strategies being employed by Novartis Oncol. to help maximize the benefits of clin. studies with buparlisib and alpelisib, including stratification according to PI3K pathway activation status, selective enrollment/target enrichment (where patients with PI3K pathway-activated tumors are specifically recruited), nonselective enrollment with mandatory tissue collection, and enrollment of patients who have progressed on previous targeted agents, such as mTOR inhibitors or endocrine therapy. An overview of Novartis-sponsored and Novartis-supported trials that are utilizing these approaches in a range of cancer types, including breast cancer, head and neck squamous cell carcinoma, non-small cell lung carcinoma, lymphoma, and glioblastoma multiforme, is also described.
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    Mishra, R.; Patel, H.; Alanazi, S.; Kilroy, M. K.; Garrett, J. T. PI3K Inhibitors in Cancer: Clinical Implications and Adverse Effects. Int. J. Mol. Sci. 2021, 22, 3464  DOI: 10.3390/ijms22073464
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    PI3K inhibitors in cancer: clinical implications and adverse effects
    Mishra, Rosalin; Patel, Hima; Alanazi, Samar; Kilroy, Mary Kate; Garrett, Joan T.
    International Journal of Molecular Sciences (2021), 22 (7), 3464CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. The phospatidylinositol-3 kinase (PI3K) pathway is a crucial intracellular signaling pathway which is mutated or amplified in a wide variety of cancers including breast, gastric, ovarian, colorectal, prostate, glioblastoma and endometrial cancers. PI3K signaling plays an important role in cancer cell survival, angiogenesis and metastasis, making it a promising therapeutic target. There are several ongoing and completed clin. trials involving PI3K inhibitors (pan, isoform-specific and dual PI3K/mTOR) with the goal to find efficient PI3K inhibitors that could overcome resistance to current therapies. This review focuses on the current landscape of various PI3K inhibitors either as monotherapy or in combination therapies and the treatment outcomes involved in various phases of clin. trials in different cancer types. There is a discussion of the drug-related toxicities, challenges assocd. with these PI3K inhibitors and the adverse events leading to treatment failure. In addn., novel PI3K drugs that have potential to be translated in the clinic are highlighted.
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    Fritsch, C.; Huang, A.; Chatenay-Rivauday, C.; Schnell, C.; Reddy, A.; Liu, M.; Kauffmann, A.; Guthy, D.; Erdmann, D.; De Pover, A.; Furet, P.; Gao, H.; Ferretti, S.; Wang, Y.; Trappe, J.; Brachmann, S. M.; Maira, S. M.; Wilson, C.; Boehm, M.; Garcia-Echeverria, C.; Chene, P.; Wiesmann, M.; Cozens, R.; Lehar, J.; Schlegel, R.; Caravatti, G.; Hofmann, F.; Sellers, W. R. Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials. Mol. Cancer Ther. 2014, 13, 1117– 1129,  DOI: 10.1158/1535-7163.Mct-13-0865
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    Characterization of the Novel and Specific PI3Kα Inhibitor NVP-BYL719 and Development of the Patient Stratification Strategy for Clinical Trials
    Fritsch, Christine; Huang, Alan; Chatenay-Rivauday, Christian; Schnell, Christian; Reddy, Anupama; Liu, Manway; Kauffmann, Audrey; Guthy, Daniel; Erdmann, Dirk; De Pover, Alain; Furet, Pascal; Gao, Hui; Ferretti, Stephane; Wang, Youzhen; Trappe, Joerg; Brachmann, Saskia M.; Maira, Sauveur-Michel; Wilson, Christopher; Boehm, Markus; Garcia-Echeverria, Carlos; Chene, Patrick; Wiesmann, Marion; Cozens, Robert; Lehar, Joseph; Schlegel, Robert; Caravatti, Giorgio; Hofmann, Francesco; Sellers, William R.
    Molecular Cancer Therapeutics (2014), 13 (5), 1117-1129CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects assocd. with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biol. properties of the 2-aminothiazole deriv. NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compd. selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclin. platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost pos. predictor of sensitivity while revealing addnl. pos. and neg. assocns. such as PIK3CA amplification and PTEN mutation, resp. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clin. trials. Mol Cancer Ther; 13(5); 1117-29. ©2014 AACR.
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    Markham, A. Alpelisib: First Global Approval. Drugs 2019, 79, 1249– 1253,  DOI: 10.1007/s40265-019-01161-6
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    Alpelisib: First Global Approval
    Markham, Anthony
    Drugs (2019), 79 (11), 1249-1253CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)
    A review. Alpelisib (Piqray)-an orally available phosphatidylinositol 3-kinase (PI3K) inhibitor with specific activity against PI3K alpha (PI3Kα)-is being developed by Novartis for the treatment of breast cancer. Alpelisib has demonstrated efficacy in combination with fulvestrant as treatment for hormone receptor (HR)-pos., human epidermal growth factor receptor-2 (HER2)-neg. breast cancer in patients with a PIK3CA mutation and was recently approved for this indication in the USA. This article summarizes the milestones in the development of alpelisib leading to this first approval.
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    Narayan, P.; Prowell, T. M.; Gao, J. J.; Fernandes, L. L.; Li, E.; Jiang, X.; Qiu, J.; Fan, J.; Song, P.; Yu, J.; Zhang, X.; King-Kallimanis, B. L.; Chen, W.; Ricks, T. K.; Gong, Y.; Wang, X.; Windsor, K.; Rhieu, S. Y.; Geiser, G.; Banerjee, A.; Chen, X.; Reyes Turcu, F.; Chatterjee, D. K.; Pathak, A.; Seidman, J.; Ghosh, S.; Philip, R.; Goldberg, K. B.; Kluetz, P. G.; Tang, S.; Amiri-Kordestani, L.; Theoret, M. R.; Pazdur, R.; Beaver, J. A. FDA Approval Summary: Alpelisib Plus Fulvestrant for Patients with HR-positive, HER2-negative, PIK3CA-mutated, Advanced or Metastatic Breast Cancer. Clin. Cancer Res. 2021, 27, 1842– 1849,  DOI: 10.1158/1078-0432.Ccr-20-3652
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    37
    FDA approval summary: alpelisib plus fulvestrant for patients with HR-positive, HER2-negative, PIK3CA-mutated, advanced or metastatic breast cancer
    Narayan, Preeti; Prowell, Tatiana M.; Gao, Jennifer J.; Fernandes, Laura L.; Li, Emily; Jiang, Xiling; Qiu, Junshan; Fan, Jianghong; Song, Pengfei; Yu, Jingyu; Zhang, Xinyuan; King-Kallimanis, Bellinda L.; Chen, Wei; Ricks, Tiffany K.; Gong, Yutao; Wang, Xing; Windsor, Katherine; Rhieu, Steve Y.; Geiser, Gerlie; Banerjee, Anamitro; Chen, Xiaohong; Turcu, Francisca Reyes; Chatterjee, Deb K.; Pathak, Anand; Seidman, Jeffrey; Ghosh, Soma; Philip, Reena; Goldberg, Kirsten B.; Kluetz, Paul G.; Tang, Shenghui; Amiri-Kordestani, Laleh; Theoret, Marc R.; Pazdur, Richard; Beaver, Julia A.
    Clinical Cancer Research (2021), 27 (7), 1842-1849CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    On May 24, 2019, the FDA granted regular approval to alpelisib in combination with fulvestrant for postmenopausal women, and men, with hormone receptor (HR)-pos., HER2-neg., phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. Approval was based on the SOLAR-1 study, a randomized, double-blind, placebo-controlled trial of alpelisib plus fulvestrant vs. placebo plus fulvestrant. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST v1.1 in the cohort of trial participants whose tumors had a PIK3CA mutation. The estd. median PFS by investigator assessment in the alpelisib plus fulvestrant arm was 11 mo [95% confidence interval (CI), 7.5-14.5] compared with 5.7 mo (95% CI, 3.7-7.4) in the placebo plus fulvestrant arm (HR, 0.65; 95% CI, 0.50-0.85; two-sided P = 0.001). The median overall survival was not yet reached for the alpelisib plus fulvestrant arm (95% CI, 28.1-NE) and was 26.9 mo (95% CI, 21.9-NE) for the fulvestrant control arm. No PFS benefit was obsd. in trial participants whose tumors did not have a PIK3CA mutation (HR, 0.85; 95% CI, 0.58-1.25). The most common adverse reactions, including lab. abnormalities, on the alpelisib plus fulvestrant arm were increased glucose, increased creatinine, diarrhea, rash, decreased lymphocyte count, increased gamma glutamyl transferase, nausea, increased alanine aminotransferase, fatigue, decreased Hb, increased lipase, decreased appetite, stomatitis, vomiting, decreased wt., decreased calcium, decreased glucose, prolonged activated partial thromboplastin time, and alopecia.
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    André, F.; Ciruelos, E. M.; Juric, D.; Loibl, S.; Campone, M.; Mayer, I. A.; Rubovszky, G.; Yamashita, T.; Kaufman, B.; Lu, Y. S.; Inoue, K.; Pápai, Z.; Takahashi, M.; Ghaznawi, F.; Mills, D.; Kaper, M.; Miller, M.; Conte, P. F.; Iwata, H.; Rugo, H. S. Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: final overall survival results from SOLAR-1. Ann. Oncol. 2021, 32, 208– 217,  DOI: 10.1016/j.annonc.2020.11.011
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    Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: final overall survival results from SOLAR-1
    Andre, F.; Ciruelos, E. M.; Juric, D.; Loibl, S.; Campone, M.; Mayer, I. A.; Rubovszky, G.; Yamashita, T.; Kaufman, B.; Lu, Y.-S.; Inoue, K.; Papai, Z.; Takahashi, M.; Ghaznawi, F.; Mills, D.; Kaper, M.; Miller, M.; Conte, P. F.; Iwata, H.; Rugo, H. S.
    Annals of Oncology (2021), 32 (2), 208-217CODEN: AONNAY; ISSN:1569-8041. (Elsevier Ltd.)
    Activation of the phosphatidylinositol-3-kinase (PI3K) pathway via PIK3CA mutations occurs in 28%-46% of hormone receptor-pos. (HR+), human epidermal growth factor receptor-2-neg. (HER2-) advanced breast cancers (ABCs) and is assocd. with poor prognosis. The SOLAR-1 trial showed that the addn. of alpelisib to fulvestrant treatment provided statistically significant and clin. meaningful progression-free survival (PFS) benefit in PIK3CA-mutated, HR+, HER2- ABC. Men and postmenopausal women with HR+, HER2- ABC whose disease progressed on or after aromatase inhibitor (AI) were randomized 1 : 1 to receive alpelisib (300 mg/day) plus fulvestrant (500 mg every 28 days and once on day 15) or placebo plus fulvestrant. Overall survival (OS) in the PIK3CA-mutant cohort was evaluated by Kaplan-Meier methodol. and a one-sided stratified log-rank test was carried out with an O'Brien-Fleming efficacy boundary of P ≤ 0.0161. In the PIK3CA-mutated cohort (n = 341), median OS [95% confidence interval (CI)] was 39.3 mo (34.1-44.9) for alpelisib-fulvestrant and 31.4 mo (26.8-41.3) for placebo-fulvestrant [hazard ratio (HR) = 0.86 (95% CI, 0.64-1.15; P = 0.15)]. OS results did not cross the prespecified efficacy boundary. Median OS (95% CI) in patients with lung and/or liver metastases was 37.2 mo (28.7-43.6) and 22.8 mo (19.0-26.8) in the alpelisib-fulvestrant and placebo-fulvestrant arms, resp. [HR = 0.68 (0.46-1.00)]. Median times to chemotherapy (95% CI) for the alpelisib-fulvestrant and placebo-fulvestrant arms were 23.3 mo (15.2-28.4) and 14.8 mo (10.5-22.6), resp. [HR = 0.72 (0.54-0.95)]. No new safety signals were obsd. with longer follow-up. Although the anal. did not cross the prespecified boundary for statistical significance, there was a 7.9-mo numeric improvement in median OS when alpelisib was added to fulvestrant treatment of patients with PIK3CA-mutated, HR+, HER2- ABC. Overall, these results further support the statistically significant prolongation of PFS obsd. with alpelisib plus fulvestrant in this population, which has a poor prognosis due to a PIK3CA mutation.
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    Turner, S.; Chia, S.; Kanakamedala, H.; Hsu, W. C.; Park, J.; Chandiwana, D.; Ridolfi, A.; Yu, C. L.; Zarate, J. P.; Rugo, H. S. Effectiveness of Alpelisib + Fulvestrant Compared with Real-World Standard Treatment Among Patients with HR+, HER2-, PIK3CA-Mutated Breast Cancer. Oncologist 2021, 26, e1133– e1142,  DOI: 10.1002/onco.13804
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    Effectiveness of Alpelisib + Fulvestrant Compared with Real-World Standard Treatment Among Patients with HR +, HER2 -, PIK3CA-Mutated Breast Cancer
    Turner, Stuart; Chia, Stephen; Kanakamedala, Hemanth; Hsu, Wei-Chun; Park, Jinhee; Chandiwana, David; Ridolfi, Antonia; Yu, Chu-Ling; Zarate, Juan Pablo; Rugo, Hope S.
    Oncologist (2021), 26 (7), e1133-e1142CODEN: OCOLF6; ISSN:1549-490X. (AlphaMed Press)
    The BYLieve trial (NCT03056755) confirmed efficacy and safety of alpelisib with fulvestrant for hormone receptor-pos. (HR+), human epidermal growth factor receptor-2-neg. (HER2-), PIK3CA-mutated advanced breast cancer (ABC), after cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) with an aromatase inhibitor (AI) as immediate prior therapy. Further analyses were performed to compare efficacy from BYLieve with effectiveness of std. treatment in the real-world setting. Patients who progressed on a CDK4/6i plus AI and were treated with alpelisib with fulvestrant in BYLieve were matched with a real-world patient cohort who received std.-of-care from a deidentified clinico-genomics database (CGDB). Primary and secondary endpoints were to compare progression-free survival (PFS), estd. by the Kaplan-Meier method, and the proportion of patients remaining progression-free at 6 mo, resp., between the two cohorts. A total of 855 patients with PIK3CA-mutant disease who had prior CDK4/6i plus hormone therapy were selected from the CGDB; further matching to 120 patients from BYLieve selected 95 patients without exposure to HER2-targeting agents, clin. study drug, or alpelisib. In unadjusted and postmatching results, primary and secondary endpoints favored treatment with alpelisib with fulvestrant in BYLieve more than std. treatments in the real-world cohort. Postadjustment, median PFS for patients treated with alpelisib in BYLieve was 7.3 vs. 3.7 mo in the real-world cohort, and 6-mo PFS was 54.6% vs. 40.1%, resp. Matched/weighted anal. comparing BYLieve with the real-world setting further supports the clin. benefit of alpelisib with fulvestrant for treatment of HR+, HER2-, PIK3CA-mutant ABC after CDK4/6i treatment. Approx. 40% of patients with hormone receptor-pos. (HR+), human epidermal growth factor receptor-2-neg. (HER2-) advanced breast cancer (ABC) have PIK3CA-mutated tumors, which have been assocd. with endocrine therapy resistance. Alpelisib, an α-selective phosphatidylinositol-3-kinase inhibitor, demonstrated significantly improved progression-free survival in SOLAR-1 and demonstrated clin. efficacy in BYLieve when combined with fulvestrant. Data are limited in comparing the efficacy of alpelisib combined with fulvestrant with effectiveness of std. therapy after CDK4/6i treatment. Using real-world data, this is the first anal. comparing alpelisib combined with fulvestrant with std. treatments for HR+, HER2-, PIK3CA-mutant ABC in the post-CDK4/6i setting.
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    Verret, B.; Cortes, J.; Bachelot, T.; Andre, F.; Arnedos, M. Efficacy of PI3K inhibitors in advanced breast cancer. Ann. Oncol. 2019, 30, x12– x20,  DOI: 10.1093/annonc/mdz381
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    Venot, Q.; Blanc, T.; Rabia, S. H.; Berteloot, L.; Ladraa, S.; Duong, J. P.; Blanc, E.; Johnson, S. C.; Hoguin, C.; Boccara, O.; Sarnacki, S.; Boddaert, N.; Pannier, S.; Martinez, F.; Magassa, S.; Yamaguchi, J.; Knebelmann, B.; Merville, P.; Grenier, N.; Joly, D.; Cormier-Daire, V.; Michot, C.; Bole-Feysot, C.; Picard, A.; Soupre, V.; Lyonnet, S.; Sadoine, J.; Slimani, L.; Chaussain, C.; Laroche-Raynaud, C.; Guibaud, L.; Broissand, C.; Amiel, J.; Legendre, C.; Terzi, F.; Canaud, G. Targeted therapy in patients with PIK3CA-related overgrowth syndrome. Nature 2018, 558, 540– 546,  DOI: 10.1038/s41586-018-0217-9
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    Targeted therapy in patients with PIK3CA-related overgrowth syndrome
    Venot, Quitterie; Blanc, Thomas; Rabia, Smail Hadj; Berteloot, Laureline; Ladraa, Sophia; Duong, Jean-Paul; Blanc, Estelle; Johnson, Simon C.; Hoguin, Clement; Boccara, Olivia; Sarnacki, Sabine; Boddaert, Nathalie; Pannier, Stephanie; Martinez, Frank; Magassa, Sato; Yamaguchi, Junna; Knebelmann, Bertrand; Merville, Pierre; Grenier, Nicolas; Joly, Dominique; Cormier-Daire, Valerie; Michot, Caroline; Bole-Feysot, Christine; Picard, Arnaud; Soupre, Veronique; Lyonnet, Stanislas; Sadoine, Jeremy; Slimani, Lotfi; Chaussain, Catherine; Laroche-Raynaud, Cecile; Guibaud, Laurent; Broissand, Christine; Amiel, Jeanne; Legendre, Christophe; Terzi, Fabiola; Canaud, Guillaume
    Nature (London, United Kingdom) (2018), 558 (7711), 540-546CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumors became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not assocd. with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.
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    Bohnacker, T.; Prota, A. E.; Beaufils, F.; Burke, J. E.; Melone, A.; Inglis, A. J.; Rageot, D.; Sele, A. M.; Cmiljanovic, V.; Cmiljanovic, N.; Bargsten, K.; Aher, A.; Akhmanova, A.; Diaz, J. F.; Fabbro, D.; Zvelebil, M.; Williams, R. L.; Steinmetz, M. O.; Wymann, M. P. Deconvolution of Buparlisib’s mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention. Nat. Commun. 2017, 8, 14683  DOI: 10.1038/ncomms14683
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    Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention
    Bohnacker Thomas; Beaufils Florent; Melone Anna; Rageot Denise; Sele Alexander M; Cmiljanovic Vladimir; Cmiljanovic Natasa; Wymann Matthias P; Prota Andrea E; Bargsten Katja; Steinmetz Michel O; Burke John E; Inglis Alison J; Williams Roger L; Aher Amol; Akhmanova Anna; Diaz J Fernando; Fabbro Doriano; Zvelebil Marketa
    Nature communications (2017), 8 (), 14683 ISSN:.
    BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.
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    Beaufils, F.; Cmiljanovic, N.; Cmiljanovic, V.; Bohnacker, T.; Melone, A.; Marone, R.; Jackson, E.; Zhang, X.; Sele, A.; Borsari, C.; Mestan, J.; Hebeisen, P.; Hillmann, P.; Giese, B.; Zvelebil, M.; Fabbro, D.; Williams, R. L.; Rageot, D.; Wymann, M. P. 5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a potent, brain-penetrant, orally bioavailable, pan-class I PI3K/mTOR inhibitor as clinical candidate in oncology. J. Med. Chem. 2017, 60, 7524– 7538,  DOI: 10.1021/acs.jmedchem.7b00930
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    5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology
    Beaufils, Florent; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Bohnacker, Thomas; Melone, Anna; Marone, Romina; Jackson, Eileen; Zhang, Xuxiao; Sele, Alexander; Borsari, Chiara; Mestan, Jurgen; Hebeisen, Paul; Hillmann, Petra; Giese, Bernd; Zvelebil, Marketa; Fabbro, Doriano; Williams, Roger L.; Rageot, Denise; Wymann, Matthias P.
    Journal of Medicinal Chemistry (2017), 60 (17), 7524-7538CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclin. characterization of compd. 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concns. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was obsd. for the structurally related 4 (BKM120, buparlisib). Compd. 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compd. 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compd.'s safety profile, identifies 1 as a clin. candidate with a broad application range in oncol., including treatment of brain tumors or CNS metastasis. Compd. 1 is currently in phase II clin. trials for advanced solid tumors and refractory lymphoma.
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    Tarantelli, C.; Gaudio, E.; Arribas, A. J.; Kwee, I.; Hillmann, P.; Rinaldi, A.; Cascione, L.; Spriano, F.; Bernasconi, E.; Guidetti, F.; Carrassa, L.; Pittau, R. B.; Beaufils, F.; Ritschard, R.; Rageot, D.; Sele, A.; Dossena, B.; Rossi, F. M.; Zucchetto, A.; Taborelli, M.; Gattei, V.; Rossi, D.; Stathis, A.; Stussi, G.; Broggini, M.; Wymann, M. P.; Wicki, A.; Zucca, E.; Cmiljanovic, V.; Fabbro, D.; Bertoni, F. PQR309 is a novel dual PI3K/mTOR inhibitor with preclinical antitumor activity in lymphomas as a single agent and in combination therapy. Clin. Cancer Res. 2018, 24, 120– 129,  DOI: 10.1158/1078-0432.CCR-17-1041
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    PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy
    Tarantelli, Chiara; Gaudio, Eugenio; Arribas, Alberto J.; Kwee, Ivo; Hillmann, Petra; Rinaldi, Andrea; Cascione, Luciano; Spriano, Filippo; Bernasconi, Elena; Guidetti, Francesca; Carrassa, Laura; Pittau, Roberta Bordone; Beaufils, Florent; Ritschard, Reto; Rageot, Denise; Sele, Alexander; Dossena, Barbara; Rossi, Francesca Maria; Zucchetto, Antonella; Taborelli, Monica; Gattei, Valter; Rossi, Davide; Stathis, Anastasios; Stussi, Georg; Broggini, Massimo; Wymann, Matthias P.; Wicki, Andreas; Zucca, Emanuele; Cmiljanovic, Vladimir; Fabbro, Doriano; Bertoni, Francesco
    Clinical Cancer Research (2018), 24 (1), 120-129CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacol. inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clin. evaluation, in preclin. lymphoma models. Exptl. Design: This study included preclin. in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation expts. on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was assocd. with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compd. that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120-9. ©2017 AACR.
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  45. 45
    Wicki, A.; Brown, N.; Xyrafas, A.; Bize, V.; Hawle, H.; Berardi, S.; Cmiljanovic, N.; Cmiljanovic, V.; Stumm, M.; Dimitrijevic, S.; Herrmann, R.; Pretre, V.; Ritschard, R.; Tzankov, A.; Hess, V.; Childs, A.; Hierro, C.; Rodon, J.; Hess, D.; Joerger, M.; von Moos, R.; Sessa, C.; Kristeleit, R. First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13). Eur. J. Cancer 2018, 96, 6– 16,  DOI: 10.1016/j.ejca.2018.03.012
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    45
    First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13)
    Wicki, Andreas; Brown, Nicholas; Xyrafas, Alexandros; Bize, Vincent; Hawle, Hanne; Berardi, Simona; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Stumm, Michael; Dimitrijevic, Sasa; Herrmann, Richard; Pretre, Vincent; Ritschard, Reto; Tzankov, Alexandar; Hess, Viviane; Childs, Alexa; Hierro, Cinta; Rodon, Jordi; Hess, Dagmar; Joerger, Markus; von Moos, Roger; Sessa, Cristiana; Kristeleit, Rebecca
    European Journal of Cancer (2018), 96 (), 6-16CODEN: EJCAEL; ISSN:0959-8049. (Elsevier Ltd.)
    PQR309 is an orally bioavailable, balanced pan-phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR) C1 and mTORC2 inhibitor. This is an accelerated titrn., 3 D 3 dose-escalation, open-label phase Itrial of continuous once-daily (OD) PQR309 administration to evaluate the safety, pharmacokinetics (PK) and pharmacodynamics in patients with advanced solid tumors. Primary objectives were to det. the max. tolerated dose (MTD) and recommended phase 2 dose (RP2D).Twenty-eight patients were included in six dosing cohorts and treated at a daily PQR309 dose ranging from 10 to 150 mg. Common adverse events (AEs; ≥30% patients) included fatigue, hyperglycemia, nausea, diarrhea, constipation, rash, anorexia and vomiting. Grade (G) 3 or 4 drug-related AEs were seen in 13 (46%) and three (11%) patients, resp. Dose-limiting toxicity (DLT) was obsd. in two patients at 100 mg OD (>14-d interruption in PQR309 due to G3 rash, G2 hyperbilirubinemia, G4 suicide attempt; dose redn. due to G3 fatigue, G2 diarrhoea, G4 transaminitis) and one patient at 80 mg (G3 hyperglycemia >7 d). PK shows fast absorption (Tmax 1-2 h) and dose proportionality for Cmax and area under the curve. A partial response in a patient with metastatic thymus cancer, 24% disease vol. redn. in a patient with sinonasal cancer and stable disease for more than 16 wk in a patient with clear cell Bartholin's gland cancer were obsd.The MTD and RP2D of PQR309 is 80 mg of orally OD. PK is dose-proportional. PD shows PI3K pathway phosphoprotein downregulation in paired tumor biopsies. Clin. activity was obsd. in patients with and without PI3K pathway dysregulation.
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  46. 46
    Borsari, C.; Rageot, D.; Beaufils, F.; Bohnacker, T.; Keles, E.; Buslov, I.; Melone, A.; Sele, A. M.; Hebeisen, P.; Fabbro, D.; Hillmann, P.; Wymann, M. P. Preclinical Development of PQR514, a Highly Potent PI3K Inhibitor Bearing a Difluoromethyl-Pyrimidine Moiety. ACS Med. Chem. Lett. 2019, 10, 1473– 1479,  DOI: 10.1021/acsmedchemlett.9b00333
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    Preclinical Development of PQR514, a Highly Potent PI3K Inhibitor Bearing a Difluoromethyl-Pyrimidine Moiety
    Borsari, Chiara; Rageot, Denise; Beaufils, Florent; Bohnacker, Thomas; Keles, Erhan; Buslov, Ivan; Melone, Anna; Sele, Alexander M.; Hebeisen, Paul; Fabbro, Doriano; Hillmann, Petra; Wymann, Matthias P.
    ACS Medicinal Chemistry Letters (2019), 10 (10), 1473-1479CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is a crit. regulator of cell growth and is frequently hyperactivated in cancer. Therefore, PI3K inhibitors represent a valuable asset in cancer therapy. Herein we have developed a novel anticancer agent, the potent pan-PI3K inhibitor PQR514 (4), which is a follow-up compd. for the phase-II clin. compd. PQR309 (1). Compd. 4 has an improved potency both in vitro and in cellular assays with respect to its predecessor compds. It shows superiority in the suppression of cancer cell proliferation and demonstrates significant antitumor activity in an OVCAR-3 xenograft model at concns. approx. eight times lower than PQR309 (1). The favorable pharmacokinetic profile and a minimal brain penetration promote PQR514 (4) as an optimized candidate for the treatment of systemic tumors.
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  47. 47
    Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60, 839– 885,  DOI: 10.1021/acs.jmedchem.6b00788
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    47
    Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions
    Jackson, Paul A.; Widen, John C.; Harki, Daniel A.; Brummond, Kay M.
    Journal of Medicinal Chemistry (2017), 60 (3), 839-885CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogs possessing this moiety. In the midst of recently approved acrylamide-contg. drugs, it is clear that a good understanding of the hetero-Michael addn. reaction and the relative reactivities of biol. thiols with Michael acceptors under physiol. conditions is needed for the design and use of these compds. as biol. tools and potential therapeutics. This Perspective provides information that will contribute to this understanding, such as kinetics of thiol addn. reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This Perspective is focused on α,β-unsatd. carbonyls given their preponderance in bioactive natural products.
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  48. 48
    Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J. Med. Chem. 2014, 57, 10072– 10079,  DOI: 10.1021/jm501412a
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    Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors
    Flanagan, Mark E.; Abramite, Joseph A.; Anderson, Dennis P.; Aulabaugh, Ann; Dahal, Upendra P.; Gilbert, Adam M.; Li, Chao; Montgomery, Justin; Oppenheimer, Stacey R.; Ryder, Tim; Schuff, Brandon P.; Uccello, Daniel P.; Walker, Gregory S.; Wu, Yan; Brown, Matthew F.; Chen, Jinshan M.; Hayward, Matthew M.; Noe, Mark C.; Obach, R. Scott; Philippe, Laurence; Shanmugasundaram, Veerabahu; Shapiro, Michael J.; Starr, Jeremy; Stroh, Justin; Che, Ye
    Journal of Medicinal Chemistry (2014), 57 (23), 10072-10079CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry addnl. risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biol. relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.
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    Martin, J. S.; MacKenzie, C. J.; Fletcher, D.; Gilbert, I. H. Characterising covalent warhead reactivity. Bioorg. Med. Chem. 2019, 27, 2066– 2074,  DOI: 10.1016/j.bmc.2019.04.002
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    Characterising covalent warhead reactivity
    Martin, James S.; MacKenzie, Claire J.; Fletcher, Daniel; Gilbert, Ian H.
    Bioorganic & Medicinal Chemistry (2019), 27 (10), 2066-2074CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    Many drugs currently used are covalent inhibitors and irreversibly inhibit their targets. Most of these were discovered through serendipity. Covalent inhibitions can have many advantages from a pharmacokinetic perspective. However, until recently most organizations have shied away from covalent compd. design due to fears of non-specific inhibition of off-target proteins leading to toxicity risks. However, there has been a renewed interest in covalent modifiers as potential drugs, as it possible to get highly selective compds. It is therefore important to know how reactive a warhead is and to be able to select the least reactive warhead possible to avoid toxicity. A robust NMR based assay was developed and used to measure the reactivity of a variety of covalent warheads against serine and cysteine - the two most common targets for covalent drugs. A selection of these warheads also had their reactivity measured against threonine, tyrosine, lysine, histidine and arginine to better understand our ability to target non-traditional residues. The reactivity was also measured at various pHs to assess what effect the environment in the active site would have on these reactions. The reactivity of a covalent modifier was found to be very dependent on the amino acid residue.
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    Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57, 3124– 3137,  DOI: 10.1021/acs.jcim.7b00553
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    Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity
    Lonsdale, Richard; Burgess, Jonathan; Colclough, Nicola; Davies, Nichola L.; Lenz, Eva M.; Orton, Alexandra L.; Ward, Richard A.
    Journal of Chemical Information and Modeling (2017), 57 (12), 3124-3137CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compds. for target validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or "warhead"). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity. The half-life to adduct formation with glutathione (GSH t1/2) is a useful assay for measuring the reactivity of cysteine-targeting covalent warheads but is limited to synthesized mols. In this manuscript the authors assess the ability of several exptl. and computational approaches to predict GSH t1/2 for a range of cysteine targeting warheads, including a novel method based on pKa. Furthermore, matched mol. pairs anal. has been performed against the internal compd. collection, revealing structure-activity relationships between a selection of different covalent warheads. These observations and methods of prediction will be valuable in the design of new covalent inhibitors with desired levels of reactivity.
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    Ma, S.; Devi-Kesavan, L. S.; Gao, J. Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K. J. Am. Chem. Soc. 2007, 129, 13633– 13645,  DOI: 10.1021/ja074222+
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    Molecular Dynamics Simulations of the Catalytic Pathway of a Cysteine Protease: A Combined QM/MM Study of Human Cathepsin K
    Ma, Shuhua; Devi-Kesavan, Lakshmi S.; Gao, Jiali
    Journal of the American Chemical Society (2007), 129 (44), 13633-13645CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Mol. dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have detd. the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aq. soln. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aq. soln. The redn. of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 Å and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.
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  52. 52
    Cusack, K. P.; Arnold, L. D.; Barberis, C. E.; Chen, H.; Ericsson, A. M.; Gaza-Bulseco, G. S.; Gordon, T. D.; Grinnell, C. M.; Harsch, A.; Pellegrini, M.; Tarcsa, E. A 13C NMR approach to categorizing potential limitations of alpha,beta-unsaturated carbonyl systems in drug-like molecules. Bioorg. Med. Chem. Lett. 2004, 14, 5503– 5507,  DOI: 10.1016/j.bmcl.2004.09.007
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    A 13C NMR approach to categorizing potential limitations of α,β-unsaturated carbonyl systems in drug-like molecules
    Cusack, Kevin P.; Arnold, Lee D.; Barberis, Claude E.; Chen, Haipeng; Ericsson, Anna M.; Gaza-Bulseco, Georgeen S.; Gordon, Thomas D.; Grinnell, Christine M.; Harsch, Andreas; Pellegrini, Maria; Tarcsa, Edit
    Bioorganic & Medicinal Chemistry Letters (2004), 14 (22), 5503-5507CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
    Compds. that contain an α,β-unsatd. carbonyl moiety are often flagged as potential Michael acceptors. All α,β-unsatd. carbonyl moieties are not equiv., however, and we sought to better understand this system and its potential implications in drug-like mols. Measurement of the 13C NMR shift of the β-carbon and correlation to in vitro results allowed compds. in our collection to be categorized as potential Michael acceptors, potential substrates for NADPH, or as photoisomerizable.
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    MacFaul, P. A.; Morley, A. D.; Crawford, J. J. A simple in vitro assay for assessing the reactivity of nitrile containing compounds. Bioorg. Med. Chem. Lett. 2009, 19, 1136– 1138,  DOI: 10.1016/j.bmcl.2008.12.105
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    A simple in vitro assay for assessing the reactivity of nitrile containing compounds
    MacFaul, Philip A.; Morley, Andrew D.; Crawford, James J.
    Bioorganic & Medicinal Chemistry Letters (2009), 19 (4), 1136-1138CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
    A quant. assay involving the reaction of nitriles with glutathione and cysteine has been used as a simple in vitro screen to assess potential toxicity risk of candidate compds. in drug discovery. Studies have indicated that, when benchmarked with selected compds., the reaction of the nitriles with glutathione can provide a useful tool for deciding whether or not to progress compds. in the absence of radiolabelling studies.
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  54. 54
    Brameld, K. A.; Owens, T. D.; Verner, E.; Venetsanakos, E.; Bradshaw, J. M.; Phan, V. T.; Tam, D.; Leung, K.; Shu, J.; LaStant, J.; Loughhead, D. G.; Ton, T.; Karr, D. E.; Gerritsen, M. E.; Goldstein, D. M.; Funk, J. O. Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors. J. Med. Chem. 2017, 60, 6516– 6527,  DOI: 10.1021/acs.jmedchem.7b00360
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    Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors
    Brameld, Ken A.; Owens, Timothy D.; Verner, Erik; Venetsanakos, Eleni; Bradshaw, J. Michael; Phan, Vernon T.; Tam, Danny; Leung, Kwan; Shu, Jin; LaStant, Jacob; Loughhead, David G.; Ton, Tony; Karr, Dane E.; Gerritsen, Mary E.; Goldstein, David M.; Funk, Jens Oliver
    Journal of Medicinal Chemistry (2017), 60 (15), 6516-6527CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clin. validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncol. indications. An irreversible covalent binding mechanism imparts many desirable pharmacol. benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chem. optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compd. 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.
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    Cee, V. J.; Volak, L. P.; Chen, Y.; Bartberger, M. D.; Tegley, C.; Arvedson, T.; McCarter, J.; Tasker, A. S.; Fotsch, C. Systematic Study of the Glutathione (GSH) Reactivity of N-Arylacrylamides: 1. Effects of Aryl Substitution. J. Med. Chem. 2015, 58, 9171– 9178,  DOI: 10.1021/acs.jmedchem.5b01018
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    Systematic Study of the Glutathione (GSH) Reactivity of N-Arylacrylamides: 1. Effects of Aryl Substitution
    Cee, Victor J.; Volak, Laurie P.; Chen, Yuping; Bartberger, Michael D.; Tegley, Chris; Arvedson, Tara; McCarter, John; Tasker, Andrew S.; Fotsch, Christopher
    Journal of Medicinal Chemistry (2015), 58 (23), 9171-9178CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relations among N-arylacrylamides, the authors detd. glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. Substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. The authors note a correlation between 1H and 13C NMR chem. shifts of the acrylamide with GSH reaction rates, suggesting that NMR chem. shifts may be a convenient surrogate measure of relative acrylamide reactivity. D. functional theory calcns. reveal a correlation between computed activation parameters and exptl. detd. reaction rates, validating the use of such methodol. for the screening of synthetic candidates in a prospective fashion.
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  56. 56
    Birkholz, A.; Kopecky, D. J.; Volak, L. P.; Bartberger, M. D.; Chen, Y.; Tegley, C. M.; Arvedson, T.; McCarter, J. D.; Fotsch, C.; Cee, V. J. Systematic Study of the Glutathione Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution. J. Med. Chem. 2020, 63, 11602– 11614,  DOI: 10.1021/acs.jmedchem.0c00749
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    56
    Systematic Study of the Glutathione Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution
    Birkholz, Adam; Kopecky, David J.; Volak, Laurie P.; Bartberger, Michael D.; Chen, Yuping; Tegley, Christopher M.; Arvedson, Tara; McCarter, John D.; Fotsch, Christopher; Cee, Victor J.
    Journal of Medicinal Chemistry (2020), 63 (20), 11602-11614CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A comprehensive understanding of structure-reactivity relationships is crit. to the design and optimization of cysteine-targeted covalent inhibitors. Herein, we report glutathione (GSH) reaction rates for N-Ph acrylamides with varied substitutions at the α- and β-positions of the acrylamide moiety. We find that the GSH reaction rates can generally be understood in terms of the electron donating or withdrawing ability of the substituent. When installed at the β-position, aminomethyl substituents with amine pKa's > 7 accelerate, while those with pKa's < 7 slow the rate of GSH addn. at pH 7.4, relative to a hydrogen substituent. Although a computational model was able to only approx. capture exptl. reactivity trends, our calcns. do not support a frequently invoked mechanism of concerted amine/thiol proton transfer and C-S bond formation and instead suggest that protonated aminomethyl functions as an electron-withdrawing group to reduce the barrier for thiolate addn. to the acrylamide.
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    Zhu, K.; Borrelli, K. W.; Greenwood, J. R.; Day, T.; Abel, R.; Farid, R. S.; Harder, E. Docking covalent inhibitors: a parameter free approach to pose prediction and scoring. J. Chem. Inf. Model. 2014, 54, 1932– 1940,  DOI: 10.1021/ci500118s
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    Docking Covalent Inhibitors: A Parameter Free Approach To Pose Prediction and Scoring
    Zhu, Kai; Borrelli, Kenneth W.; Greenwood, Jeremy R.; Day, Tyler; Abel, Robert; Farid, Ramy S.; Harder, Edward
    Journal of Chemical Information and Modeling (2014), 54 (7), 1932-1940CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    Although many popular docking programs include a facility to account for covalent ligands, large-scale systematic docking validation studies of covalent inhibitors have been sparse. In this paper, we present the development and validation of a novel approach for docking and scoring covalent inhibitors, which consists of conventional noncovalent docking, heuristic formation of the covalent attachment point, and structural refinement of the protein-ligand complex. This approach combines the strengths of the docking program Glide and the protein structure modeling program Prime and does not require any parameter fitting for the study of addnl. covalent reaction types. We first test this method by predicting the native binding geometry of 38 covalently bound complexes. The av. RMSD of the predicted poses is 1.52 Å, and 76% of test set inhibitors have an RMSD of less than 2.0 Å. In addn., the apparent affinity score constructed herein is tested on a virtual screening study and the characterization of the SAR properties of two different series of congeneric compds. with satisfactory success.
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    Toledo Warshaviak, D.; Golan, G.; Borrelli, K. W.; Zhu, K.; Kalid, O. Structure-based virtual screening approach for discovery of covalently bound ligands. J. Chem. Inf. Model. 2014, 54, 1941– 1950,  DOI: 10.1021/ci500175r
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    Structure-Based Virtual Screening Approach for Discovery of Covalently Bound Ligands
    Toledo Warshaviak, Dora; Golan, Gali; Borrelli, Kenneth W.; Zhu, Kai; Kalid, Ori
    Journal of Chemical Information and Modeling (2014), 54 (7), 1941-1950CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    We present a fast and effective covalent docking approach suitable for large-scale virtual screening (VS). We applied this method to four targets (HCV NS3 protease, Cathepsin K, EGFR, and XPO1) with known crystal structures and known covalent inhibitors. We implemented a customized "VS mode" of the Schrodinger Covalent Docking algorithm (CovDock), which we refer to as CovDock-VS. Known actives and target-specific sets of decoys were docked to selected X-ray structures, and poses were filtered based on noncovalent protein-ligand interactions known to be important for activity. We were able to retrieve 71%, 72%, and 77% of the known actives for Cathepsin K, HCV NS3 protease, and EGFR within 5% of the decoy library, resp. With the more challenging XPO1 target, where no specific interactions with the protein could be used for postprocessing of the docking results, we were able to retrieve 95% of the actives within 30% of the decoy library and achieved an early enrichment factor (EF1%) of 33. The poses of the known actives bound to existing crystal structures of 4 targets were predicted with an av. RMSD of 1.9 Å. To the best of our knowledge, CovDock-VS is the first fully automated tool for efficient virtual screening of covalent inhibitors. Importantly, CovDock-VS can handle multiple chem. reactions within the same library, only requiring a generic SMARTS-based predefinition of the reaction. CovDock-VS provides a fast and accurate way of differentiating actives from decoys without significantly deteriorating the accuracy of the predicted poses for covalent protein-ligand complexes. Therefore, we propose CovDock-VS as an efficient structure-based virtual screening method for discovery of novel and diverse covalent ligands.
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  59. 59
    Rageot, D.; Bohnacker, T.; Keles, E.; McPhail, J. A.; Hoffmann, R. M.; Melone, A.; Borsari, C.; Sriramaratnam, R.; Sele, A. M.; Beaufils, F.; Hebeisen, P.; Fabbro, D.; Hillmann, P.; Burke, J. E.; Wymann, M. P. (S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a potent, orally bioavailable, and brain-penetrable dual inhibitor of class I PI3K and mTOR kinase. J. Med. Chem. 2019, 62, 6241– 6261,  DOI: 10.1021/acs.jmedchem.9b00525
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    (S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a Potent, Orally Bioavailable, and Brain-Penetrable Dual Inhibitor of Class I PI3K and mTOR Kinase
    Rageot, Denise; Bohnacker, Thomas; Keles, Erhan; McPhail, Jacob A.; Hoffmann, Reece M.; Melone, Anna; Borsari, Chiara; Sriramaratnam, Rohitha; Sele, Alexander M.; Beaufils, Florent; Hebeisen, Paul; Fabbro, Doriano; Hillmann, Petra; Burke, John E.; Wymann, Matthias P.
    Journal of Medicinal Chemistry (2019), 62 (13), 6241-6261CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure-activity relationship study, which led to the discovery of a drug-like ATP-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530, compd. 6), which qualifies as a clin. candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compd. 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compd. 6 prevented cell growth in a cancer cell line panel. The preclin. in vivo characterization of compd. 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncol.
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  60. 60
    Goel, P.; Alam, O.; Naim, M. J.; Nawaz, F.; Iqbal, M.; Alam, M. I. Recent advancement of piperidine moiety in treatment of cancer- A review. Eur. J. Med. Chem. 2018, 157, 480– 502,  DOI: 10.1016/j.ejmech.2018.08.017
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    Recent advancement of piperidine moiety in treatment of cancer- A review
    Goel, Pallavi; Alam, Ozair; Naim, Mohd Javed; Nawaz, Farah; Iqbal, Muzaffar; Alam, Md Iqbal
    European Journal of Medicinal Chemistry (2018), 157 (), 480-502CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    A review. Piperidine is an important pharmacophore, a privileged scaffold and an excellent heterocyclic system in the field of drug discovery which provides numerous opportunities in studying/exploring this moiety as an anticancer agent by acting on various receptors of utmost importance. Cancer is an uncontrolled division of cells that results in the formation of tumor which have the potential to migrate to other parts of the body (metastasis) finally becoming a major threat to human population. Since piperidine displayed great potential in this area it is being further probed to get novel entities for the treatment of cancer. This review throws light on recent biol. expansions of piperidine along with structure activity relationships to deliver assocn. between various synthesized newer/novel derivs. and receptor sites.
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    Copeland, R. A. Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 2005, 46, 1– 265
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    Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists
    Copeland Robert A
    Methods of biochemical analysis (2005), 46 (), 1-265 ISSN:0076-6941.
    There is no expanded citation for this reference.
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    Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discovery 2017, 22, 3– 20,  DOI: 10.1177/1087057116671509
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    A perspective on the kinetics of covalent and irreversible inhibition
    Strelow, John M.
    SLAS Discovery (2017), 22 (1), 3-20CODEN: SDLIBT; ISSN:2472-5560. (Sage Publications)
    The clin. and com. success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochem., cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochem. assay, the IC50 may simply reflect the target protein concn. in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI. The kinact/KI is a rate const. describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the max. potential rate (kinact) of inactivation. In this perspective, it is proposed that the kinact/KI should be employed as a crit. parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochem. assays to the cell, and more accurately define selectivity. It is also proposed that a physiol. relevant kinact/KI and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two crit. determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.
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    Dale, N. C.; Johnstone, E. K. M.; White, C. W.; Pfleger, K. D. G. NanoBRET: The Bright Future of Proximity-Based Assays. Front. Bioeng. Biotechnol. 2019, 7, 56  DOI: 10.3389/fbioe.2019.00056
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    NanoBRET: The Bright Future of Proximity-Based Assays
    Dale Natasha C; Johnstone Elizabeth K M; White Carl W; Pfleger Kevin D G; Dale Natasha C; Johnstone Elizabeth K M; White Carl W; Pfleger Kevin D G; Dale Natasha C; Johnstone Elizabeth K M; White Carl W; Pfleger Kevin D G; Pfleger Kevin D G
    Frontiers in bioengineering and biotechnology (2019), 7 (), 56 ISSN:2296-4185.
    Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.
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    Vasta, J. D.; Corona, C. R.; Wilkinson, J.; Zimprich, C. A.; Hartnett, J. R.; Ingold, M. R.; Zimmerman, K.; Machleidt, T.; Kirkland, T. A.; Huwiler, K. G.; Ohana, R. F.; Slater, M.; Otto, P.; Cong, M.; Wells, C. I.; Berger, B. T.; Hanke, T.; Glas, C.; Ding, K.; Drewry, D. H.; Huber, K. V. M.; Willson, T. M.; Knapp, S.; Muller, S.; Meisenheimer, P. L.; Fan, F.; Wood, K. V.; Robers, M. B. Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem. Biol. 2018, 25, 206.e11– 214.e11,  DOI: 10.1016/j.chembiol.2017.10.010
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    Ikediobi, O. N.; Davies, H.; Bignell, G.; Edkins, S.; Stevens, C.; O’Meara, S.; Santarius, T.; Avis, T.; Barthorpe, S.; Brackenbury, L.; Buck, G.; Butler, A.; Clements, J.; Cole, J.; Dicks, E.; Forbes, S.; Gray, K.; Halliday, K.; Harrison, R.; Hills, K.; Hinton, J.; Hunter, C.; Jenkinson, A.; Jones, D.; Kosmidou, V.; Lugg, R.; Menzies, A.; Mironenko, T.; Parker, A.; Perry, J.; Raine, K.; Richardson, D.; Shepherd, R.; Small, A.; Smith, R.; Solomon, H.; Stephens, P.; Teague, J.; Tofts, C.; Varian, J.; Webb, T.; West, S.; Widaa, S.; Yates, A.; Reinhold, W.; Weinstein, J. N.; Stratton, M. R.; Futreal, P. A.; Wooster, R. Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol. Cancer Ther. 2006, 5, 2606– 2612,  DOI: 10.1158/1535-7163.Mct-06-0433
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    Mutation analysis of 24 known cancer genes in the NCI-60 cell line set
    Ikediobi, Ogechi N.; Davies, Helen; Bignell, Graham; Edkins, Sarah; Stevens, Claire; O'Meara, Sarah; Santarius, Thomas; Avis, Tim; Barthorpe, Syd; Brackenbury, Lisa; Buck, Gemma; Butler, Adam; Clements, Jody; Cole, Jennifer; Dicks, Ed; Forbes, Simon; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathan; Hunter, Chris; Jenkinson, Andy; Jones, David; Kosmidou, Vivienne; Lugg, Richard; Menzies, Andrew; Mironenko, Tatiana; Parker, Adrian; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alex; Smith, Raffaella; Solomon, Helen; Stephens, Philip; Teague, Jon; Tofts, Calli; Varian, Jennifer; Webb, Tony; West, Sofie; Widaa, Sara; Yates, Andy; Reinhold, William; Weinstein, John N.; Stratton, Michael R.; Futreal, P. Andrew; Wooster, Richard
    Molecular Cancer Therapeutics (2006), 5 (11), 2606-2612CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacol. and at the mol. level more extensively than any other set of cell lines. However, no systematic mutation anal. of genes causally implicated in oncogenesis has been reported. This study reports the sequence anal. of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examd., with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacol. and mol. profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.
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  66. 66
    Gall, V. A.; Philips, A. V.; Qiao, N.; Clise-Dwyer, K.; Perakis, A. A.; Zhang, M.; Clifton, G. T.; Sukhumalchandra, P.; Ma, Q.; Reddy, S. M.; Yu, D.; Molldrem, J. J.; Peoples, G. E.; Alatrash, G.; Mittendorf, E. A. Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells. Cancer Res. 2017, 77, 5374– 5383,  DOI: 10.1158/0008-5472.Can-16-2774
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    trastuzumab increases her2 uptake and cross-presentation by dendritic cells
    Gall, Victor A.; Philips, Anne V.; Qiao, Na; Clise-Dwyer, Karen; Perakis, Alexander A.; Zhang, Mao; Clifton, Guy T.; Sukhumalchandra, Pariya; Ma, Qing; Reddy, Sangeetha M.; Yu, Dihua; Molldrem, Jeffrey J.; Peoples, George E.; Alatrash, Gheath; Mittendorf, Elizabeth A.
    Cancer Research (2017), 77 (19), 5374-5383CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Early-phase clin. trials evaluating CD8+ T cell-eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clin. trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of std. therapy in the per treatment analyses conducted after a median follow-up of greater than 34 mo. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-pos. breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival obsd. with combination therapy.
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  67. 67
    Chaussade, C.; Rewcastle, G. W.; Kendall, J. D.; Denny, W. A.; Cho, K.; Grønning, L. M.; Chong, M. L.; Anagnostou, S. H.; Jackson, S. P.; Daniele, N.; Shepherd, P. R. Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem. J. 2007, 404, 449– 458,  DOI: 10.1042/BJ20070003
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    Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling
    Chaussade, Claire; Rewcastle, Gordon W.; Kendall, Jackie D.; Denny, William A.; Cho, Kitty; Gronning, Line M.; Chong, Mei Ling; Anagnostou, Sasha H.; Jackson, Shaun P.; Daniele, Nathalie; Shepherd, Peter R.
    Biochemical Journal (2007), 404 (3), 449-458CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Recent genetic knock-in and pharmacol. approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110α isoform (PIK3CA) that plays the predominant role in insulin signaling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signaling. These include a p110α-specific inhibitor (PIK-75), a p110α-selective inhibitor (PI-103), a p110β-specific inhibitor (TGX-221) and a p110δ-specific inhibitor (IC87114). Although we find that p110α is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110β or p110δ alone was also not sufficient to block insulin signaling to PKB in these cells, but, when added in combination with p110α inhibitors, they are able to significantly attenuate insulin signaling. Surprisingly, in J774.2 macrophage cells, insulin signaling to PKB was inhibited to a similar extent by inhibitors of p110α, p110β or p110δ. These results provide evidence that p110β and p110δ can play a role in insulin signaling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.
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    Freeman-Cook, K. D.; Hoffman, R. L.; Johnson, T. W. Lipophilic efficiency: the most important efficiency metric in medicinal chemistry. Future Med. Chem. 2013, 5, 113– 115,  DOI: 10.4155/fmc.12.208
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    Lipophilic efficiency: the most important efficiency metric in medicinal chemistry
    Freeman-Cook, Kevin D.; Hoffman, Robert L.; Johnson, Ted W.
    Future Medicinal Chemistry (2013), 5 (2), 113-115CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)
    A review is given on the crit. role of lipophilicity in drug discovery and optimization of general absorption, distribution, metab. and excretion properties, toxicol. profiles and ultimately pharmacol. response. Math. equations are presented concerning the impact of lipophilicity on the dose and the calcn. of the lipophilic ligand efficiency (LipE).
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    Leeson, P. D.; Springthorpe, B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nat. Rev. Drug Discovery 2007, 6, 881– 890,  DOI: 10.1038/nrd2445
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    The influence of drug-like concepts on decision-making in medicinal chemistry
    Leeson, Paul D.; Springthorpe, Brian
    Nature Reviews Drug Discovery (2007), 6 (11), 881-890CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. Despite the wide acceptance of drug-like principles such as the 'rule of five', this anal. of mols. currently being synthesized in leading pharmaceutical companies reveals that their phys. properties differ significantly from those of recently discovered oral drugs. The marked increase in lipophilicity in particular could increase the likelihood of attrition in drug development. The application of guidelines linked to the concept of drug-likeness, such as the 'rule of five', has gained wide acceptance as an approach to reduce attrition in drug discovery and development. However, despite this acceptance, anal. of recent trends reveals that the phys. properties of mols. that are currently being synthesized in leading drug discovery companies differ significantly from those of recently discovered oral drugs and compds. in clin. development. The consequences of the marked increase in lipophilicity - the most important drug-like phys. property - include a greater likelihood of lack of selectivity and attrition in drug development. Tackling the threat of compd.-related toxicol. attrition needs to move to the mainstream of medicinal chem. decision-making.
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    Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The Cysteinome of Protein Kinases as a Target in Drug Development. Angew. Chem., Int. Ed. 2018, 57, 4372– 4385,  DOI: 10.1002/anie.201707875
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    The Cysteinome of Protein Kinases as a Target in Drug Development
    Chaikuad, Apirat; Koch, Pierre; Laufer, Stefan A.; Knapp, Stefan
    Angewandte Chemie, International Edition (2018), 57 (16), 4372-4385CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Drugs that function through covalent bond formation represent a considerable fraction of the authors' repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clin. applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Herein, the authors review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.
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    Zhang, T.; Inesta-Vaquera, F.; Niepel, M.; Zhang, J.; Ficarro, S. B.; Machleidt, T.; Xie, T.; Marto, J. A.; Kim, N.; Sim, T.; Laughlin, J. D.; Park, H.; LoGrasso, P. V.; Patricelli, M.; Nomanbhoy, T. K.; Sorger, P. K.; Alessi, D. R.; Gray, N. S. Discovery of potent and selective covalent inhibitors of JNK. Chem. Biol. 2012, 19, 140– 154,  DOI: 10.1016/j.chembiol.2011.11.010
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    Discovery of Potent and Selective Covalent Inhibitors of JNK
    Zhang, Tinghu; Inesta-Vaquera, Francisco; Niepel, Mario; Zhang, Jianming; Ficarro, Scott B.; Machleidt, Thomas; Xie, Ting; Marto, Jarrod A.; Kim, Nam Doo; Sim, Taebo; Laughlin, John D.; Park, Hajeung; LoGrasso, Philip V.; Patricelli, Matt; Nomanbhoy, Tyzoon K.; Sorger, Peter K.; Alessi, Dario R.; Gray, Nathanael S.
    Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (1), 140-154CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.60 and 2.97 Å resoln. that show the compds. form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK, in cells exposed to submicromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochem., cellular, and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggests that the compd. will be broadly useful as a pharmacol. probe of JNK-dependent signal transduction. Potential lead compds. have also been identified for kinases, including IRAK1, PIK3C3, PIP4K2C, and PIP5K3.
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    Zhang, T.; Hatcher, J. M.; Teng, M.; Gray, N. S.; Kostic, M. Recent Advances in Selective and Irreversible Covalent Ligand Development and Validation. Cell Chem. Biol. 2019, 26, 1486– 1500,  DOI: 10.1016/j.chembiol.2019.09.012
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    Recent Advances in Selective and Irreversible Covalent Ligand Development and Validation
    Zhang, Tinghu; Hatcher, John M.; Teng, Mingxing; Gray, Nathanael S.; Kostic, Milka
    Cell Chemical Biology (2019), 26 (11), 1486-1500CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
    A review. Some of the most widely used drugs, such as aspirin and penicillin, are covalent drugs. Covalent binding can improve potency, selectivity, and duration of the effects, but the intrinsic reactivity represents a potential liability and may result in idiosyncratic toxicity. For decades, the cons were believed to outweigh the pros, and covalent targeting was deprioritized in drug discovery. Recently, several covalent inhibitors have been approved for cancer treatment, thus rebooting the field. In this review, we briefly reflect on the history of selective covalent targeting, and provide a comprehensive overview of emerging developments from a chem. biol. stand-point. Our discussion will reflect on efforts to validate irreversible covalent ligands, expand the scope of targets, and discover new ligands and warheads. We conclude with a brief commentary of remaining limitations and emerging opportunities in selective covalent targeting.
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    We describe a new dynamic kinetic simulation program that allows multiple data sets to be fit simultaneously to a single model based on numerical integration of the rate equations describing the reaction mechanism. Unlike other programs that allow fitting based on numerical integration of rate equations, in the dynamic simulation rate consts., output factors, and starting concns. of reactants can be scrolled while observing the change in the shape of the simulated reaction curves. Fast dynamic simulation facilitates the exploration of initial parameters that serve as the starting point for nonlinear regression in fitting data and facilitates exploration of the relationships between individual consts. and observable reactions. The exploration of parameter space by dynamic simulation provides a powerful tool for learning kinetics and for evaluating the extent to which parameters are constrained by the data. This feature is crit. to avoid overly complex models that are not supported by the data.
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    Protein Science (2014), 23 (10), 1332-1340CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    PI3Kα remains an attractive target for the development of anticancer targeted therapy. A no. of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small mol. co-crystal structures, but the utilization of this crystal form is limited by low diffraction resoln. and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing prodn. of isolated stable p110α subunit missing the adapter-binding domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Addnl., we detd. apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, resp. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family.
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    Structure (Oxford, United Kingdom) (2013), 21 (2), 209-219CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    The oncogenicity of the L858R mutant form of the epidermal growth factor receptor (EGFR) in non-small-cell lung cancer is thought to be due to the constitutive activation of its kinase domain. The selectivity of the marketed drugs gefitinib and erlotinib for L858R mutant is attributed to their specific recognition of the active kinase and to weaker ATP binding by L858R EGFR. We present crystal structures showing that neither L858R nor the drug-resistant L858R+T790M EGFR kinase domain is in the constitutively active conformation. Addnl. co-crystal structures show that gefitinib and dacomitinib, an irreversible anilinoquinazoline deriv. currently in clin. development, may not be conformation specific for the active state of the enzyme. Structural data further reveal the potential mode of recognition of one of the autophosphorylation sites in the C-terminal tail, Tyr-1016, by the kinase domain. Biochem. and biophys. evidence suggest that the oncogenic mutations impact the conformational dynamics of the enzyme.
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    Glutathione is present in millimolar concns. in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equil. const., we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously detd. value for [GSH]2:[GSSG] in the ER of 83 mM, this translates into a total glutathione concn. in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ∼7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.
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    Beyond the Michaelis-Menten equation: Accurate and efficient estimation of enzyme kinetic parameters
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    Examining enzyme kinetics is critical for understanding cellular systems and for using enzymes in industry. The Michaelis-Menten equation has been widely used for over a century to estimate the enzyme kinetic parameters from reaction progress curves of substrates, which is known as the progress curve assay. However, this canonical approach works in limited conditions, such as when there is a large excess of substrate over enzyme. Even when this condition is satisfied, the identifiability of parameters is not always guaranteed, and often not verifiable in practice. To overcome such limitations of the canonical approach for the progress curve assay, here we propose a Bayesian approach based on an equation derived with the total quasi-steady-state approximation. In contrast to the canonical approach, estimates obtained with this proposed approach exhibit little bias for any combination of enzyme and substrate concentrations. Importantly, unlike the canonical approach, an optimal experiment to identify parameters with certainty can be easily designed without any prior information. Indeed, with this proposed design, the kinetic parameters of diverse enzymes with disparate catalytic efficiencies, such as chymotrypsin, fumarase, and urease, can be accurately and precisely estimated from a minimal amount of timecourse data. A publicly accessible computational package performing such accurate and efficient Bayesian inference for enzyme kinetics is provided.
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    Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance
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    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (1), 173-178CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chem. reactivity in overall potency are not well-understood. To characterize the underlying mol. processes at a microscopic level and det. the appropriate kinetic consts., specialized exptl. design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 105-107 M-1s-1), despite their low specific reactivity (kinact ≤ 2.1 × 10-3 s-1), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissocn. and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidn. on enzyme catalysis and inhibitor pharmacol. are characterized. Oxidn. of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidn. (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.
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    PyrrolylBODIPYs: Syntheses, Properties, and Application as Environment-Sensitive Fluorescence Probes
    Yu, Changjiang; Miao, Wei; Wang, Jun; Hao, Erhong; Jiao, Lijuan
    ACS Omega (2017), 2 (7), 3551-3561CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    Four pyrrole B-ring functionalized pyrrolylBODIPYs and their B-ring unsubstituted analogs were synthesized from easily accessible starting 5-halo-2-formylpyrroles and were characterized by NMR, HRMS, X-ray anal., optical/electronic properties. In great contrast to the substitution(s) at the other two pyrrolic units, electron donating substituent(s) at pyrrole B-ring bring significantly blue-shift of the absorption and emission bands. Cyclic voltammetry and DFT calcns. indicate that this blue-shift may be attributed to the increased HOMO and the LUMO energy levels and the overall increase of the energy band gaps. These pyrrolylBODIPYs generally show intense absorption (centered at 570-624 nm) and fluorescence emission (582-654 nm) in nonpolar solvents. A gradual decrease of the fluorescence intensity was obsd. for these dyes with the increase of solvent dipolar moment, which combines with the red to far-red absorption/emission rendering these pyrrolylBODIPYs potential applications as environment-sensitive fluorescence probes as demonstrated in this work for BSA.
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  • Abstract

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    Figure 1

    Figure 1. (a, c) Design strategy for the development of irreversible inhibitors targeting (a) BTK and (c) EGFR. X-ray crystallographic structure of (a) BTK (colored wheat) in complex with PCI-29732 (teal; PDB ID 3GEN) (78) and (c) EGFR (cyan) in complex with gefitinib (brown, PDB ID 4I22). (79) (b, d) Chemical structure of the reversible-inhibitor scaffolds and covalent derivatives are depicted. The incorporation of the warhead on PCI-29732 led to acabrutinib and ibrutinib, and gefitinib served as a scaffold for dacomitinib and afatinib. (e) PQR514 (46) (magenta) bound to phosphoinositide 3-kinase α (PI3Kα) (gray; based on PDB ID 6OAC (59)). Cys862 positions from eight superimposed PI3Kα-inhibitor complexes are shown (see Table S1 for details). The position of the cysteine thiols (yellow) is conserved across the different complexes and deviates less than 2.6 Å. (f) Schematic design of the covalent PI3Kαi library. The distances between reversible inhibitor skeletons and the targeted nucleophiles are shown as dashed green lines (∼4 Å for BTK and EGFR vs ∼11 Å for PI3Kα).

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    Figure 2

    Figure 2. (a) Chemical structure of a set of nine warhead-containing compounds (1–9). (b) General reaction of warhead-containing compounds with βME. (c) High-performance liquid chromatography (HPLC) reaction monitoring of inhibitor (3) and its βME adduct (3-βME) formation. (d) Time-dependent inhibitor consumption curves used to calculate kchem. Values are mean ± standard deviation (SD) (n = 3). Error bars are not shown when smaller than symbols. (e) Chemical shift of the α-proton in 1H NMR spectra of compounds bearing an unsubstituted double bond (7) and β-methyl (8)- and β,β-dimethyl (9)-substituted warheads. (f) Intrinsic reactivity of the inhibitors (kchem) plotted against the chemical shift of the α-protons of the indicated compounds. Raw data and details for kchem calculations are reported in Table S2 and in the Materials and Methods section in the Supporting Information. The second-order rate constant kchem could not be measured for compounds 6 and 9 due to lack of reactivity with 12 M βME, and also not for 1 due to its extremely fast reaction with 1 mM βME. kchem for compound 8 was lower than 1 × 10–6 M–1·s–1 and was thus considered nonreactive. These three values are therefore “out-off scale” (denoted with red arrows) for kchem. (g) Representation of energy of the lowest-unoccupied molecular orbital (LUMO) electron density (top) and LUMO map (bottom) for compound 1. Electron deficiency ranges from blue (high) to red (low). Given that the LUMO designates space available for a pair of electrons, color tints toward blue in the LUMO map indicate where a nucleophilic attack would likely occur. Calculations were performed using Spartan 18, Wavefunction, Inc. (h) ELUMO values plotted against the intrinsic reactivity of the inhibitors (kchem). Raw data and details for kchem calculations are reported in Table S2 and in the Materials and Methods section in the Supporting Information. As in (f), values for kchem denoted with red arrows are “out-off scale”. (i) Chemical shift of α-protons of indicated compounds plotted against ELUMO values. (j) Model for cellular on- and off-target reactions of covalent inhibitors. In the on-target reaction (green), enzyme E and inhibitor I form first a reversible E∼I complex (equilibrium dependent on Ki), which is then converted to a covalent EI complex (controlled by kinact). Competing off-target reactions (red) with cellular sulfhydryls (S) consume the inhibitor and form a stable SI adduct. (k) On- and off-target covalent reactions of inhibitors as depicted in (j) were modeled using KinTek Global Kinetic Explorer: concentrations were set to 7 mM for intracellular sulfhydryls (for intracellular reduced glutathione (GSH) concentrations, see ref (80)); for the targeted enzyme (PI3Kα) to 10 nM; and for indicated covalent inhibitors to 100 nM. Experimental values for kinact and kchem used for the modeling are listed in Table S2 and Figure S1. Details for the determination of kinact are given in Figure 3 and associated materials.

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    Figure 3

    Figure 3. (a) Chemical structure of a collection of nine compounds with pairwise matched linkers and either fast (2, 10–12) or moderately (3, 13–15a) reactive Michael acceptors, and of 15b and 15c. (b) The reactive volume (Vr) of the warhead was estimated by a sphere around the sulfur atom of Cys862, with a radius equal to the distance (d) between the Cys862 sulfur and the β-carbon of the Michael acceptor (Vr = 4/3·π·d3); see Table S4 for values. The modeled locations of the β-carbon of the Michael acceptor are indicated by spheres colored as in (a). (c) Correlation between kinact and the local warhead concentration (see Table S4 for calculation). (d, e) Time-dependent IC50 determinations derived from time- and concentration-dependent time-resolved fluorescence resonance energy transfer (TR-FRET) ratios curves depicted in Figure S2 (IC50s for all compounds with error bars, see Figure S13). Strong (2, 10–12 (d)) and weak (3, 13–15a (e)) electrophiles were investigated. (f) Compound-specific intrinsic reactivity (kchem) determined as in Figure 2; (g) dissociation constants (Ki) calculated from concentration and time-dependent TR-FRET experiments as shown in Figure S2 (h) rate constants for covalent binding to PI3Kα (kinact); (i) second-order rate constant used to characterize covalent binding of irreversible inhibitors to the target protein (kinact/Ki). The calculation of kinetic parameters was carried out through global fitting for numerical integration to a kinetic model using KinTek Global Kinetic Explorer modeling software. (73−76,81,82) All values are shown as mean ± SD (n = 3). Error bars are not shown when smaller than the symbols. Orange, strong electrophiles; green, weak electrophiles. The distance between the Michael acceptor (β-carbon) and Cys862 thiol was calculated using PyMOL 2.3.5 Schrödinger, Inc. (see Figure S3 and Table S3). (j) TR-FRET experiments for the reversible analogue of compound 2 (2r). (k) Covalent docking of compounds 2, 10–12 using CovDock Schrödinger. Representation of the shift of the morpholine oxygen (spheres, in hinge region defined by Val851) and of the NH2 (spheres, in binding affinity region defined by Asp810) with respect to PQR530 scaffold (magenta) is displayed. (l) CovDock Schrödinger modeling has been used to investigate the displacement of the PI3K core module of the indicated compounds when covalently bound to Cys862 of PI3Kα. The distances of (i) the morpholine oxygen atom (O17) and the N nitrogen atom of Val851 backbone amide (O17-V851, red) in the hinge region, and (ii) the amino group (N26) of the aminopyrimidine and the closest oxygen atom in COO– side chain of Asp810 (N26-D810, light blue) in the affinity region were determined from CovDock modeling coordinates from 50 predicted poses, and average and SD were calculated. As a control for a structural distortion of the PI3K-binding module, the distances between the O17 and N26 atoms (O17-N26, black) were determined. Numbering of inhibitor atoms can be found in (a) and (k). Distances are reported in angstrom. Modeling was performed using PDB ID 6OAC (59) as a starting point. All compounds are depicted in Figure S6.

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    Figure 4

    Figure 4. (a) Library of acrylamide-containing compounds (16–24). (b) Intrinsic reactivity (kchem) of compounds 16–24, ibrutinib and CNX-1351. Experiments performed with HPLC (1 mM inhibitor and 600 mM βME, n = 3). All values are reported as mean ± SD. Error bars are not shown when smaller than the symbols. (c) Efficiency in covalent bond formation (kinact/Ki) plotted against distance from Cys862. Zero on the x-axis corresponds to Cys862 positioning; zero to left: shorter linkers; zero to right: longer linkers. Kinetic parameters are reported in Table S5. The distances between the Michael acceptors (β-carbons) and Cys862 thiol were calculated using Maestro 11.1 and PyMOL 2.3.5 Schrödinger LLC (see Figure S5). (d) Plot of kchem/kinact ratios vs the reactive volume Vr of compounds 12, 2, 11, 10 (red circles), 15a, 3, 14, 13 (green squares), 19, 22, 18, 17, 16 (blue circles), and 20, 23, 21, 24 (orange diamonds). The top x-axis shows the Vr·NA term utilized as in eq S14b to determine the scaling factor σ for each compound group as shown in Figure S7 and Table S6. The compound group-specific σ factors were used to normalize the kchem/kinact ratios by 1/σ. The (blue) regression line includes all compounds listed above except for 20, 23, 21, 24, with a linker reaching beyond the target Cys862. (e) Normalized kcat reaction rates were calculated according to eq 15 with a spherical Vr approximation using the modeled distance to the target d. For Ki, the indicated values were used. (f) Normalized kcat reaction rates were calculated according to eq 17a as a function of d and the inhibitor concentration [I]. For both graphs in (e) and (f), a mean kchem averaged over the values from compounds 19, 22, 18, 17, and 16 was used (kchem (19, 22, 18, 17, 16) = 3.38 × 10–4 M–1·s–1). (g) LC-SRM quantification of Cys862-modification by 19, 22, and 22r. (h, i) X-ray structure of (h) 19 (deep teal) bound to PI3Kα (PDB ID 7R9V, resolution 2.69 Å) and (i) 22 (deep purple) bound to PI3Kα (PDB ID 7R9Y, resolution 2.85 Å). H-bonds are depicted as dashed black lines. As for the CHF2-group, a H-bond either with (i) Lys802 or with (ii) the triazine core (intramolecular interaction) is possible based on the proton positioning. (j, k) Electron density maps of the inhibitor (2Fo–Fc map contoured at 1σ, blue mesh) fit the structure of (j) 19 and (k) 22, and the electron density clearly shows that the acrylamide forms a covalent bond with the thiol group of Cys862.

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    Figure 5

    Figure 5. (a–d) Residence time experiment using BRET in intact HEK293 cells expressing NanoLuc fused to (a) PI3Kα, (b) PI3Kα Cys862Ser, (c) PI3Kβ, and (d) PI3Kδ. The cells were incubated with inhibitors (3 μM) for 2 h. Subsequently, free inhibitor was washed out twice using Opti-MEM medium (for 10 min at 37 °C and 5% CO2), before the cell-permeable BRET tracer was added (0.2 μM final concentration). A tracer bearing a pyrrolylBODIPY fluorescent moiety (83) (excitation at 460 nm and emission at 618 nm) was used to determine the on-target residence time of the inhibitors after drug washout. (64) Displacement of the inhibitors by the BRET tracer from the ATP-binding pocket in the indicated PI3K isoforms was monitored by the recovery of the BRET signal between the NanoLuc-fused PI3K (donor) and the BRET tracer (acceptor). A prolonged residence time of inhibitors (after drug washout) diminishes the BRET signal and points to a covalent interaction. Data shown are mean ± standard error of the mean (SEM) (n = 3). Results for CNX-1351 are shown in Figure S10.

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    Figure 6

    Figure 6. (a–f) Cellular washout experiments in cancer cells with a mutated and constitutively activated PI3Kα. (a, b) MCF7 (PIK3CA E545K), (c, d) T47D (PIK3CA H1047R), and (e, f) SKOV3 (PIK3CA H1047R; HER2+) cancer cell lines were treated with 3 μM of the indicated compounds for 2 h, followed by drug washout (twice with fully supplemented Dulbecco’s modified Eagle’s medium (DMEM) for 10 min each). The cells were then incubated at 37 °C and 5% CO2 for the indicated times before they were fixed, and the phosphorylation status of PKB/Akt was determined by in-cell western; t = 0 values indicate reference measurements in the presence of inhibitor (i.e., immediately after the 2 h treatment window and without removal of drug). Where indicated, TGX221 (3 μM) was added 1 h before the fixation of cells, to investigate a potential cell-line-dependent further contribution of PI3Kβ to PKB/Akt phosphorylation. Data shown are mean ± SEM (n = 4; two independent measurements). Error bars are not shown when smaller than symbols. (g, h) Plot of clog D vs biological activity ((g) pKi; (h) pIC50) of compounds 19, 22, and CNX-1351. Lipophilic ligand efficiency (LipE) values higher than 5 are considered to be the threshold for lead compounds and high-quality chemical probes. LipE calculations are shown in Table S9. (i) On-target covalent modification modeled after 40 min using KinTek Global Kinetic Explorer. See Figure S1c,d for additional details. (j) Metabolic stability of 19, 22, and CNX-1351 using rat liver microsomes fortified with phase I metabolism cofactor NADPH. The time-dependent degradation of test items (starting concentration 1 μM) in the presence of rat liver microsomes (0.5 mg microsomal protein/mL) was measured (mean ± SD; n = 2). Error bars are not shown when smaller than symbols. Corresponding raw data are reported in Table S10.

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    Figure 7

    Figure 7. (a) Analysis of putative off-target reactions of covalent PI3Kα inhibitors with protein kinases. Coordinates for a cysteinome protein kinase template were kindly provided by Chaikuad and Knapp. Their template was generated based on the PDB ID 1ATP of the catalytic subunit of cAMP-dependent protein kinase (PKA), onto which cysteines throughout the kinome have been projected as reported in ref (70). These coordinates were aligned with PI3K crystallographic data from PDB IDs 5OQ4 (PQR309/PI3Kγ complex), 1E8X (ATP/PI3Kγ complex), and structures of compound 19 bound to PI3Kα provided here, to dock 19 into the PKA template’s ATP-binding site. In the depicted compound 19 (dark blue), the sulfur atom of Cys862 (oversize yellow sphere) is located at a distance of 8.0 Å from Cys F3 (Cys annotation as in ref (70)), which corresponds to a Cys in JNK1, 2, and 3. Closer is only Cys F4 at a distance of 4.0 Å, which is the Cys862 of PI3Kα projected onto the PKA template. The rest of the projected protein kinase Cys residues are ≫10 Å away from the warhead of 19 and cannot be targeted without displacement of the ATP-binding module. (b) Validation of off-target binding of 19 at 1 μM to protein kinases in KINOMEScan experiments (comparisons with results from PQR514, PQR309, BYL719, GDC-0980, and PKI-587, and lipid kinase interactions are shown in Figure S11 and quantitative data are listed in Table S13). The three red spots in the kinase tree represent CSF1R (autoinhibited; juxtamembrane domains of some protein tyrosine kinase receptors stabilize the kinase domain in an inactive state), JAK1 (JH2 domain-pseudokinase), and KIT (autoinhibited) with remaining binding of 20, 32, and 35%, respectively. CSF1R, JAK1, and KIT (and mutant) catalytic domains (in active conformations) showed no relevant interactions with 19.

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  • References

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      A review. Owing to the dysregulation of protein kinase activity in many diseases including cancer, this enzyme family has become one of the most important drug targets in the 21st century. There are 68 FDA-approved therapeutic agents that target about two dozen different protein kinases and six of these drugs were approved in 2021. Of the approved drugs, twelve target protein-serine/threonine protein kinases, four are directed against dual specificity protein kinases (MEK1/2), thirteen block nonreceptor protein-tyrosine kinases, and 39 target receptor protein-tyrosine kinases. The data indicate that 58 of these drugs are prescribed for the treatment of neoplasms (49 against solid tumors including breast, lung, and colon, five against nonsolid tumors such as leukemias, and four against both solid and nonsolid tumors: acalabrutinib, ibrutinib, imatinib, and midostaurin). Three drugs (baricitinib, tofacitinib, upadacitinib) are used for the treatment of inflammatory diseases including rheumatoid arthritis. Of the 68 approved drugs, eighteen are used in the treatment of multiple diseases. The following six drugs received FDA approval in 2021 for the treatment of these specified diseases: belumosudil (graft vs. host disease), infigratinib (cholangiocarcinomas), mobocertinib and tepotinib (specific forms of non-small cell lung cancer), tivozanib (renal cell carcinoma), and trilaciclib (to decrease chemotherapy-induced myelosuppression). All of the FDA-approved drugs are orally effective with the exception of netarsudil, temsirolimus, and the newly approved trilaciclib. This review summarizes the physicochem. properties of all 68 FDA-approved small mol. protein kinase inhibitors including lipophilic efficiency and ligand efficiency.
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    9. 9
      Riely, G. J.; Neal, J. W.; Camidge, D. R.; Spira, A. I.; Piotrowska, Z.; Costa, D. B.; Tsao, A. S.; Patel, J. D.; Gadgeel, S. M.; Bazhenova, L.; Zhu, V. W.; West, H. L.; Mekhail, T.; Gentzler, R. D.; Nguyen, D.; Vincent, S.; Zhang, S.; Lin, J.; Bunn, V.; Jin, S.; Li, S.; Jänne, P. A. Activity and Safety of Mobocertinib (TAK-788) in Previously Treated Non-Small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations from a Phase I/II Trial. Cancer Discovery 2021, 11, 1688– 1699,  DOI: 10.1158/2159-8290.Cd-20-1598
      9
      Activity and safety of mobocertinib (TAK-788) in previousiytreated non-small cell lung cancer with EGFR exon 20 insertion mutations from a phase I/ll trial
      Riely, Gregory J.; Neal, Joel W.; Ross Camidge, D.; Spira, Alexander I.; Piotrowska, Zofia; Costa, Daniel B.; Tsao, Anne S.; Patel, Jyoti D.; Gadgeel, Shirish M.; Bazhenova, Lyudmila; Zhu, Viola W.; West, Howard L.; Mekhail, Tarek; Gentzler, Ryan D.; Nguyen, Danny; Vincent, Sylvie; Zhang, Steven; Lin, Jianchang; Bunn, Veronica; Jin, Shu; Li, Shuanglian; Janne, Pasi A.
      Cancer Discovery (2021), 11 (7), 1688-1699CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)
      Mobocertinib, an oral epidermal growth factor receptor (EGFR) inhibitor targeting EGFR gene mutations, including exon 20 insertions (EGFRex20ins), in nonsmall cell lung cancer, was evaluated in a phase I/ll dose-escalation/expansion trial (ClinicalTrials. gov NCT02716116). Dose escalation identified 160 mg/d as the recommended phase 2 dose and max. tolerated dose. Among 136 patients treated with 160 mg/d, the most common any-grade treatment-related adverse events (TRAE; >25%) were diarrhea (83%), nausea (43%), rash (33%), and vomiting (26%), with diarrhea (21%) the only grade ≥3 TRAE >5%. Among 28 EGFRex20ins patients treated at 160 mg/d, the investigator-assessed confirmed response rate was 43% (12/28; 95% confidence interval, 24%-63%) with median duration of response of 14 mo (5.0-not reached) and median progression-free survival of 7.3 mo (4.4-15.6). Mobocertinib demonstrated antitumor activity in patients with diverse EGFRex20ins variants with a safety profile consistent with other EGFR inhibitors. SIGNIFICANCE: No oral EGFR-targeted therapies are currently approved for patients with EGFRex- 20ins NSCLC. Mobocertinib demonstrated antitumor activity with manageable toxicity in patients with advanced EGFRex20ins NSCLC in this study, supporting addnl. development of mobocertinib in this patient population.
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    10. 10
      Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307– 317,  DOI: 10.1038/nrd3410
      10
      The resurgence of covalent drugs
      Singh, Juswinder; Petter, Russell C.; Baillie, Thomas A.; Whitty, Adrian
      Nature Reviews Drug Discovery (2011), 10 (4), 307-317CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
      A review. Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacol. advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.
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    11. 11
      London, N.; Miller, R. M.; Krishnan, S.; Uchida, K.; Irwin, J. J.; Eidam, O.; Gibold, L.; Cimermančič, P.; Bonnet, R.; Shoichet, B. K.; Taunton, J. Covalent docking of large libraries for the discovery of chemical probes. Nat. Chem. Biol. 2014, 10, 1066– 1072,  DOI: 10.1038/nchembio.1666
      11
      Covalent docking of large libraries for the discovery of chemical probes
      London, Nir; Miller, Rand M.; Krishnan, Shyam; Uchida, Kenji; Irwin, John J.; Eidam, Oliv; Gibold, Lucie; Cimermancic, Peter; Bonnet, Richard; Shoichet, Brian K.; Taunton, Jack
      Nature Chemical Biology (2014), 10 (12), 1066-1072CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Chem. probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biol. studies. Despite these advantages, protein-reactive compds. are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small mols. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chem. probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).
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    12. 12
      Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60, 2879– 2889,  DOI: 10.1021/acs.jmedchem.6b01815
      12
      Determining Cysteines Available for Covalent Inhibition Across the Human Kinome
      Zhao, Zheng; Liu, Qingsong; Bliven, Spencer; Xie, Lei; Bourne, Philip E.
      Journal of Medicinal Chemistry (2017), 60 (7), 2879-2889CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Covalently bound protein kinase inhibitors have been frequently designed to target non-catalytic Cys residues at the ATP binding site. Thus, it is important to know if a given Cys residue can form a covalent bond. Here, we combined a function-site interaction fingerprint method and DFT calcns. to det. the potential of Cys residues to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site Cys dataset was assembled. The orientation of the Cys SH group indicated which Cys residues could potentially form covalent bonds. These covalent inhibitor accessible Cys residues were located within 5 kinase regions (P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop and DFG-3 close to the DFG peptide). In an independent test set, these Cys residues covered 95% of covalent kinase inhibitors. Thus, this study provides new insights into Cys reactivity and preference which is important for the prospective development of covalent kinase inhibitors.
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    13. 13
      Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 2013, 20, 146– 159,  DOI: 10.1016/j.chembiol.2012.12.006
      13
      Developing Irreversible Inhibitors of the Protein Kinase Cysteinome
      Liu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.
      Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 146-159CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      A review. Protein kinases are a large family of approx. 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clin. success of a no. of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large no. of kinases may represent therapeutically relevant targets. To date, the majority of clin. and preclin. kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a no. of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacol. specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biol. activity profiles. In addn., we provided an informatics assessment of the potential "kinase cysteinome" and discuss strategies for the efficient development of new covalent inhibitors.
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    14. 14
      Barf, T.; Covey, T.; Izumi, R.; van de Kar, B.; Gulrajani, M.; van Lith, B.; van Hoek, M.; de Zwart, E.; Mittag, D.; Demont, D.; Verkaik, S.; Krantz, F.; Pearson, P. G.; Ulrich, R.; Kaptein, A. Acalabrutinib (ACP-196): A Covalent Bruton Tyrosine Kinase Inhibitor with a Differentiated Selectivity and In Vivo Potency Profile. J. Pharmacol. Exp. Ther. 2017, 363, 240– 252,  DOI: 10.1124/jpet.117.242909
      14
      Acalabrutinib (ACP-196): a covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile
      Barf, Tjeerd; Covey, Todd; Izumi, Raquel; van de Kar, Bas; Gulrajani, Michael; van Lith, Bart; van Hoek, Maaike; de Zwart, Edwin; Mittag, Diana; Demont, Dennis; Verkaik, Saskia; Krantz, Fanny; Pearson, Paul G.; Ulrich, Roger; Kaptein, Allard
      Journal of Pharmacology and Experimental Therapeutics (2017), 363 (2), 240-252CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)
      Several small-mol. Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacol. characterization of BTK inhibitor acalabrutinib [compd. 1, ACP-196 (4-[8- amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin1-yl]-N-(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochem. and cellular selectivity than ibrutinib and spebrutinib (compds. 2 and 3, resp.). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Detn. of the inhibitory potential of anti-IgM-induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclin. and clin. studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approx. 99% median target coverage at 3 and 12 h and around 90% at 24 h in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacol. differentiators vs. ibrutinib and spebrutinib and is currently being evaluated in clin. trials.
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    15. 15
      Cheng, H.; Planken, S. Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer. ACS Med. Chem. Lett. 2018, 9, 861– 863,  DOI: 10.1021/acsmedchemlett.8b00311
      15
      Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer
      Cheng, Hengmiao; Planken, Simon
      ACS Medicinal Chemistry Letters (2018), 9 (9), 861-863CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are leading causes for lung cancer. Extensive drug discovery efforts targeting EGFR have led to the discovery and FDA approval of both reversible and covalent inhibitors. Second and third generation covalent inhibitors for EGFR have also been described, with the latter targeting specific emerging mutations. After decades of extensive effort, KRAS is widely regarded as an intractable therapeutic target; however, recent publications suggest covalent inhibition is a promising strategy to deliver inhibitors of the KRASG12C mutation.
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    16. 16
      Tomassi, S.; Lategahn, J.; Engel, J.; Keul, M.; Tumbrink, H. L.; Ketzer, J.; Mühlenberg, T.; Baumann, M.; Schultz-Fademrecht, C.; Bauer, S.; Rauh, D. Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor. J. Med. Chem. 2017, 60, 2361– 2372,  DOI: 10.1021/acs.jmedchem.6b01626
      16
      Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor
      Tomassi, Stefano; Lategahn, Jonas; Engel, Julian; Keul, Marina; Tumbrink, Hannah L.; Ketzer, Julia; Muehlenberg, Thomas; Baumann, Matthias; Schultz-Fademrecht, Carsten; Bauer, Sebastian; Rauh, Daniel
      Journal of Medicinal Chemistry (2017), 60 (6), 2361-2372CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chem. entities that efficiently inhibit drug-resistant EGFR. Herein, the authors report the optimization process for a hit compd. that has emerged from a phenotypic screen resulting in indazole-based compds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug resistant EGFR-L858R/T790M and covalently alkylate Cys 797. Western blot anal., as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates the authors' approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.
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    17. 17
      Planken, S.; Behenna, D. C.; Nair, S. K.; Johnson, T. O.; Nagata, A.; Almaden, C.; Bailey, S.; Ballard, T. E.; Bernier, L.; Cheng, H.; Cho-Schultz, S.; Dalvie, D.; Deal, J. G.; Dinh, D. M.; Edwards, M. P.; Ferre, R. A.; Gajiwala, K. S.; Hemkens, M.; Kania, R. S.; Kath, J. C.; Matthews, J.; Murray, B. W.; Niessen, S.; Orr, S. T.; Pairish, M.; Sach, N. W.; Shen, H.; Shi, M.; Solowiej, J.; Tran, K.; Tseng, E.; Vicini, P.; Wang, Y.; Weinrich, S. L.; Zhou, R.; Zientek, M.; Liu, L.; Luo, Y.; Xin, S.; Zhang, C.; Lafontaine, J. Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR. J. Med. Chem. 2017, 60, 3002– 3019,  DOI: 10.1021/acs.jmedchem.6b01894
      17
      Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR
      Planken, Simon; Behenna, Douglas C.; Nair, Sajiv K.; Johnson, Theodore O.; Nagata, Asako; Almaden, Chau; Bailey, Simon; Ballard, T. Eric; Bernier, Louise; Cheng, Hengmiao; Cho-Schultz, Sujin; Dalvie, Deepak; Deal, Judith G.; Dinh, Dac M.; Edwards, Martin P.; Ferre, Rose Ann; Gajiwala, Ketan S.; Hemkens, Michelle; Kania, Robert S.; Kath, John C.; Matthews, Jean; Murray, Brion W.; Niessen, Sherry; Orr, Suvi T. M.; Pairish, Mason; Sach, Neal W.; Shen, Hong; Shi, Manli; Solowiej, James; Tran, Khanh; Tseng, Elaine; Vicini, Paolo; Wang, Yuli; Weinrich, Scott L.; Zhou, Ru; Zientek, Michael; Liu, Longqing; Luo, Yiqin; Xin, Shuibo; Zhang, Chengyi; Lafontaine, Jennifer
      Journal of Medicinal Chemistry (2017), 60 (7), 3002-3019CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approx. 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed. Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clin. candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compd. 21 is currently being evaluated in phase-I clin. trials of mutant EGFR driven NSCLC.
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    18. 18
      Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent inhibitors of interleukin-2 inducible T cell kinase (itk) with nanomolar potency in a whole-blood assay. J. Med. Chem. 2012, 55, 10047– 10063,  DOI: 10.1021/jm301190s
      18
      Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay
      Zapf, Christoph W.; Gerstenberger, Brian S.; Xing, Li; Limburg, David C.; Anderson, David R.; Caspers, Nicole; Han, Seungil; Aulabaugh, Ann; Kurumbail, Ravi; Shakya, Subarna; Li, Xin; Spaulding, Vikki; Czerwinski, Robert M.; Seth, Nilufer; Medley, Quintus G.
      Journal of Medicinal Chemistry (2012), 55 (22), 10047-10063CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concns. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogs are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approx. 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
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    19. 19
      Ward, R. A.; Colclough, N.; Challinor, M.; Debreczeni, J. E.; Eckersley, K.; Fairley, G.; Feron, L.; Flemington, V.; Graham, M. A.; Greenwood, R.; Hopcroft, P.; Howard, T. D.; James, M.; Jones, C. D.; Jones, C. R.; Renshaw, J.; Roberts, K.; Snow, L.; Tonge, M.; Yeung, K. Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2. J. Med. Chem. 2015, 58, 4790– 4801,  DOI: 10.1021/acs.jmedchem.5b00466
      19
      Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2
      Ward, Richard A.; Colclough, Nicola; Challinor, Mairi; Debreczeni, Judit E.; Eckersley, Kay; Fairley, Gary; Feron, Lyman; Flemington, Vikki; Graham, Mark A.; Greenwood, Ryan; Hopcroft, Philip; Howard, Tina D.; James, Michael; Jones, Clifford D.; Jones, Christopher R.; Renshaw, Jonathan; Roberts, Karen; Snow, Lindsay; Tonge, Michael; Yeung, Kay
      Journal of Medicinal Chemistry (2015), 58 (11), 4790-4801CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The RAS/RAF/MEK/ERK signaling pathway has been targeted with a no. of small mol. inhibitors in oncol. clin. development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small mol. inhibitors. There are a no. of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogs) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chem. series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compds. for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.
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    20. 20
      Angst, D.; Gessier, F.; Janser, P.; Vulpetti, A.; Wälchli, R.; Beerli, C.; Littlewood-Evans, A.; Dawson, J.; Nuesslein-Hildesheim, B.; Wieczorek, G.; Gutmann, S.; Scheufler, C.; Hinniger, A.; Zimmerlin, A.; Funhoff, E. G.; Pulz, R.; Cenni, B. Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase. J. Med. Chem. 2020, 63, 5102– 5118,  DOI: 10.1021/acs.jmedchem.9b01916
      20
      Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase
      Angst, Daniela; Gessier, Francois; Janser, Philipp; Vulpetti, Anna; Walchli, Rudolf; Beerli, Christian; Littlewood-Evans, Amanda; Dawson, Janet; Nuesslein-Hildesheim, Barbara; Wieczorek, Grazyna; Gutmann, Sascha; Scheufler, Clemens; Hinniger, Alexandra; Zimmerlin, Alfred; Funhoff, Enrico G.; Pulz, Robert; Cenni, Bruno
      Journal of Medicinal Chemistry (2020), 63 (10), 5102-5118CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncol. indications based on their suboptimal kinase selectivity. We describe the discovery and preclin. profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clin. studies for chronic spontaneous urticaria and Sjoegren's syndrome.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjs1KiurY%253D&md5=9122b310c294d6071ae7cd73cdb91b3d
    21. 21
      Gurbani, D.; Du, G.; Henning, N. J.; Rao, S.; Bera, A. K.; Zhang, T.; Gray, N. S.; Westover, K. D. Structure and Characterization of a Covalent Inhibitor of Src Kinase. Front. Mol. Biosci. 2020, 7, 81  DOI: 10.3389/fmolb.2020.00081
      21
      Structure and characterization of a covalent inhibitor of Src kinase
      Gurbani, Deepak; Du, Guangyan; Henning, Nathaniel J.; Rao, Suman; Bera, Asim K.; Zhang, Tinghu; Gray, Nathanael S.; Westover, Kenneth D.
      Frontiers in Molecular Biosciences (2020), 7 (), 00081CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
      Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clin., possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochem. and biophys. characterization of this compd. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic anal. shows a slow inactivation rate compared to other clin. approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.
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    22. 22
      Abdeldayem, A.; Raouf, Y. S.; Constantinescu, S. N.; Moriggl, R.; Gunning, P. T. Advances in covalent kinase inhibitors. Chem. Soc. Rev. 2020, 49, 2617– 2687,  DOI: 10.1039/c9cs00720b
      22
      Advances in covalent kinase inhibitors
      Abdeldayem, Ayah; Raouf, Yasir S.; Constantinescu, Stefan N.; Moriggl, Richard; Gunning, Patrick T.
      Chemical Society Reviews (2020), 49 (9), 2617-2687CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Over the past decade, covalent kinase inhibitors (CKI) have seen a resurgence in drug discovery. Covalency affords a unique set of advantages as well as challenges relative to their non-covalent counterpart. After reversible protein target recognition and binding, covalent inhibitors irreversibly modify a proximal nucleophilic residue on the protein via reaction with an electrophile. To date, the acrylamide group remains the predominantly employed electrophile in CKI development, with its incorporation in the majority of clin. candidates and FDA approved covalent therapies. Nonetheless, in recent years considerable efforts have ensued to characterize alternative electrophiles that exhibit irreversible or reversibly covalent binding mechanisms towards cysteine thiols and other amino acids. This review article provides a comprehensive overview of CKIs reported in the literature over a decade period, 2007-2018. Emphasis is placed on the rationale behind warhead choice, optimization approach, and inhibitor design. Current FDA approved CKIs are also highlighted, in addn. to a detailed anal. of the common trends and themes obsd. within the listed data set.
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    23. 23
      Yan, G.; Zhong, X.; Pu, C.; Yue, L.; Shan, H.; Lan, S.; Zhou, M.; Hou, X.; Yang, J.; Li, D.; Fan, S.; Li, R. Targeting Cysteine Located Outside the Active Site: An Effective Strategy for Covalent ALKi Design. J. Med. Chem. 2021, 64, 1558– 1569,  DOI: 10.1021/acs.jmedchem.0c01707
      23
      Targeting Cysteine Located Outside the Active Site: An Effective Strategy for Covalent ALKi Design
      Yan, Guoyi; Zhong, Xinxin; Pu, Chunlan; Yue, Lin; Shan, Huifang; Lan, Suke; Zhou, Meng; Hou, Xueyan; Yang, Jie; Li, Deyu; Fan, Shilong; Li, Rui
      Journal of Medicinal Chemistry (2021), 64 (3), 1558-1569CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Potent inhibitors of ALK are highly desired because of the occurrence of drug resistance. We herein firstly report the development of a rationally designed inhibitor, Con B-1, which can covalently bind to Cys1259, a cysteine located outside the ALK active site by linking a warhead with Ceritinib through a 2,2'-Oxybis(ethylamine) linker. The in vitro and in vivo assays showed ConB-1 is a potent selective ALKi with low toxicity to normal cells. In addn., the mol. showed significant improvement of anticancer activities and potential antidrug resistant activity compared with Ceritinib, demonstrating the covalent inhibitor of ALK can be a promising drug candidate for the treatment of NSCLC. This work may provide a novel perspective on the design of covalent inhibitors.
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    24. 24
      Jiang, B.; Jiang, J.; Kaltheuner, I. H.; Iniguez, A. B.; Anand, K.; Ferguson, F. M.; Ficarro, S. B.; Seong, B. K. A.; Greifenberg, A. K.; Dust, S.; Kwiatkowski, N. P.; Marto, J. A.; Stegmaier, K.; Zhang, T.; Geyer, M.; Gray, N. S. Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma. Eur. J. Med. Chem. 2021, 221, 113481  DOI: 10.1016/j.ejmech.2021.113481
      24
      Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma
      Jiang, Baishan; Jiang, Jie; Kaltheuner, Ines H.; Iniguez, Amanda Balboni; Anand, Kanchan; Ferguson, Fleur M.; Ficarro, Scott B.; Seong, Bo Kyung Alex; Greifenberg, Ann Katrin; Dust, Sofia; Kwiatkowski, Nicholas P.; Marto, Jarrod A.; Stegmaier, Kimberly; Zhang, Tinghu; Geyer, Matthias; Gray, Nathanael S.
      European Journal of Medicinal Chemistry (2021), 221 (), 113481CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compds. inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chem. campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, i.p. administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.
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    25. 25
      Zhang, T.; Kwiatkowski, N.; Olson, C. M.; Dixon-Clarke, S. E.; Abraham, B. J.; Greifenberg, A. K.; Ficarro, S. B.; Elkins, J. M.; Liang, Y.; Hannett, N. M.; Manz, T.; Hao, M.; Bartkowiak, B.; Greenleaf, A. L.; Marto, J. A.; Geyer, M.; Bullock, A. N.; Young, R. A.; Gray, N. S. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 2016, 12, 876– 884,  DOI: 10.1038/nchembio.2166
      25
      Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors
      Zhang, Tinghu; Kwiatkowski, Nicholas; Olson, Calla M.; Dixon-Clarke, Sarah E.; Abraham, Brian J.; Greifenberg, Ann K.; Ficarro, Scott B.; Elkins, Jonathan M.; Liang, Yanke; Hannett, Nancy M.; Manz, Theresa; Hao, Mingfeng; Bartkowiak, Bartlomiej; Greenleaf, Arno L.; Marto, Jarrod A.; Geyer, Matthias; Bullock, Alex N.; Young, Richard A.; Gray, Nathanael S.
      Nature Chemical Biology (2016), 12 (10), 876-884CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play crit. roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystn. of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-assocd. transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small mols. capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.
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    26. 26
      Kwiatkowski, N.; Zhang, T.; Rahl, P. B.; Abraham, B. J.; Reddy, J.; Ficarro, S. B.; Dastur, A.; Amzallag, A.; Ramaswamy, S.; Tesar, B.; Jenkins, C. E.; Hannett, N. M.; McMillin, D.; Sanda, T.; Sim, T.; Kim, N. D.; Look, T.; Mitsiades, C. S.; Weng, A. P.; Brown, J. R.; Benes, C. H.; Marto, J. A.; Young, R. A.; Gray, N. S. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 2014, 511, 616– 620,  DOI: 10.1038/nature13393
      26
      Targeting transcription regulation in cancer with a covalent CDK7 inhibitor
      Kwiatkowski, Nicholas; Zhang, Tinghu; Rahl, Peter B.; Abraham, Brian J.; Reddy, Jessica; Ficarro, Scott B.; Dastur, Anahita; Amzallag, Arnaud; Ramaswamy, Sridhar; Tesar, Bethany; Jenkins, Catherine E.; Hannett, Nancy M.; McMillin, Douglas; Sanda, Takaomi; Sim, Taebo; Kim, Nam Doo; Look, Thomas; Mitsiades, Constantine S.; Weng, Andrew P.; Brown, Jennifer R.; Benes, Cyril H.; Marto, Jarrod A.; Young, Richard A.; Gray, Nathanael S.
      Nature (London, United Kingdom) (2014), 511 (7511), 616-620CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacol. inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here the authors present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide anal. in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumor cells. Pharmacol. modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types that are dependent on transcription for maintenance of the oncogenic state.
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    27. 27
      Nacht, M.; Qiao, L.; Sheets, M. P.; St Martin, T.; Labenski, M.; Mazdiyasni, H.; Karp, R.; Zhu, Z.; Chaturvedi, P.; Bhavsar, D.; Niu, D.; Westlin, W.; Petter, R. C.; Medikonda, A. P.; Singh, J. Discovery of a Potent and Isoform-Selective Targeted Covalent Inhibitor of the Lipid Kinase PI3Kα. J. Med. Chem. 2013, 56, 712– 721,  DOI: 10.1021/jm3008745
      27
      Discovery of a Potent and Isoform-Selective Targeted Covalent Inhibitor of the Lipid Kinase PI3Kα
      Nacht, Mariana; Qiao, Lixin; Sheets, Michael P.; St. Martin, Thia; Labenski, Matthew; Mazdiyasni, Hormoz; Karp, Russell; Zhu, Zhendong; Chaturvedi, Prasoon; Bhavsar, Deepa; Niu, Deqiang; Westlin, William; Petter, Russell C.; Medikonda, Aravind Prasad; Singh, Juswinder
      Journal of Medicinal Chemistry (2013), 56 (3), 712-721CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallog., that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC50 < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI50 < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biol. impact of selectively targeting PI3Kα.
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    28. 28
      Wymann, M. P.; Schneiter, R. Lipid signalling in disease. Nat. Rev. Mol. Cell Biol. 2008, 9, 162– 176,  DOI: 10.1038/nrm2335
      28
      Lipid signalling in disease
      Wymann, Matthias P.; Schneiter, Roger
      Nature Reviews Molecular Cell Biology (2008), 9 (2), 162-176CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. Signaling lipids such as eicosanoids, phosphoinositides, sphingolipids and fatty acids control important cellular processes, including cell proliferation, apoptosis, metab. and migration. Extracellular signals from cytokines, growth factors and nutrients control the activity of a key set of lipid-modifying enzymes: phospholipases, prostaglandin synthase, 5-lipoxygenase, phosphoinositide 3-kinase, sphingosine kinase and sphingomyelinase. These enzymes and their downstream targets constitute a complex lipid signaling network with multiple nodes of interaction and cross-regulation. Imbalances in this network contribute to the pathogenesis of human disease. Although the function of a particular signaling lipid is traditionally studied in isolation, this review attempts a more integrated overview of the key role of these signaling lipids in inflammation, cancer and metabolic disease, and discusses emerging strategies for therapeutic intervention.
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    29. 29
      Fruman, D. A.; Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discovery 2014, 13, 140– 156,  DOI: 10.1038/nrd4204
      29
      PI3K and cancer: lessons, challenges and opportunities
      Fruman, David A.; Rommel, Christian
      Nature Reviews Drug Discovery (2014), 13 (2), 140-156CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
      A review. The central role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biol. has prompted a sizeable effort to target PI3K and/or downstream kinases such as AKT and mammalian target of rapamycin (mTOR) in cancer. However, emerging clin. data show limited single-agent activity of inhibitors targeting PI3K, AKT or mTOR at tolerated doses. One exception is the response to PI3Kδ inhibitors in chronic lymphocytic leukemia, where a combination of cell-intrinsic and -extrinsic activities drive efficacy. Here, we review key challenges and opportunities for the clin. development of inhibitors targeting the PI3K-AKT-mTOR pathway. Through a greater focus on patient selection, increased understanding of immune modulation and strategic application of rational combinations, it should be possible to realize the potential of this promising class of targeted anticancer agents.
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    30. 30
      Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.; Gazdar, A.; Powell, S. M.; Riggins, G. J.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.; Velculescu, V. E. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004, 304, 554,  DOI: 10.1126/science.1096502
      30
      Brevia: High frequency of mutations of the PIK3Ca gene in human cancers
      Samuels, Yardena; Wang, Zhenghe; Bardelli, Alberto; Silliman, Natalie; Ptak, Janine; Szabo, Steve; Yan, Hai; Gazdar, Adi; Powell, Steven M.; Riggins, Gregory J.; Willson, James K. V.; Markowitz, Sanford; Kinzler, Kenneth W.; Vogelstein, Bert; Velculescu, Victor E.
      Science (Washington, DC, United States) (2004), 304 (5670), 554CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      There is no expanded citation for this reference.
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    31. 31
      Vanhaesebroeck, B.; Perry, M. W. D.; Brown, J. R.; André, F.; Okkenhaug, K. PI3K inhibitors are finally coming of age. Nat. Rev. Drug Discovery 2021, 20, 741– 769,  DOI: 10.1038/s41573-021-00209-1
      31
      Phosphoinositide 3-kinase inhibitors are finally coming of age
      Vanhaesebroeck, Bart; Perry, Matthew W. D.; Brown, Jennifer R.; Andre, Fabrice; Okkenhaug, Klaus
      Nature Reviews Drug Discovery (2021), 20 (10), 741-769CODEN: NRDDAG; ISSN:1474-1776. (Nature Portfolio)
      A review. Overactive phosphoinositide 3-kinase (PI3K) in cancer and immune dysregulation has spurred extensive efforts to develop therapeutic PI3K inhibitors. Although progress has been hampered by issues such as poor drug tolerance and drug resistance, several PI3K inhibitors have now received regulatory approval-the PI3Kα isoform-selective inhibitor alpelisib for the treatment of breast cancer and inhibitors mainly aimed at the leukocyte-enriched PI3Kδ in B cell malignancies. In addn. to targeting cancer cell-intrinsic PI3K activity, emerging evidence highlights the potential of PI3K inhibitors in cancer immunotherapy. This Review summarizes key discoveries that aid the clin. translation of PI3Kα and PI3Kδ inhibitors, highlighting lessons learnt and future opportunities.
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    32. 32
      Yang, J.; Nie, J.; Ma, X.; Wei, Y.; Peng, Y.; Wei, X. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol. Cancer 2019, 18, 26  DOI: 10.1186/s12943-019-0954-x
      32
      Targeting PI3K in cancer: mechanisms and advances in clinical trials
      Yang Jing; Nie Ji; Ma Xuelei; Wei Yuquan; Peng Yong; Wei Xiawei
      Molecular cancer (2019), 18 (1), 26 ISSN:.
      Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is one of the most important intracellular pathways, which can be considered as a master regulator for cancer. Enormous efforts have been dedicated to the development of drugs targeting PI3K signaling, many of which are currently employed in clinical trials evaluation, and it is becoming increasingly clear that PI3K inhibitors are effective in inhibiting tumor progression. PI3K inhibitors are subdivided into dual PI3K/mTOR inhibitors, pan-PI3K inhibitors and isoform-specific inhibitors. In this review, we performed a critical review to summarize the role of the PI3K pathway in tumor development, recent PI3K inhibitors development based on clinical trials, and the mechanisms of resistance to PI3K inhibition.
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    33. 33
      Massacesi, C.; Di Tomaso, E.; Urban, P.; Germa, C.; Quadt, C.; Trandafir, L.; Aimone, P.; Fretault, N.; Dharan, B.; Tavorath, R.; Hirawat, S. PI3K inhibitors as new cancer therapeutics: implications for clinical trial design. OncoTargets Ther. 2016, 9, 203– 210,  DOI: 10.2147/OTT.S89967
      33
      PI3K inhibitors as new cancer therapeutics: implications for clinical trial design
      Massacesi, Cristian; Di Tomaso, Emmanuelle; Urban, Patrick; Germa, Caroline; Quadt, Cornelia; Trandafir, Lucia; Aimone, Paola; Fretault, Nathalie; Dharan, Bharani; Tavorath, Ranjana; Hirawat, Samit
      OncoTargets and Therapy (2016), 9 (), 203-210CODEN: OTNHAZ; ISSN:1178-6930. (Dove Medical Press Ltd.)
      The PI3K-AKT-mTOR pathway is frequently activated in cancer. PI3K inhibitors, including the pan-PI3K inhibitor buparlisib (BKM120) and the PI3Ka-selective inhibitor alpelisib (BYL719), currently in clin. development by Novartis Oncol., may therefore be effective as anticancer agents. Early clin. studies with PI3K inhibitors have demonstrated preliminary antitumor activity and acceptable safety profiles. However, a no. of unanswered questions regarding PI3K inhibition in cancer remain, including: what is the best approach for different tumor types, and which biomarkers will accurately identify the patient populations most likely to benefit from specific PI3K inhibitors. This review summarizes the strategies being employed by Novartis Oncol. to help maximize the benefits of clin. studies with buparlisib and alpelisib, including stratification according to PI3K pathway activation status, selective enrollment/target enrichment (where patients with PI3K pathway-activated tumors are specifically recruited), nonselective enrollment with mandatory tissue collection, and enrollment of patients who have progressed on previous targeted agents, such as mTOR inhibitors or endocrine therapy. An overview of Novartis-sponsored and Novartis-supported trials that are utilizing these approaches in a range of cancer types, including breast cancer, head and neck squamous cell carcinoma, non-small cell lung carcinoma, lymphoma, and glioblastoma multiforme, is also described.
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    34. 34
      Mishra, R.; Patel, H.; Alanazi, S.; Kilroy, M. K.; Garrett, J. T. PI3K Inhibitors in Cancer: Clinical Implications and Adverse Effects. Int. J. Mol. Sci. 2021, 22, 3464  DOI: 10.3390/ijms22073464
      34
      PI3K inhibitors in cancer: clinical implications and adverse effects
      Mishra, Rosalin; Patel, Hima; Alanazi, Samar; Kilroy, Mary Kate; Garrett, Joan T.
      International Journal of Molecular Sciences (2021), 22 (7), 3464CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. The phospatidylinositol-3 kinase (PI3K) pathway is a crucial intracellular signaling pathway which is mutated or amplified in a wide variety of cancers including breast, gastric, ovarian, colorectal, prostate, glioblastoma and endometrial cancers. PI3K signaling plays an important role in cancer cell survival, angiogenesis and metastasis, making it a promising therapeutic target. There are several ongoing and completed clin. trials involving PI3K inhibitors (pan, isoform-specific and dual PI3K/mTOR) with the goal to find efficient PI3K inhibitors that could overcome resistance to current therapies. This review focuses on the current landscape of various PI3K inhibitors either as monotherapy or in combination therapies and the treatment outcomes involved in various phases of clin. trials in different cancer types. There is a discussion of the drug-related toxicities, challenges assocd. with these PI3K inhibitors and the adverse events leading to treatment failure. In addn., novel PI3K drugs that have potential to be translated in the clinic are highlighted.
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    35. 35
      Fritsch, C.; Huang, A.; Chatenay-Rivauday, C.; Schnell, C.; Reddy, A.; Liu, M.; Kauffmann, A.; Guthy, D.; Erdmann, D.; De Pover, A.; Furet, P.; Gao, H.; Ferretti, S.; Wang, Y.; Trappe, J.; Brachmann, S. M.; Maira, S. M.; Wilson, C.; Boehm, M.; Garcia-Echeverria, C.; Chene, P.; Wiesmann, M.; Cozens, R.; Lehar, J.; Schlegel, R.; Caravatti, G.; Hofmann, F.; Sellers, W. R. Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials. Mol. Cancer Ther. 2014, 13, 1117– 1129,  DOI: 10.1158/1535-7163.Mct-13-0865
      35
      Characterization of the Novel and Specific PI3Kα Inhibitor NVP-BYL719 and Development of the Patient Stratification Strategy for Clinical Trials
      Fritsch, Christine; Huang, Alan; Chatenay-Rivauday, Christian; Schnell, Christian; Reddy, Anupama; Liu, Manway; Kauffmann, Audrey; Guthy, Daniel; Erdmann, Dirk; De Pover, Alain; Furet, Pascal; Gao, Hui; Ferretti, Stephane; Wang, Youzhen; Trappe, Joerg; Brachmann, Saskia M.; Maira, Sauveur-Michel; Wilson, Christopher; Boehm, Markus; Garcia-Echeverria, Carlos; Chene, Patrick; Wiesmann, Marion; Cozens, Robert; Lehar, Joseph; Schlegel, Robert; Caravatti, Giorgio; Hofmann, Francesco; Sellers, William R.
      Molecular Cancer Therapeutics (2014), 13 (5), 1117-1129CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects assocd. with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biol. properties of the 2-aminothiazole deriv. NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compd. selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclin. platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost pos. predictor of sensitivity while revealing addnl. pos. and neg. assocns. such as PIK3CA amplification and PTEN mutation, resp. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clin. trials. Mol Cancer Ther; 13(5); 1117-29. ©2014 AACR.
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    36. 36
      Markham, A. Alpelisib: First Global Approval. Drugs 2019, 79, 1249– 1253,  DOI: 10.1007/s40265-019-01161-6
      36
      Alpelisib: First Global Approval
      Markham, Anthony
      Drugs (2019), 79 (11), 1249-1253CODEN: DRUGAY; ISSN:0012-6667. (Springer International Publishing AG)
      A review. Alpelisib (Piqray)-an orally available phosphatidylinositol 3-kinase (PI3K) inhibitor with specific activity against PI3K alpha (PI3Kα)-is being developed by Novartis for the treatment of breast cancer. Alpelisib has demonstrated efficacy in combination with fulvestrant as treatment for hormone receptor (HR)-pos., human epidermal growth factor receptor-2 (HER2)-neg. breast cancer in patients with a PIK3CA mutation and was recently approved for this indication in the USA. This article summarizes the milestones in the development of alpelisib leading to this first approval.
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    37. 37
      Narayan, P.; Prowell, T. M.; Gao, J. J.; Fernandes, L. L.; Li, E.; Jiang, X.; Qiu, J.; Fan, J.; Song, P.; Yu, J.; Zhang, X.; King-Kallimanis, B. L.; Chen, W.; Ricks, T. K.; Gong, Y.; Wang, X.; Windsor, K.; Rhieu, S. Y.; Geiser, G.; Banerjee, A.; Chen, X.; Reyes Turcu, F.; Chatterjee, D. K.; Pathak, A.; Seidman, J.; Ghosh, S.; Philip, R.; Goldberg, K. B.; Kluetz, P. G.; Tang, S.; Amiri-Kordestani, L.; Theoret, M. R.; Pazdur, R.; Beaver, J. A. FDA Approval Summary: Alpelisib Plus Fulvestrant for Patients with HR-positive, HER2-negative, PIK3CA-mutated, Advanced or Metastatic Breast Cancer. Clin. Cancer Res. 2021, 27, 1842– 1849,  DOI: 10.1158/1078-0432.Ccr-20-3652
      37
      FDA approval summary: alpelisib plus fulvestrant for patients with HR-positive, HER2-negative, PIK3CA-mutated, advanced or metastatic breast cancer
      Narayan, Preeti; Prowell, Tatiana M.; Gao, Jennifer J.; Fernandes, Laura L.; Li, Emily; Jiang, Xiling; Qiu, Junshan; Fan, Jianghong; Song, Pengfei; Yu, Jingyu; Zhang, Xinyuan; King-Kallimanis, Bellinda L.; Chen, Wei; Ricks, Tiffany K.; Gong, Yutao; Wang, Xing; Windsor, Katherine; Rhieu, Steve Y.; Geiser, Gerlie; Banerjee, Anamitro; Chen, Xiaohong; Turcu, Francisca Reyes; Chatterjee, Deb K.; Pathak, Anand; Seidman, Jeffrey; Ghosh, Soma; Philip, Reena; Goldberg, Kirsten B.; Kluetz, Paul G.; Tang, Shenghui; Amiri-Kordestani, Laleh; Theoret, Marc R.; Pazdur, Richard; Beaver, Julia A.
      Clinical Cancer Research (2021), 27 (7), 1842-1849CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      On May 24, 2019, the FDA granted regular approval to alpelisib in combination with fulvestrant for postmenopausal women, and men, with hormone receptor (HR)-pos., HER2-neg., phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. Approval was based on the SOLAR-1 study, a randomized, double-blind, placebo-controlled trial of alpelisib plus fulvestrant vs. placebo plus fulvestrant. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST v1.1 in the cohort of trial participants whose tumors had a PIK3CA mutation. The estd. median PFS by investigator assessment in the alpelisib plus fulvestrant arm was 11 mo [95% confidence interval (CI), 7.5-14.5] compared with 5.7 mo (95% CI, 3.7-7.4) in the placebo plus fulvestrant arm (HR, 0.65; 95% CI, 0.50-0.85; two-sided P = 0.001). The median overall survival was not yet reached for the alpelisib plus fulvestrant arm (95% CI, 28.1-NE) and was 26.9 mo (95% CI, 21.9-NE) for the fulvestrant control arm. No PFS benefit was obsd. in trial participants whose tumors did not have a PIK3CA mutation (HR, 0.85; 95% CI, 0.58-1.25). The most common adverse reactions, including lab. abnormalities, on the alpelisib plus fulvestrant arm were increased glucose, increased creatinine, diarrhea, rash, decreased lymphocyte count, increased gamma glutamyl transferase, nausea, increased alanine aminotransferase, fatigue, decreased Hb, increased lipase, decreased appetite, stomatitis, vomiting, decreased wt., decreased calcium, decreased glucose, prolonged activated partial thromboplastin time, and alopecia.
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    38. 38
      André, F.; Ciruelos, E. M.; Juric, D.; Loibl, S.; Campone, M.; Mayer, I. A.; Rubovszky, G.; Yamashita, T.; Kaufman, B.; Lu, Y. S.; Inoue, K.; Pápai, Z.; Takahashi, M.; Ghaznawi, F.; Mills, D.; Kaper, M.; Miller, M.; Conte, P. F.; Iwata, H.; Rugo, H. S. Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: final overall survival results from SOLAR-1. Ann. Oncol. 2021, 32, 208– 217,  DOI: 10.1016/j.annonc.2020.11.011
      38
      Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: final overall survival results from SOLAR-1
      Andre, F.; Ciruelos, E. M.; Juric, D.; Loibl, S.; Campone, M.; Mayer, I. A.; Rubovszky, G.; Yamashita, T.; Kaufman, B.; Lu, Y.-S.; Inoue, K.; Papai, Z.; Takahashi, M.; Ghaznawi, F.; Mills, D.; Kaper, M.; Miller, M.; Conte, P. F.; Iwata, H.; Rugo, H. S.
      Annals of Oncology (2021), 32 (2), 208-217CODEN: AONNAY; ISSN:1569-8041. (Elsevier Ltd.)
      Activation of the phosphatidylinositol-3-kinase (PI3K) pathway via PIK3CA mutations occurs in 28%-46% of hormone receptor-pos. (HR+), human epidermal growth factor receptor-2-neg. (HER2-) advanced breast cancers (ABCs) and is assocd. with poor prognosis. The SOLAR-1 trial showed that the addn. of alpelisib to fulvestrant treatment provided statistically significant and clin. meaningful progression-free survival (PFS) benefit in PIK3CA-mutated, HR+, HER2- ABC. Men and postmenopausal women with HR+, HER2- ABC whose disease progressed on or after aromatase inhibitor (AI) were randomized 1 : 1 to receive alpelisib (300 mg/day) plus fulvestrant (500 mg every 28 days and once on day 15) or placebo plus fulvestrant. Overall survival (OS) in the PIK3CA-mutant cohort was evaluated by Kaplan-Meier methodol. and a one-sided stratified log-rank test was carried out with an O'Brien-Fleming efficacy boundary of P ≤ 0.0161. In the PIK3CA-mutated cohort (n = 341), median OS [95% confidence interval (CI)] was 39.3 mo (34.1-44.9) for alpelisib-fulvestrant and 31.4 mo (26.8-41.3) for placebo-fulvestrant [hazard ratio (HR) = 0.86 (95% CI, 0.64-1.15; P = 0.15)]. OS results did not cross the prespecified efficacy boundary. Median OS (95% CI) in patients with lung and/or liver metastases was 37.2 mo (28.7-43.6) and 22.8 mo (19.0-26.8) in the alpelisib-fulvestrant and placebo-fulvestrant arms, resp. [HR = 0.68 (0.46-1.00)]. Median times to chemotherapy (95% CI) for the alpelisib-fulvestrant and placebo-fulvestrant arms were 23.3 mo (15.2-28.4) and 14.8 mo (10.5-22.6), resp. [HR = 0.72 (0.54-0.95)]. No new safety signals were obsd. with longer follow-up. Although the anal. did not cross the prespecified boundary for statistical significance, there was a 7.9-mo numeric improvement in median OS when alpelisib was added to fulvestrant treatment of patients with PIK3CA-mutated, HR+, HER2- ABC. Overall, these results further support the statistically significant prolongation of PFS obsd. with alpelisib plus fulvestrant in this population, which has a poor prognosis due to a PIK3CA mutation.
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    39. 39
      Turner, S.; Chia, S.; Kanakamedala, H.; Hsu, W. C.; Park, J.; Chandiwana, D.; Ridolfi, A.; Yu, C. L.; Zarate, J. P.; Rugo, H. S. Effectiveness of Alpelisib + Fulvestrant Compared with Real-World Standard Treatment Among Patients with HR+, HER2-, PIK3CA-Mutated Breast Cancer. Oncologist 2021, 26, e1133– e1142,  DOI: 10.1002/onco.13804
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      Effectiveness of Alpelisib + Fulvestrant Compared with Real-World Standard Treatment Among Patients with HR +, HER2 -, PIK3CA-Mutated Breast Cancer
      Turner, Stuart; Chia, Stephen; Kanakamedala, Hemanth; Hsu, Wei-Chun; Park, Jinhee; Chandiwana, David; Ridolfi, Antonia; Yu, Chu-Ling; Zarate, Juan Pablo; Rugo, Hope S.
      Oncologist (2021), 26 (7), e1133-e1142CODEN: OCOLF6; ISSN:1549-490X. (AlphaMed Press)
      The BYLieve trial (NCT03056755) confirmed efficacy and safety of alpelisib with fulvestrant for hormone receptor-pos. (HR+), human epidermal growth factor receptor-2-neg. (HER2-), PIK3CA-mutated advanced breast cancer (ABC), after cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) with an aromatase inhibitor (AI) as immediate prior therapy. Further analyses were performed to compare efficacy from BYLieve with effectiveness of std. treatment in the real-world setting. Patients who progressed on a CDK4/6i plus AI and were treated with alpelisib with fulvestrant in BYLieve were matched with a real-world patient cohort who received std.-of-care from a deidentified clinico-genomics database (CGDB). Primary and secondary endpoints were to compare progression-free survival (PFS), estd. by the Kaplan-Meier method, and the proportion of patients remaining progression-free at 6 mo, resp., between the two cohorts. A total of 855 patients with PIK3CA-mutant disease who had prior CDK4/6i plus hormone therapy were selected from the CGDB; further matching to 120 patients from BYLieve selected 95 patients without exposure to HER2-targeting agents, clin. study drug, or alpelisib. In unadjusted and postmatching results, primary and secondary endpoints favored treatment with alpelisib with fulvestrant in BYLieve more than std. treatments in the real-world cohort. Postadjustment, median PFS for patients treated with alpelisib in BYLieve was 7.3 vs. 3.7 mo in the real-world cohort, and 6-mo PFS was 54.6% vs. 40.1%, resp. Matched/weighted anal. comparing BYLieve with the real-world setting further supports the clin. benefit of alpelisib with fulvestrant for treatment of HR+, HER2-, PIK3CA-mutant ABC after CDK4/6i treatment. Approx. 40% of patients with hormone receptor-pos. (HR+), human epidermal growth factor receptor-2-neg. (HER2-) advanced breast cancer (ABC) have PIK3CA-mutated tumors, which have been assocd. with endocrine therapy resistance. Alpelisib, an α-selective phosphatidylinositol-3-kinase inhibitor, demonstrated significantly improved progression-free survival in SOLAR-1 and demonstrated clin. efficacy in BYLieve when combined with fulvestrant. Data are limited in comparing the efficacy of alpelisib combined with fulvestrant with effectiveness of std. therapy after CDK4/6i treatment. Using real-world data, this is the first anal. comparing alpelisib combined with fulvestrant with std. treatments for HR+, HER2-, PIK3CA-mutant ABC in the post-CDK4/6i setting.
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    40. 40
      Verret, B.; Cortes, J.; Bachelot, T.; Andre, F.; Arnedos, M. Efficacy of PI3K inhibitors in advanced breast cancer. Ann. Oncol. 2019, 30, x12– x20,  DOI: 10.1093/annonc/mdz381
      There is no corresponding record for this reference.
    41. 41
      Venot, Q.; Blanc, T.; Rabia, S. H.; Berteloot, L.; Ladraa, S.; Duong, J. P.; Blanc, E.; Johnson, S. C.; Hoguin, C.; Boccara, O.; Sarnacki, S.; Boddaert, N.; Pannier, S.; Martinez, F.; Magassa, S.; Yamaguchi, J.; Knebelmann, B.; Merville, P.; Grenier, N.; Joly, D.; Cormier-Daire, V.; Michot, C.; Bole-Feysot, C.; Picard, A.; Soupre, V.; Lyonnet, S.; Sadoine, J.; Slimani, L.; Chaussain, C.; Laroche-Raynaud, C.; Guibaud, L.; Broissand, C.; Amiel, J.; Legendre, C.; Terzi, F.; Canaud, G. Targeted therapy in patients with PIK3CA-related overgrowth syndrome. Nature 2018, 558, 540– 546,  DOI: 10.1038/s41586-018-0217-9
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      Targeted therapy in patients with PIK3CA-related overgrowth syndrome
      Venot, Quitterie; Blanc, Thomas; Rabia, Smail Hadj; Berteloot, Laureline; Ladraa, Sophia; Duong, Jean-Paul; Blanc, Estelle; Johnson, Simon C.; Hoguin, Clement; Boccara, Olivia; Sarnacki, Sabine; Boddaert, Nathalie; Pannier, Stephanie; Martinez, Frank; Magassa, Sato; Yamaguchi, Junna; Knebelmann, Bertrand; Merville, Pierre; Grenier, Nicolas; Joly, Dominique; Cormier-Daire, Valerie; Michot, Caroline; Bole-Feysot, Christine; Picard, Arnaud; Soupre, Veronique; Lyonnet, Stanislas; Sadoine, Jeremy; Slimani, Lotfi; Chaussain, Catherine; Laroche-Raynaud, Cecile; Guibaud, Laurent; Broissand, Christine; Amiel, Jeanne; Legendre, Christophe; Terzi, Fabiola; Canaud, Guillaume
      Nature (London, United Kingdom) (2018), 558 (7711), 540-546CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumors became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not assocd. with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.
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    42. 42
      Bohnacker, T.; Prota, A. E.; Beaufils, F.; Burke, J. E.; Melone, A.; Inglis, A. J.; Rageot, D.; Sele, A. M.; Cmiljanovic, V.; Cmiljanovic, N.; Bargsten, K.; Aher, A.; Akhmanova, A.; Diaz, J. F.; Fabbro, D.; Zvelebil, M.; Williams, R. L.; Steinmetz, M. O.; Wymann, M. P. Deconvolution of Buparlisib’s mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention. Nat. Commun. 2017, 8, 14683  DOI: 10.1038/ncomms14683
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      Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention
      Bohnacker Thomas; Beaufils Florent; Melone Anna; Rageot Denise; Sele Alexander M; Cmiljanovic Vladimir; Cmiljanovic Natasa; Wymann Matthias P; Prota Andrea E; Bargsten Katja; Steinmetz Michel O; Burke John E; Inglis Alison J; Williams Roger L; Aher Amol; Akhmanova Anna; Diaz J Fernando; Fabbro Doriano; Zvelebil Marketa
      Nature communications (2017), 8 (), 14683 ISSN:.
      BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.
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    43. 43
      Beaufils, F.; Cmiljanovic, N.; Cmiljanovic, V.; Bohnacker, T.; Melone, A.; Marone, R.; Jackson, E.; Zhang, X.; Sele, A.; Borsari, C.; Mestan, J.; Hebeisen, P.; Hillmann, P.; Giese, B.; Zvelebil, M.; Fabbro, D.; Williams, R. L.; Rageot, D.; Wymann, M. P. 5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a potent, brain-penetrant, orally bioavailable, pan-class I PI3K/mTOR inhibitor as clinical candidate in oncology. J. Med. Chem. 2017, 60, 7524– 7538,  DOI: 10.1021/acs.jmedchem.7b00930
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      5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology
      Beaufils, Florent; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Bohnacker, Thomas; Melone, Anna; Marone, Romina; Jackson, Eileen; Zhang, Xuxiao; Sele, Alexander; Borsari, Chiara; Mestan, Jurgen; Hebeisen, Paul; Hillmann, Petra; Giese, Bernd; Zvelebil, Marketa; Fabbro, Doriano; Williams, Roger L.; Rageot, Denise; Wymann, Matthias P.
      Journal of Medicinal Chemistry (2017), 60 (17), 7524-7538CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclin. characterization of compd. 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concns. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was obsd. for the structurally related 4 (BKM120, buparlisib). Compd. 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compd. 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compd.'s safety profile, identifies 1 as a clin. candidate with a broad application range in oncol., including treatment of brain tumors or CNS metastasis. Compd. 1 is currently in phase II clin. trials for advanced solid tumors and refractory lymphoma.
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    44. 44
      Tarantelli, C.; Gaudio, E.; Arribas, A. J.; Kwee, I.; Hillmann, P.; Rinaldi, A.; Cascione, L.; Spriano, F.; Bernasconi, E.; Guidetti, F.; Carrassa, L.; Pittau, R. B.; Beaufils, F.; Ritschard, R.; Rageot, D.; Sele, A.; Dossena, B.; Rossi, F. M.; Zucchetto, A.; Taborelli, M.; Gattei, V.; Rossi, D.; Stathis, A.; Stussi, G.; Broggini, M.; Wymann, M. P.; Wicki, A.; Zucca, E.; Cmiljanovic, V.; Fabbro, D.; Bertoni, F. PQR309 is a novel dual PI3K/mTOR inhibitor with preclinical antitumor activity in lymphomas as a single agent and in combination therapy. Clin. Cancer Res. 2018, 24, 120– 129,  DOI: 10.1158/1078-0432.CCR-17-1041
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      PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy
      Tarantelli, Chiara; Gaudio, Eugenio; Arribas, Alberto J.; Kwee, Ivo; Hillmann, Petra; Rinaldi, Andrea; Cascione, Luciano; Spriano, Filippo; Bernasconi, Elena; Guidetti, Francesca; Carrassa, Laura; Pittau, Roberta Bordone; Beaufils, Florent; Ritschard, Reto; Rageot, Denise; Sele, Alexander; Dossena, Barbara; Rossi, Francesca Maria; Zucchetto, Antonella; Taborelli, Monica; Gattei, Valter; Rossi, Davide; Stathis, Anastasios; Stussi, Georg; Broggini, Massimo; Wymann, Matthias P.; Wicki, Andreas; Zucca, Emanuele; Cmiljanovic, Vladimir; Fabbro, Doriano; Bertoni, Francesco
      Clinical Cancer Research (2018), 24 (1), 120-129CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacol. inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clin. evaluation, in preclin. lymphoma models. Exptl. Design: This study included preclin. in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation expts. on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was assocd. with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compd. that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120-9. ©2017 AACR.
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    45. 45
      Wicki, A.; Brown, N.; Xyrafas, A.; Bize, V.; Hawle, H.; Berardi, S.; Cmiljanovic, N.; Cmiljanovic, V.; Stumm, M.; Dimitrijevic, S.; Herrmann, R.; Pretre, V.; Ritschard, R.; Tzankov, A.; Hess, V.; Childs, A.; Hierro, C.; Rodon, J.; Hess, D.; Joerger, M.; von Moos, R.; Sessa, C.; Kristeleit, R. First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13). Eur. J. Cancer 2018, 96, 6– 16,  DOI: 10.1016/j.ejca.2018.03.012
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      First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13)
      Wicki, Andreas; Brown, Nicholas; Xyrafas, Alexandros; Bize, Vincent; Hawle, Hanne; Berardi, Simona; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Stumm, Michael; Dimitrijevic, Sasa; Herrmann, Richard; Pretre, Vincent; Ritschard, Reto; Tzankov, Alexandar; Hess, Viviane; Childs, Alexa; Hierro, Cinta; Rodon, Jordi; Hess, Dagmar; Joerger, Markus; von Moos, Roger; Sessa, Cristiana; Kristeleit, Rebecca
      European Journal of Cancer (2018), 96 (), 6-16CODEN: EJCAEL; ISSN:0959-8049. (Elsevier Ltd.)
      PQR309 is an orally bioavailable, balanced pan-phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR) C1 and mTORC2 inhibitor. This is an accelerated titrn., 3 D 3 dose-escalation, open-label phase Itrial of continuous once-daily (OD) PQR309 administration to evaluate the safety, pharmacokinetics (PK) and pharmacodynamics in patients with advanced solid tumors. Primary objectives were to det. the max. tolerated dose (MTD) and recommended phase 2 dose (RP2D).Twenty-eight patients were included in six dosing cohorts and treated at a daily PQR309 dose ranging from 10 to 150 mg. Common adverse events (AEs; ≥30% patients) included fatigue, hyperglycemia, nausea, diarrhea, constipation, rash, anorexia and vomiting. Grade (G) 3 or 4 drug-related AEs were seen in 13 (46%) and three (11%) patients, resp. Dose-limiting toxicity (DLT) was obsd. in two patients at 100 mg OD (>14-d interruption in PQR309 due to G3 rash, G2 hyperbilirubinemia, G4 suicide attempt; dose redn. due to G3 fatigue, G2 diarrhoea, G4 transaminitis) and one patient at 80 mg (G3 hyperglycemia >7 d). PK shows fast absorption (Tmax 1-2 h) and dose proportionality for Cmax and area under the curve. A partial response in a patient with metastatic thymus cancer, 24% disease vol. redn. in a patient with sinonasal cancer and stable disease for more than 16 wk in a patient with clear cell Bartholin's gland cancer were obsd.The MTD and RP2D of PQR309 is 80 mg of orally OD. PK is dose-proportional. PD shows PI3K pathway phosphoprotein downregulation in paired tumor biopsies. Clin. activity was obsd. in patients with and without PI3K pathway dysregulation.
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    46. 46
      Borsari, C.; Rageot, D.; Beaufils, F.; Bohnacker, T.; Keles, E.; Buslov, I.; Melone, A.; Sele, A. M.; Hebeisen, P.; Fabbro, D.; Hillmann, P.; Wymann, M. P. Preclinical Development of PQR514, a Highly Potent PI3K Inhibitor Bearing a Difluoromethyl-Pyrimidine Moiety. ACS Med. Chem. Lett. 2019, 10, 1473– 1479,  DOI: 10.1021/acsmedchemlett.9b00333
      46
      Preclinical Development of PQR514, a Highly Potent PI3K Inhibitor Bearing a Difluoromethyl-Pyrimidine Moiety
      Borsari, Chiara; Rageot, Denise; Beaufils, Florent; Bohnacker, Thomas; Keles, Erhan; Buslov, Ivan; Melone, Anna; Sele, Alexander M.; Hebeisen, Paul; Fabbro, Doriano; Hillmann, Petra; Wymann, Matthias P.
      ACS Medicinal Chemistry Letters (2019), 10 (10), 1473-1479CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is a crit. regulator of cell growth and is frequently hyperactivated in cancer. Therefore, PI3K inhibitors represent a valuable asset in cancer therapy. Herein we have developed a novel anticancer agent, the potent pan-PI3K inhibitor PQR514 (4), which is a follow-up compd. for the phase-II clin. compd. PQR309 (1). Compd. 4 has an improved potency both in vitro and in cellular assays with respect to its predecessor compds. It shows superiority in the suppression of cancer cell proliferation and demonstrates significant antitumor activity in an OVCAR-3 xenograft model at concns. approx. eight times lower than PQR309 (1). The favorable pharmacokinetic profile and a minimal brain penetration promote PQR514 (4) as an optimized candidate for the treatment of systemic tumors.
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    47. 47
      Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60, 839– 885,  DOI: 10.1021/acs.jmedchem.6b00788
      47
      Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions
      Jackson, Paul A.; Widen, John C.; Harki, Daniel A.; Brummond, Kay M.
      Journal of Medicinal Chemistry (2017), 60 (3), 839-885CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogs possessing this moiety. In the midst of recently approved acrylamide-contg. drugs, it is clear that a good understanding of the hetero-Michael addn. reaction and the relative reactivities of biol. thiols with Michael acceptors under physiol. conditions is needed for the design and use of these compds. as biol. tools and potential therapeutics. This Perspective provides information that will contribute to this understanding, such as kinetics of thiol addn. reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This Perspective is focused on α,β-unsatd. carbonyls given their preponderance in bioactive natural products.
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    48. 48
      Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J. Med. Chem. 2014, 57, 10072– 10079,  DOI: 10.1021/jm501412a
      48
      Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors
      Flanagan, Mark E.; Abramite, Joseph A.; Anderson, Dennis P.; Aulabaugh, Ann; Dahal, Upendra P.; Gilbert, Adam M.; Li, Chao; Montgomery, Justin; Oppenheimer, Stacey R.; Ryder, Tim; Schuff, Brandon P.; Uccello, Daniel P.; Walker, Gregory S.; Wu, Yan; Brown, Matthew F.; Chen, Jinshan M.; Hayward, Matthew M.; Noe, Mark C.; Obach, R. Scott; Philippe, Laurence; Shanmugasundaram, Veerabahu; Shapiro, Michael J.; Starr, Jeremy; Stroh, Justin; Che, Ye
      Journal of Medicinal Chemistry (2014), 57 (23), 10072-10079CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry addnl. risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biol. relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.
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    49. 49
      Martin, J. S.; MacKenzie, C. J.; Fletcher, D.; Gilbert, I. H. Characterising covalent warhead reactivity. Bioorg. Med. Chem. 2019, 27, 2066– 2074,  DOI: 10.1016/j.bmc.2019.04.002
      49
      Characterising covalent warhead reactivity
      Martin, James S.; MacKenzie, Claire J.; Fletcher, Daniel; Gilbert, Ian H.
      Bioorganic & Medicinal Chemistry (2019), 27 (10), 2066-2074CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      Many drugs currently used are covalent inhibitors and irreversibly inhibit their targets. Most of these were discovered through serendipity. Covalent inhibitions can have many advantages from a pharmacokinetic perspective. However, until recently most organizations have shied away from covalent compd. design due to fears of non-specific inhibition of off-target proteins leading to toxicity risks. However, there has been a renewed interest in covalent modifiers as potential drugs, as it possible to get highly selective compds. It is therefore important to know how reactive a warhead is and to be able to select the least reactive warhead possible to avoid toxicity. A robust NMR based assay was developed and used to measure the reactivity of a variety of covalent warheads against serine and cysteine - the two most common targets for covalent drugs. A selection of these warheads also had their reactivity measured against threonine, tyrosine, lysine, histidine and arginine to better understand our ability to target non-traditional residues. The reactivity was also measured at various pHs to assess what effect the environment in the active site would have on these reactions. The reactivity of a covalent modifier was found to be very dependent on the amino acid residue.
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    50. 50
      Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57, 3124– 3137,  DOI: 10.1021/acs.jcim.7b00553
      50
      Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity
      Lonsdale, Richard; Burgess, Jonathan; Colclough, Nicola; Davies, Nichola L.; Lenz, Eva M.; Orton, Alexandra L.; Ward, Richard A.
      Journal of Chemical Information and Modeling (2017), 57 (12), 3124-3137CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compds. for target validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or "warhead"). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity. The half-life to adduct formation with glutathione (GSH t1/2) is a useful assay for measuring the reactivity of cysteine-targeting covalent warheads but is limited to synthesized mols. In this manuscript the authors assess the ability of several exptl. and computational approaches to predict GSH t1/2 for a range of cysteine targeting warheads, including a novel method based on pKa. Furthermore, matched mol. pairs anal. has been performed against the internal compd. collection, revealing structure-activity relationships between a selection of different covalent warheads. These observations and methods of prediction will be valuable in the design of new covalent inhibitors with desired levels of reactivity.
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    51. 51
      Ma, S.; Devi-Kesavan, L. S.; Gao, J. Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K. J. Am. Chem. Soc. 2007, 129, 13633– 13645,  DOI: 10.1021/ja074222+
      51
      Molecular Dynamics Simulations of the Catalytic Pathway of a Cysteine Protease: A Combined QM/MM Study of Human Cathepsin K
      Ma, Shuhua; Devi-Kesavan, Lakshmi S.; Gao, Jiali
      Journal of the American Chemical Society (2007), 129 (44), 13633-13645CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Mol. dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have detd. the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aq. soln. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aq. soln. The redn. of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 Å and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.
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    52. 52
      Cusack, K. P.; Arnold, L. D.; Barberis, C. E.; Chen, H.; Ericsson, A. M.; Gaza-Bulseco, G. S.; Gordon, T. D.; Grinnell, C. M.; Harsch, A.; Pellegrini, M.; Tarcsa, E. A 13C NMR approach to categorizing potential limitations of alpha,beta-unsaturated carbonyl systems in drug-like molecules. Bioorg. Med. Chem. Lett. 2004, 14, 5503– 5507,  DOI: 10.1016/j.bmcl.2004.09.007
      52
      A 13C NMR approach to categorizing potential limitations of α,β-unsaturated carbonyl systems in drug-like molecules
      Cusack, Kevin P.; Arnold, Lee D.; Barberis, Claude E.; Chen, Haipeng; Ericsson, Anna M.; Gaza-Bulseco, Georgeen S.; Gordon, Thomas D.; Grinnell, Christine M.; Harsch, Andreas; Pellegrini, Maria; Tarcsa, Edit
      Bioorganic & Medicinal Chemistry Letters (2004), 14 (22), 5503-5507CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      Compds. that contain an α,β-unsatd. carbonyl moiety are often flagged as potential Michael acceptors. All α,β-unsatd. carbonyl moieties are not equiv., however, and we sought to better understand this system and its potential implications in drug-like mols. Measurement of the 13C NMR shift of the β-carbon and correlation to in vitro results allowed compds. in our collection to be categorized as potential Michael acceptors, potential substrates for NADPH, or as photoisomerizable.
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    53. 53
      MacFaul, P. A.; Morley, A. D.; Crawford, J. J. A simple in vitro assay for assessing the reactivity of nitrile containing compounds. Bioorg. Med. Chem. Lett. 2009, 19, 1136– 1138,  DOI: 10.1016/j.bmcl.2008.12.105
      53
      A simple in vitro assay for assessing the reactivity of nitrile containing compounds
      MacFaul, Philip A.; Morley, Andrew D.; Crawford, James J.
      Bioorganic & Medicinal Chemistry Letters (2009), 19 (4), 1136-1138CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      A quant. assay involving the reaction of nitriles with glutathione and cysteine has been used as a simple in vitro screen to assess potential toxicity risk of candidate compds. in drug discovery. Studies have indicated that, when benchmarked with selected compds., the reaction of the nitriles with glutathione can provide a useful tool for deciding whether or not to progress compds. in the absence of radiolabelling studies.
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    54. 54
      Brameld, K. A.; Owens, T. D.; Verner, E.; Venetsanakos, E.; Bradshaw, J. M.; Phan, V. T.; Tam, D.; Leung, K.; Shu, J.; LaStant, J.; Loughhead, D. G.; Ton, T.; Karr, D. E.; Gerritsen, M. E.; Goldstein, D. M.; Funk, J. O. Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors. J. Med. Chem. 2017, 60, 6516– 6527,  DOI: 10.1021/acs.jmedchem.7b00360
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      Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors
      Brameld, Ken A.; Owens, Timothy D.; Verner, Erik; Venetsanakos, Eleni; Bradshaw, J. Michael; Phan, Vernon T.; Tam, Danny; Leung, Kwan; Shu, Jin; LaStant, Jacob; Loughhead, David G.; Ton, Tony; Karr, Dane E.; Gerritsen, Mary E.; Goldstein, David M.; Funk, Jens Oliver
      Journal of Medicinal Chemistry (2017), 60 (15), 6516-6527CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clin. validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncol. indications. An irreversible covalent binding mechanism imparts many desirable pharmacol. benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chem. optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compd. 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.
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    55. 55
      Cee, V. J.; Volak, L. P.; Chen, Y.; Bartberger, M. D.; Tegley, C.; Arvedson, T.; McCarter, J.; Tasker, A. S.; Fotsch, C. Systematic Study of the Glutathione (GSH) Reactivity of N-Arylacrylamides: 1. Effects of Aryl Substitution. J. Med. Chem. 2015, 58, 9171– 9178,  DOI: 10.1021/acs.jmedchem.5b01018
      55
      Systematic Study of the Glutathione (GSH) Reactivity of N-Arylacrylamides: 1. Effects of Aryl Substitution
      Cee, Victor J.; Volak, Laurie P.; Chen, Yuping; Bartberger, Michael D.; Tegley, Chris; Arvedson, Tara; McCarter, John; Tasker, Andrew S.; Fotsch, Christopher
      Journal of Medicinal Chemistry (2015), 58 (23), 9171-9178CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relations among N-arylacrylamides, the authors detd. glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. Substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. The authors note a correlation between 1H and 13C NMR chem. shifts of the acrylamide with GSH reaction rates, suggesting that NMR chem. shifts may be a convenient surrogate measure of relative acrylamide reactivity. D. functional theory calcns. reveal a correlation between computed activation parameters and exptl. detd. reaction rates, validating the use of such methodol. for the screening of synthetic candidates in a prospective fashion.
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    56. 56
      Birkholz, A.; Kopecky, D. J.; Volak, L. P.; Bartberger, M. D.; Chen, Y.; Tegley, C. M.; Arvedson, T.; McCarter, J. D.; Fotsch, C.; Cee, V. J. Systematic Study of the Glutathione Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution. J. Med. Chem. 2020, 63, 11602– 11614,  DOI: 10.1021/acs.jmedchem.0c00749
      56
      Systematic Study of the Glutathione Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution
      Birkholz, Adam; Kopecky, David J.; Volak, Laurie P.; Bartberger, Michael D.; Chen, Yuping; Tegley, Christopher M.; Arvedson, Tara; McCarter, John D.; Fotsch, Christopher; Cee, Victor J.
      Journal of Medicinal Chemistry (2020), 63 (20), 11602-11614CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A comprehensive understanding of structure-reactivity relationships is crit. to the design and optimization of cysteine-targeted covalent inhibitors. Herein, we report glutathione (GSH) reaction rates for N-Ph acrylamides with varied substitutions at the α- and β-positions of the acrylamide moiety. We find that the GSH reaction rates can generally be understood in terms of the electron donating or withdrawing ability of the substituent. When installed at the β-position, aminomethyl substituents with amine pKa's > 7 accelerate, while those with pKa's < 7 slow the rate of GSH addn. at pH 7.4, relative to a hydrogen substituent. Although a computational model was able to only approx. capture exptl. reactivity trends, our calcns. do not support a frequently invoked mechanism of concerted amine/thiol proton transfer and C-S bond formation and instead suggest that protonated aminomethyl functions as an electron-withdrawing group to reduce the barrier for thiolate addn. to the acrylamide.
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    57. 57
      Zhu, K.; Borrelli, K. W.; Greenwood, J. R.; Day, T.; Abel, R.; Farid, R. S.; Harder, E. Docking covalent inhibitors: a parameter free approach to pose prediction and scoring. J. Chem. Inf. Model. 2014, 54, 1932– 1940,  DOI: 10.1021/ci500118s
      57
      Docking Covalent Inhibitors: A Parameter Free Approach To Pose Prediction and Scoring
      Zhu, Kai; Borrelli, Kenneth W.; Greenwood, Jeremy R.; Day, Tyler; Abel, Robert; Farid, Ramy S.; Harder, Edward
      Journal of Chemical Information and Modeling (2014), 54 (7), 1932-1940CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      Although many popular docking programs include a facility to account for covalent ligands, large-scale systematic docking validation studies of covalent inhibitors have been sparse. In this paper, we present the development and validation of a novel approach for docking and scoring covalent inhibitors, which consists of conventional noncovalent docking, heuristic formation of the covalent attachment point, and structural refinement of the protein-ligand complex. This approach combines the strengths of the docking program Glide and the protein structure modeling program Prime and does not require any parameter fitting for the study of addnl. covalent reaction types. We first test this method by predicting the native binding geometry of 38 covalently bound complexes. The av. RMSD of the predicted poses is 1.52 Å, and 76% of test set inhibitors have an RMSD of less than 2.0 Å. In addn., the apparent affinity score constructed herein is tested on a virtual screening study and the characterization of the SAR properties of two different series of congeneric compds. with satisfactory success.
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    58. 58
      Toledo Warshaviak, D.; Golan, G.; Borrelli, K. W.; Zhu, K.; Kalid, O. Structure-based virtual screening approach for discovery of covalently bound ligands. J. Chem. Inf. Model. 2014, 54, 1941– 1950,  DOI: 10.1021/ci500175r
      58
      Structure-Based Virtual Screening Approach for Discovery of Covalently Bound Ligands
      Toledo Warshaviak, Dora; Golan, Gali; Borrelli, Kenneth W.; Zhu, Kai; Kalid, Ori
      Journal of Chemical Information and Modeling (2014), 54 (7), 1941-1950CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      We present a fast and effective covalent docking approach suitable for large-scale virtual screening (VS). We applied this method to four targets (HCV NS3 protease, Cathepsin K, EGFR, and XPO1) with known crystal structures and known covalent inhibitors. We implemented a customized "VS mode" of the Schrodinger Covalent Docking algorithm (CovDock), which we refer to as CovDock-VS. Known actives and target-specific sets of decoys were docked to selected X-ray structures, and poses were filtered based on noncovalent protein-ligand interactions known to be important for activity. We were able to retrieve 71%, 72%, and 77% of the known actives for Cathepsin K, HCV NS3 protease, and EGFR within 5% of the decoy library, resp. With the more challenging XPO1 target, where no specific interactions with the protein could be used for postprocessing of the docking results, we were able to retrieve 95% of the actives within 30% of the decoy library and achieved an early enrichment factor (EF1%) of 33. The poses of the known actives bound to existing crystal structures of 4 targets were predicted with an av. RMSD of 1.9 Å. To the best of our knowledge, CovDock-VS is the first fully automated tool for efficient virtual screening of covalent inhibitors. Importantly, CovDock-VS can handle multiple chem. reactions within the same library, only requiring a generic SMARTS-based predefinition of the reaction. CovDock-VS provides a fast and accurate way of differentiating actives from decoys without significantly deteriorating the accuracy of the predicted poses for covalent protein-ligand complexes. Therefore, we propose CovDock-VS as an efficient structure-based virtual screening method for discovery of novel and diverse covalent ligands.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpslegtbw%253D&md5=0ff8b6e682473abd2e2dadb49d95539a
    59. 59
      Rageot, D.; Bohnacker, T.; Keles, E.; McPhail, J. A.; Hoffmann, R. M.; Melone, A.; Borsari, C.; Sriramaratnam, R.; Sele, A. M.; Beaufils, F.; Hebeisen, P.; Fabbro, D.; Hillmann, P.; Burke, J. E.; Wymann, M. P. (S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a potent, orally bioavailable, and brain-penetrable dual inhibitor of class I PI3K and mTOR kinase. J. Med. Chem. 2019, 62, 6241– 6261,  DOI: 10.1021/acs.jmedchem.9b00525
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      (S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a Potent, Orally Bioavailable, and Brain-Penetrable Dual Inhibitor of Class I PI3K and mTOR Kinase
      Rageot, Denise; Bohnacker, Thomas; Keles, Erhan; McPhail, Jacob A.; Hoffmann, Reece M.; Melone, Anna; Borsari, Chiara; Sriramaratnam, Rohitha; Sele, Alexander M.; Beaufils, Florent; Hebeisen, Paul; Fabbro, Doriano; Hillmann, Petra; Burke, John E.; Wymann, Matthias P.
      Journal of Medicinal Chemistry (2019), 62 (13), 6241-6261CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure-activity relationship study, which led to the discovery of a drug-like ATP-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530, compd. 6), which qualifies as a clin. candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compd. 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compd. 6 prevented cell growth in a cancer cell line panel. The preclin. in vivo characterization of compd. 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncol.
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    60. 60
      Goel, P.; Alam, O.; Naim, M. J.; Nawaz, F.; Iqbal, M.; Alam, M. I. Recent advancement of piperidine moiety in treatment of cancer- A review. Eur. J. Med. Chem. 2018, 157, 480– 502,  DOI: 10.1016/j.ejmech.2018.08.017
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      Recent advancement of piperidine moiety in treatment of cancer- A review
      Goel, Pallavi; Alam, Ozair; Naim, Mohd Javed; Nawaz, Farah; Iqbal, Muzaffar; Alam, Md Iqbal
      European Journal of Medicinal Chemistry (2018), 157 (), 480-502CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      A review. Piperidine is an important pharmacophore, a privileged scaffold and an excellent heterocyclic system in the field of drug discovery which provides numerous opportunities in studying/exploring this moiety as an anticancer agent by acting on various receptors of utmost importance. Cancer is an uncontrolled division of cells that results in the formation of tumor which have the potential to migrate to other parts of the body (metastasis) finally becoming a major threat to human population. Since piperidine displayed great potential in this area it is being further probed to get novel entities for the treatment of cancer. This review throws light on recent biol. expansions of piperidine along with structure activity relationships to deliver assocn. between various synthesized newer/novel derivs. and receptor sites.
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    61. 61
      Copeland, R. A. Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 2005, 46, 1– 265
      61
      Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists
      Copeland Robert A
      Methods of biochemical analysis (2005), 46 (), 1-265 ISSN:0076-6941.
      There is no expanded citation for this reference.
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    62. 62
      Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discovery 2017, 22, 3– 20,  DOI: 10.1177/1087057116671509
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      A perspective on the kinetics of covalent and irreversible inhibition
      Strelow, John M.
      SLAS Discovery (2017), 22 (1), 3-20CODEN: SDLIBT; ISSN:2472-5560. (Sage Publications)
      The clin. and com. success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochem., cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochem. assay, the IC50 may simply reflect the target protein concn. in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI. The kinact/KI is a rate const. describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the max. potential rate (kinact) of inactivation. In this perspective, it is proposed that the kinact/KI should be employed as a crit. parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochem. assays to the cell, and more accurately define selectivity. It is also proposed that a physiol. relevant kinact/KI and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two crit. determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.
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    63. 63
      Dale, N. C.; Johnstone, E. K. M.; White, C. W.; Pfleger, K. D. G. NanoBRET: The Bright Future of Proximity-Based Assays. Front. Bioeng. Biotechnol. 2019, 7, 56  DOI: 10.3389/fbioe.2019.00056
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      NanoBRET: The Bright Future of Proximity-Based Assays
      Dale Natasha C; Johnstone Elizabeth K M; White Carl W; Pfleger Kevin D G; Dale Natasha C; Johnstone Elizabeth K M; White Carl W; Pfleger Kevin D G; Dale Natasha C; Johnstone Elizabeth K M; White Carl W; Pfleger Kevin D G; Pfleger Kevin D G
      Frontiers in bioengineering and biotechnology (2019), 7 (), 56 ISSN:2296-4185.
      Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.
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    64. 64
      Vasta, J. D.; Corona, C. R.; Wilkinson, J.; Zimprich, C. A.; Hartnett, J. R.; Ingold, M. R.; Zimmerman, K.; Machleidt, T.; Kirkland, T. A.; Huwiler, K. G.; Ohana, R. F.; Slater, M.; Otto, P.; Cong, M.; Wells, C. I.; Berger, B. T.; Hanke, T.; Glas, C.; Ding, K.; Drewry, D. H.; Huber, K. V. M.; Willson, T. M.; Knapp, S.; Muller, S.; Meisenheimer, P. L.; Fan, F.; Wood, K. V.; Robers, M. B. Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem. Biol. 2018, 25, 206.e11– 214.e11,  DOI: 10.1016/j.chembiol.2017.10.010
      There is no corresponding record for this reference.
    65. 65
      Ikediobi, O. N.; Davies, H.; Bignell, G.; Edkins, S.; Stevens, C.; O’Meara, S.; Santarius, T.; Avis, T.; Barthorpe, S.; Brackenbury, L.; Buck, G.; Butler, A.; Clements, J.; Cole, J.; Dicks, E.; Forbes, S.; Gray, K.; Halliday, K.; Harrison, R.; Hills, K.; Hinton, J.; Hunter, C.; Jenkinson, A.; Jones, D.; Kosmidou, V.; Lugg, R.; Menzies, A.; Mironenko, T.; Parker, A.; Perry, J.; Raine, K.; Richardson, D.; Shepherd, R.; Small, A.; Smith, R.; Solomon, H.; Stephens, P.; Teague, J.; Tofts, C.; Varian, J.; Webb, T.; West, S.; Widaa, S.; Yates, A.; Reinhold, W.; Weinstein, J. N.; Stratton, M. R.; Futreal, P. A.; Wooster, R. Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol. Cancer Ther. 2006, 5, 2606– 2612,  DOI: 10.1158/1535-7163.Mct-06-0433
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      Mutation analysis of 24 known cancer genes in the NCI-60 cell line set
      Ikediobi, Ogechi N.; Davies, Helen; Bignell, Graham; Edkins, Sarah; Stevens, Claire; O'Meara, Sarah; Santarius, Thomas; Avis, Tim; Barthorpe, Syd; Brackenbury, Lisa; Buck, Gemma; Butler, Adam; Clements, Jody; Cole, Jennifer; Dicks, Ed; Forbes, Simon; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathan; Hunter, Chris; Jenkinson, Andy; Jones, David; Kosmidou, Vivienne; Lugg, Richard; Menzies, Andrew; Mironenko, Tatiana; Parker, Adrian; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alex; Smith, Raffaella; Solomon, Helen; Stephens, Philip; Teague, Jon; Tofts, Calli; Varian, Jennifer; Webb, Tony; West, Sofie; Widaa, Sara; Yates, Andy; Reinhold, William; Weinstein, John N.; Stratton, Michael R.; Futreal, P. Andrew; Wooster, Richard
      Molecular Cancer Therapeutics (2006), 5 (11), 2606-2612CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacol. and at the mol. level more extensively than any other set of cell lines. However, no systematic mutation anal. of genes causally implicated in oncogenesis has been reported. This study reports the sequence anal. of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examd., with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacol. and mol. profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.
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    66. 66
      Gall, V. A.; Philips, A. V.; Qiao, N.; Clise-Dwyer, K.; Perakis, A. A.; Zhang, M.; Clifton, G. T.; Sukhumalchandra, P.; Ma, Q.; Reddy, S. M.; Yu, D.; Molldrem, J. J.; Peoples, G. E.; Alatrash, G.; Mittendorf, E. A. Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells. Cancer Res. 2017, 77, 5374– 5383,  DOI: 10.1158/0008-5472.Can-16-2774
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      trastuzumab increases her2 uptake and cross-presentation by dendritic cells
      Gall, Victor A.; Philips, Anne V.; Qiao, Na; Clise-Dwyer, Karen; Perakis, Alexander A.; Zhang, Mao; Clifton, Guy T.; Sukhumalchandra, Pariya; Ma, Qing; Reddy, Sangeetha M.; Yu, Dihua; Molldrem, Jeffrey J.; Peoples, George E.; Alatrash, Gheath; Mittendorf, Elizabeth A.
      Cancer Research (2017), 77 (19), 5374-5383CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Early-phase clin. trials evaluating CD8+ T cell-eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clin. trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of std. therapy in the per treatment analyses conducted after a median follow-up of greater than 34 mo. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-pos. breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival obsd. with combination therapy.
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    67. 67
      Chaussade, C.; Rewcastle, G. W.; Kendall, J. D.; Denny, W. A.; Cho, K.; Grønning, L. M.; Chong, M. L.; Anagnostou, S. H.; Jackson, S. P.; Daniele, N.; Shepherd, P. R. Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem. J. 2007, 404, 449– 458,  DOI: 10.1042/BJ20070003
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      Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling
      Chaussade, Claire; Rewcastle, Gordon W.; Kendall, Jackie D.; Denny, William A.; Cho, Kitty; Gronning, Line M.; Chong, Mei Ling; Anagnostou, Sasha H.; Jackson, Shaun P.; Daniele, Nathalie; Shepherd, Peter R.
      Biochemical Journal (2007), 404 (3), 449-458CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Recent genetic knock-in and pharmacol. approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110α isoform (PIK3CA) that plays the predominant role in insulin signaling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signaling. These include a p110α-specific inhibitor (PIK-75), a p110α-selective inhibitor (PI-103), a p110β-specific inhibitor (TGX-221) and a p110δ-specific inhibitor (IC87114). Although we find that p110α is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110β or p110δ alone was also not sufficient to block insulin signaling to PKB in these cells, but, when added in combination with p110α inhibitors, they are able to significantly attenuate insulin signaling. Surprisingly, in J774.2 macrophage cells, insulin signaling to PKB was inhibited to a similar extent by inhibitors of p110α, p110β or p110δ. These results provide evidence that p110β and p110δ can play a role in insulin signaling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.
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    68. 68
      Freeman-Cook, K. D.