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Sweet and Blind Spots in E3 Ligase Ligand Space Revealed by a Thermophoresis-Based AssayClick to copy article linkArticle link copied!
- Samuel MaiwaldSamuel MaiwaldDepartment of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, GermanyMore by Samuel Maiwald
- Christopher HeimChristopher HeimDepartment of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, GermanyMore by Christopher Heim
- Birte Hernandez AlvarezBirte Hernandez AlvarezDepartment of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, GermanyMore by Birte Hernandez Alvarez
- Marcus D. Hartmann*Marcus D. Hartmann*Tel. +49 7071 601 323. Email: [email protected]Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, GermanyMore by Marcus D. Hartmann
Abstract
Repurposing E3 ubiquitin ligases for targeted protein degradation via customized molecular glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable molecular glues and PROTACs is our fragmentary understanding of E3 ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of cereblon (CRBN).
The contemporary concept of targeted protein degradation is exponentially gaining attention as an important strategy in various clinical settings, most prominently in cancer therapy. (1) One of the most promising approaches in this area is to target proteins of interest (POI) for ubiquitination via E3 ubiquitin ligases. (2) The human genome encodes for several hundreds of such ligases, which are specialized in their substrate specificity and their spatiotemporal expression. Of these hundreds of ligases, only a handful are characterized, of which the VHL (von Hippel–Lindau) and CRBN (Cereblon) substrate receptors are among those that have gained the most attention. (3−6)
For repurposing E3 ligases to ubiquitinate POIs, two different major strategies are currently being employed, which are based on two conceptually different classes of small molecules. The first approach relies on small molecules called molecular glues, which mediate interactions between proteins that would not interact without the glue molecule. (7) For CRBN, such molecules are typically based on the thalidomide scaffold and constitute the class of immunomodulatory drugs termed IMiDs. (8) While natural substrates of CRBN are still largely elusive, the binding of IMiDs is known to modulate CRBN substrate specificity to recognize “neo-substrates”, in particular a group of zinc-finger transcription factors, which is thought to be the main driving force for the efficacy of the FDA-approved IMiD lenalidomide in multiple myeloma. (9) Here, the substrate specificity can be adjusted by small changes to the IMiD structure. (10,11) However, the specificity of IMiDs appears to be rather broad, and also largely unrelated small molecules that are able to bind to CRBN can yield degradation profiles and induce in vivo effects comparable to those of IMiDs. (12−14)
The other major strategy is the proteolysis targeting chimera (PROTAC) approach, which was coined by Crews and co-workers in 2001. (15) PROTACs are rather large bifunctional molecules, in which a ligand for an E3 ligase is connected to a specific binder of a POI via a flexible linker. While their larger molecular weight may pose a disadvantage, their chimeric nature allows for high target specificity. Their E3 ligase ligand is typically derived from the natural recognition motif of the ligase, e.g., a hydroxyproline-containing peptide for VHL, which can bind to a groove on its surface (Figure 1) (16) or, when the natural ligand is unknown, from a small-molecule binder like thalidomide, which binds to a conserved tri-tryptophan pocket in CRBN (Figure 1). (12,17) The warhead for the POI can often be derived from an existing binder or inhibitor of the POI. The third component, the linker, is the least constrained part and can be of any length that brings the POI into an orientation that allows for ubiquitination via the E3 ligase. (1) In practice, the development of a PROTAC for efficient ubiquitination of a certain POI can involve extensive screening of different linker types and of different E3 ligands for different E3 ligases, until a functional combination is identified. (18)
Figure 1
Figure 1. Overall structure and ligand binding modes for E3 ligase substrate receptors VHL and CRBN. (A) Structure of VHL-Elongin B-Elongin C (VBC) complex with bound HIF1-α peptide (PDB: 1LM8). (B) Illustrative surface representation of the VHL binding groove with bound 11mer reporter, based on the same structure, highlighting the position of the fluorophore and hydroxyproline (Hyp). (C) Structure of the bacterial CRBN homologue MsCI4 bound to thalidomide (PDB: 4V2Y). (D) Illustrative surface representation of MsCI4 bound to BODIPY-uracil reporter. (E) Proposed clash of nitrofurantoin in the binding pocket of CRBN. Nitrofurantoin (green) was aligned with a structure of hydantoin in complex with MsCI4 (PDB: 5OH7) and shown in comparison to thalidomide (red). The protruding moiety of nitrofurantoin comes in close proximity to W99 of MsCI4. (F) Depiction of the pharmacophore-based nomenclature for 5- and 6-membered rings. R refers to the protruding moiety with the canonical exit vector.
Currently, both the molecular glue/IMiD and the PROTAC approach are limited by the number of available E3 ligases and ligands, pointing at the need for the characterization of further ligases and the discovery of new ligands. For the latter, only a limited number of methods can be used to distinguish between unspecific interactions and the specific binding of a ligand to the specific binding pocket of the ligase. This is often realized by following the behavior of a fluorescent reporter ligand, which competes for the same binding site. For VHL, this has been implemented with a FAM-labeled substrate-peptide reporter that is monitored in a fluorescence polarization (FP) assay. (19) While FAM FP is constant while the reporter is bound to VHL, its displacement by a test compound leads to a drop in FP, which can be used to determine affinities in a titration experiment. The same approach has also been used for CRBN, with thalidomide coupled to a Cy5 fluorophore as a reporter. (17) An alternative specific for CRBN is a FRET (Förster resonance energy transfer) assay exploiting the presence of three strictly conserved tryptophan residues that are lining the ligand binding pocket. Here, the reporter ligand MANT-uracil carries a MANT (N-methyl-anthraniloyl) fluorophore, which forms a FRET pair with these tryptophans, linked to uracil as the actual binding moiety, such that the displacement of MANT-uracil from the binding pocket can be followed by the loss of the FRET effect. (20)
As both the FP and the FRET assay rely on changes of fluorescent properties that are initiated by the displacement of the respective reporter ligand, these assays can be hampered by overlapping optical properties such as autofluorescence of the test compounds. We therefore sought to establish a universal competitive assay that is orthogonal to these approaches, based on microscale thermophoresis (MST). A competitive MST-based assay relies on the behavior of a reporter ligand in a thermal gradient that is induced at a defined time using an infrared laser. This behavior is dominated by the thermal migration of the reporter along (positive thermophoresis) or against the temperature gradient (negative thermophoresis) and by a temperature-related intensity change (TRIC) effect, both of which can differ greatly between the free and the E3 ligase-bound state. (21,22) While also in this MST assay the reporter is monitored via its fluorescence, the assay is not based on fluorescence changes initiated by the displacement of the reporter from the ligase during sample preparation but on changes transiently induced by the application of the thermal gradient during the experiment. (23) Here, we implemented this assay both for an established VHL reporter ligand and for a CRBN reporter that we customized for this study.
For VHL, we employed the complex consisting of VHL, elongin B, and elongin C, together with two FAM-coupled reporter peptides with a length of 11 and 19 amino acids, which correspond to the reporter ligands previously used in FP experiments (Figure 2, see also Figure 1). (19) To determine their affinity, we performed initial titration experiments, varying the VHL concentration. The normalized MST traces show that the reporters exhibit positive thermophoretic behavior, i.e., move out of the heated spot induced during the measurement (Figure 2). Progressive addition of, and thus the binding of the reporter to, VHL dampens this effect, yielding a sigmoidal dose–response curve corresponding to Kd values of 434 and 53 nM for the short and long reporter peptide, respectively. For comparison, we also performed FP experiments with these reporters, obtaining Kd values of 280 and 20 nM, which is also in reasonable agreement with the values reported previously for comparable FP experiments (560 and 36 nM). (19) Having determined the affinity of the reporter peptides, we performed competition experiments with unlabeled versions of the same peptides, in which the latter were titrated as test ligands against a constant concentration of VHL and reporter, for each reporter peptide. The normalized MST traces show that the progressive addition of the test ligands, and thus the out-competition and dissociation of the reporters from VHL, leads to a restoration of their thermophoretic behavior in unbound state; a fit of the resulting sigmoidal dose–response curves yields Ki values of 535 or 604 nM for the short and 167 or 203 nM for the long unlabeled peptide (Figure 2). These experiments show that the assay can yield meaningful results using established fluorescent reporters. However, optimized reporters can improve the sensitivity dramatically, as we demonstrate in the following.
Figure 2
Figure 2. MST traces and dose–response curves for Kd determination of short (FAM-11mer) and long (FAM-19mer) reporter peptide to VHL, and competition experiments with the short (11mer) and long (19mer) peptides. IC50 and derived Ki values are shown in blue and black, respectively, together with their confidence intervals. All values are in nM. FAM, fluorescein amidite; Hyp, hydroxyproline.
For the adaptation for CRBN, we designed the custom reporter ligand BODIPY-uracil, which has a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) 493/503 fluorophore linked to uracil as the CRBN-binding moiety (Figure 3, see also Figure 1), inspired by the MANT-uracil reporter used in the FRET assay. (20) As the ligase construct, we tested both the human CRBN (hCRBN) thalidomide binding domain (hTBD), which can be expressed in E. coli with high yields, (24) as well as the bacterial CRBN homologue Magnetospirillum gryphiswaldense cereblon isoform 4 (MsCI4) that we previously employed in the FRET assay. (20) Initial titration experiments of these CRBN constructs against the reporter revealed several advantages of this customized setup. First, the initial fluorescence of the reporter was quenched by CRBN binding, which allowed for the determination of the dissociation constant prior to the MST measurement to Kd = 3.8 μM for MsCI4 and 11 μM for hTBD (Figure 3). However, more important are two major advantages concerning the actual MST experiment. The first one lies in the unusual thermophoretic behavior of the BODIPY-uracil::CRBN system, which changes sign between complexation states. While the free BODIPY-uracil shows positive thermophoresis, it is negative for both the BODIPY-uracil::MsCI4 and the BODIPY-uracil::hTBD complex: The MST traces reveal that the reporter is depleted from the heated spot in unbound state (decreasing fluorescence), but enriched when in complex with the ligase (increasing fluorescence). The second major advantage is a strong TRIC effect of the BODIPY fluorophore, which manifests in a fast initial response of the fluorophore at the onset of the measurement when the jump in temperature is applied, long before thermophoresis is approaching equilibrium. (22) The MST traces of the BODIPY-uracil::MsCI4 and BODIPY-uracil::hTBD titrations reveal that both the thermophoretic behavior and the TRIC effect change sign in the same direction between free and complexed state, and thus are cumulative, leading to an exceptional sensitivity (Figure 3).
Figure 3
Figure 3. MST traces and affinities of the BODIPY-uracil reporter and reference compounds to the bacterial MsCI4 and the thalidomide binding domain of human CRBN (hTBD). The affinity of BODIPY-uracil was determined via initial fluorescence measurements. The MST traces show that binding or unbinding of the reporter yields an inversion of the thermophoretic behavior. The affinities of DMSO, succinimide (without DMSO), and thalidomide (with 0.5% DMSO) were determined via out-competition of the reporter in MST measurements, for which the MST traces and derived dose–response curves are shown. IC50 and derived Ki values are shown in blue and black, respectively, together with their confidence intervals. All values are in μM.
In a next step, we determined the affinities of a set of reference compounds in competitive titrations, which includes thalidomide, succinimide, and DMSO. The latter is of special importance, as DMSO itself is competitive with CRBN binders. (6,20) In the MST assay with MsCI4, the competitive DMSO titration yielded a Ki of 11 mM, corresponding to a concentration of 0.08% (v/v) DMSO, which is in good agreement with the value obtained in the FRET assay (5.7 mM/0.06%). In contrast, the DMSO titration with the hTBD yielded a Ki of 137 mM, corresponding to a concentration of 1.0% (v/v) DMSO, which is virtually identical to the Ki obtained in the FRET assay for a similar hTBD construct, and significantly higher than that for MsCI4. (20) This led us to test the applicability of the assay in the absence and presence of DMSO. To this end, we performed and evaluated competitive titration experiments with the water-soluble binder succinimide without DMSO, and with thalidomide at a constant DMSO concentration of 0.5% (v/v). For succinimide, in the absence of DMSO, we obtained ideal MST traces for both constructs and Ki values of 0.64 and 8.2 μM for MsCI4 and hTBD, respectively (Figure 3). For thalidomide, however, the difference in DMSO tolerance between the two constructs was readily apparent from the traces. For MsCI4, the characteristic reversal of the thermophoretic behavior was not observed, because the 0.5% DMSO were well above the Ki value of the DMSO::MsCI4 interaction, such that most of the reporter was already out-competed by DMSO at the lowest thalidomide concentrations; a quantitative analysis was not attempted (Figure 3). In contrast, the experiment with hTBD yielded largely unperturbed traces and a Ki value of 8.5 μM; a detailed analysis and discussion of the impact of DMSO competition on the characterization of CRBN ligands can be found in the Supporting Information.
These results indicate that the hTBD-based MST assay is significantly more robust and HTS-amenable than the MsCI4-based FRET assay. Therefore, we tested the assay’s applicability for the HTS of compound libraries, which relies on single-concentration measurements instead of titration series. To this end, we determined the Z′ factor of the assay in the presence of 0.5% DMSO, once focused on the TRIC effect (on-time 5s) and once including the full MST behavior (on-time 20s) (Figure 4). The two evaluations resulted in Z′ factors of 0.85 and 0.83, suggesting that the evaluation of the TRIC effect with 5s per sample is sufficient for HTS, (25) which can be performed with the commercially available MST- or TRIC-based devices that use a 384-well SBS plate format.
Figure 4
Figure 4. Determination of Z′ factor for hTBD. MST traces of solvent (0.5% DMSO) and positive control thalidomide (top panel) and scatter plot and resulting Z′ values at on-time 5s and on-time 20s (bottom panel). Means of positive and negative controls are shown by solid lines; dashed lines indicate 3-fold standard deviations.
With the established hTBD-based assay, we sought to characterize the binding of a number of compounds, with a focus on those that were not measurable in the MsCI4-based FRET assay due to overlapping optical properties or solubility issues. This includes the third generation IMiD avadomide (Figure S1) as well as a number of only remotely related pharmaceuticals. We had previously identified lactams and cyclic imides as minimal CRBN-binding moieties and predicted several pharmaceuticals based on these moieties as CRBN binders, some of which could not be examined in the FRET assay due to solubility issues. This includes the antidepressant γ-lactam rolipram and the antiviral uracil dasabuvir, of which only the binding of rolipram could be validated so far via crystallography. (13) Further, we included the antibiotic hydantoin nitrofurantoin, which was previously predicted but not yet tested as a binder. All these mentioned pharmaceuticals and IMiDs are single-branched in the same position of their core binding moieties. We had previously defined this as position a of the binding moiety, and define everything attached to this a position the “protruding” moiety (13) (Figure 1). Consequently, this position marks the canonical exit vector from the binding pocket, which is an important determinant when assembling linkers and ligase ligands in PROTAC design. (18,26)
We first ran a series of experiments to determine the affinities of the established binders, the IMiDs lenalidomide, pomalidomide, avadomide, and iberdomide and also rolipram. For the IMiDs, the resulting affinities are in the expected range, with iberdomide having the highest affinity (Ki = 4.9 μM) (27) (Figure 5). Rolipram, for which the binding had not been quantified so far, bound with a relatively low affinity of Ki = 102 μM, substantiating the notion that cyclic imides pose higher-affinity CRBN-binding moieties than lactams. (13) While these measurements were of rather confirmatory nature, we were surprised by the results we obtained for nitrofurantoin and dasabuvir.
Figure 5
Figure 5. Chemical structures, dose–response curves and affinity values for (A) IMiDs and rolipram, (B) small hydantoins and hydantoins branched via hydrazo groups, and (C) uracils to hTBD. IC50 and derived Ki values are shown in blue and black, respectively, together with their confidence intervals. All values are in μM. n.b., no binding; 2-NP-AHD, 1-(2-nitrobenzylideneamino)hydantoin; NF-DB, 1-(3-(5-nitrofuran-2-yl)allylidene)amino)hydantoin.
Unexpectedly, nitrofurantoin did not show any binding in the MST assay, a result that we could also confirm in the MsCI4-based FRET assay (Figures 5 and S1). To understand the reasons for this lack of affinity, we tested a number of related N1-substituted hydantoins, i.e., hydantoins branched in position a. We started with the minimal binder hydantoin (Ki = 216 μM) itself, N1-methylhydantoin (68 μM) and N1-aminohydantoin (229 μM). The higher affinity of N1-methylhydantoin supports our previous finding that single-branching in position a leads to increased affinity, while the result for N1-aminohydantoin indicates that this does not hold true for the branching with an amino group, possibly due to the protonation of the latter. We continued with commercially available hydantoins with larger protruding moieties, which were all branched via a hydrazo group as in nitrofurantoin (Figure 5). Intriguingly, all showed the same lack of affinity to hTBD; for two of them, 1-benzylideneaminohydantoin and 1-(2-nitrobenzylideneamino)hydantoin, we also confirmed this absence of affinity in the FRET assay with MsCI4, ruling out that it is due to an artifact of one specific assay or protein construct. Consequently, the reasons for the lack of binding are presumably to be ascribed to the hydrazo group, which is coplanar with the hydantoin ring due to the resonance of the free electron pairs of the nitrogen atoms in the hydantoin moiety. With this particular geometry, binding is presumably precluded as it would potentially force the hydrazo carbon into a steric clash with one of the tryptophans of the binding pocket (Figure 1).
Furthermore, as a part of the hydantoin series, we tested the importance of the carbonyl in position b (see Figure 1) in the context of hTBD by comparing the affinity of hydantoin to that of 4-imidazolidinone, in which this group is absent, and 2-thiohydantoin, in which it is replaced with a thiocarbonyl. As reported for MsCI4, (13) the lack of the group led to a reduction, while the thiocarbonyl variant led to an increase of affinity, suggesting that branching in this position should be tested more systematically (Figure 5).
The surprising result for dasabuvir, a large uracil branched in position a, (28) was an unexpectedly high affinity (Ki = 4.7 μM) that is competitive with that of the third generation IMiD iberdomide and the highest affinity of all hTBD binders determined in this study (Figure 5). Consequently, we tested a range of related N1-substituted uracils, starting with parent uracil (212 μM). As expected, single branching with a methyl group in position a led to an improved affinity for N1-methyluracil (31 μM), while the larger substitution in uridine led to a drop (390 μM). The further addition of a phosphate group to that protruding moiety in uridine-5′-monophosphate (UMP) even abolished binding completely. As the 5′-OH group of uridine is outside the binding pocket and not recognized in a specific manner, (12) this suggests that a negative charge on the linking or protruding moiety as the phosphate group in UMP could generally be detrimental for binding. For confirmation, we also tested UMP binding in the MsCI4-based FRET assay, where uridine showed a high affinity, (13) and could indeed also not see any residual affinity for UMP (Figure S1). Finally, we tested sofosbuvir, another large uracil that is chemically distant to dasabuvir. Although binding to hTBD was observable, the affinity to the human domain is very weak (>1 mM). We had observed a significantly higher affinity of sofosbuvir for MsCI4 in the FRET assay (22 μM), (13) which we could also confirm in the MST assay using MsCI4 (27 μM). This is the first time that we observe a larger discrepancy in the affinity of compounds between MsCI4 and hTBD. As sofosbuvir carries a complex protruding moiety, for which secondary interactions more distant to the canonical binding pocket may not be properly reflected by the bacterial homologue, this points at limitations of MsCI4 as a surrogate system for hCRBN.
In conclusion, we have shown that the competitive MST-based assay poses a promising tool for the characterization of the ligand space of potentially any ligase for which a suitable reporter ligand can be synthesized. It is a complementary alternative to currently employed fluorescence-based assays that is largely unaffected by possibly overlapping optical properties of test ligands. As demonstrated for VHL, the assay can be performed with an existing reporter ligand, but the careful selection of the fluorophore can significantly improve sensitivity, as demonstrated for CRBN, which also makes it promising for HTS. Here, the BODIPY-uracil::hTBD system has proven to be very robust, especially as the association or dissociation of this particular reporter::ligase complex yields an inversion of the thermophoretic behavior; we could use it to evaluate a number of compounds that were not manageable in the MsCI4-based FRET assay, and for a systematic analysis of the influence of DMSO on CRBN binding assays. Thus, we have gained novel insight into the CRBN ligand space, revealing steric and charge restraints for the branching at the canonical branching position a of the core binding moiety, and suggesting to further explore branching at position b, which could yield novel exit vectors for the design of IMiDs and PROTACs. Further, with dasabuvir, we discovered a largely unrelated FDA-approved pharmaceutical that competes even with the affinity of the third generation IMiD iberdomide. In fact, dasabuvir is the highest-affinity hTBD binder we have tested so far. It seems likely that it benefits from additional interactions of its rather bulky protruding moiety with the immediate surroundings of the core binding pocket, which may encourage a more systematic screening for such interactions for the design of protruding or linking moieties that contribute to the affinity of IMiDs and CRBN-based PROTACs.
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00440.
Analysis and discussion of the impact of DMSO competition on the characterization of CRBN ligands; analysis and discussion of the influence of fluorescence quenching and autofluorescent test compounds; experimental procedures; FRET assay data for avadomide, nitrofurantoin and UMP; influence of DMSO; influence of initial fluorescence on MST behavior (PDF)
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Author Information
- Marcus D. Hartmann - Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany;
http://orcid.org/0000-0001-6937-5677;
S.M. and C.H. contributed equally.
This work was supported by institutional funds of the Max Planck Society.
Acknowledgments
We thank Andrei Lupas for continuing support, Iuliia Boichenko for initial contributions, Reinhard Albrecht for the cloning of hTBD, and Kerstin Bär and Maxim Drömer for excellent technical assistance.
| BODIPY | 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene |
| CRBN | cereblon |
| FAM | fluorescein amidite |
| FP | fluorescence polarization |
| FRET | Förster resonance energy transfer |
| IMiD | immunomodulatory drug |
| MANT | N-methyl-anthraniloyl |
| MsCI4 | Magnetospirillum gryphiswaldense cereblon isoform 4 |
| MST | microscale thermophoresis |
| POI | protein of interest |
| PROTAC | proteolysis-targeting chimera |
| TBD | thalidomide binding domain |
| TRIC | temperature-related intensity change |
| VHL | von Hippel–Lindau |
References
This article references 28 other publications.
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- 3Schapira, M.; Calabrese, M. F.; Bullock, A. N.; Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discovery 2019, 18 (12), 949– 963, DOI: 10.1038/s41573-019-0047-yGoogle Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVKrtLrM&md5=9bfd48c6c47fc1cce15129495fea7079Targeted protein degradation: expanding the toolboxSchapira, Matthieu; Calabrese, Matthew F.; Bullock, Alex N.; Crews, Craig M.Nature Reviews Drug Discovery (2019), 18 (12), 949-963CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. Proteolysis-targeting chimeras (PROTACs) and related mols. that induce targeted protein degrdn. by the ubiquitin-proteasome system represent a new therapeutic modality and are the focus of great interest, owing to potential advantages over traditional occupancy-based inhibitors with respect to dosing, side effects, drug resistance and modulating 'undruggable' targets. However, the technol. is still maturing, and the design elements for successful PROTAC-based drugs are currently being elucidated. Importantly, fewer than 10 of the more than 600 E3 ubiquitin ligases have so far been exploited for targeted protein degrdn., and expansion of knowledge in this area is a key opportunity. Here, we briefly discuss lessons learned about targeted protein degrdn. in chem. biol. and drug discovery and systematically review the expression profile, domain architecture and chem. tractability of human E3 ligases that could expand the toolbox for PROTAC discovery.
- 4Steinebach, C.; Kehm, H.; Lindner, S.; Vu, L. P.; Kopff, S.; Marmol, A. L.; Weiler, C.; Wagner, K. G.; Reichenzeller, M.; Kronke, J.; Gutschow, M. PROTAC-mediated crosstalk between E3 ligases. Chem. Commun. 2019, 55 (12), 1821– 1824, DOI: 10.1039/C8CC09541HGoogle Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFSmtLY%253D&md5=17ed8abc73d45081c73f1995205713cfPROTAC-mediated crosstalk between E3 ligasesSteinebach, Christian; Kehm, Hannes; Lindner, Stefanie; Vu, Lan Phuong; Koepff, Simon; Lopez Marmol, Alvaro; Weiler, Corinna; Wagner, Karl G.; Reichenzeller, Michaela; Kroenke, Jan; Guetschow, MichaelChemical Communications (Cambridge, United Kingdom) (2019), 55 (12), 1821-1824CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Small-mol. heterobifunctional degraders can effectively control protein levels and are useful research tools. We assembled proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN) and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a PROTAC-induced heterodimerization of the two E3 ligases leading to unidirectional and efficient degrdn. of CRBN.
- 5Girardini, M.; Maniaci, C.; Hughes, S. J.; Testa, A.; Ciulli, A. Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs. Bioorg. Med. Chem. 2019, 27 (12), 2466– 2479, DOI: 10.1016/j.bmc.2019.02.048Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktV2rsb8%253D&md5=ef00788b6428899cf4dfe1c8d357dd64Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACsGirardini, Miriam; Maniaci, Chiara; Hughes, Scott J.; Testa, Andrea; Ciulli, AlessioBioorganic & Medicinal Chemistry (2019), 27 (12), 2466-2479CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biol. important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degrdn. of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degrdn. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compd. 14a induced potent, rapid and profound preferential degrdn. of CRBN over VHL in cancer cell lines. At lower concns., weaker degrdn. of VHL was instead obsd. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.
- 6Hartmann, M. D.; Boichenko, I.; Coles, M.; Lupas, A. N.; Hernandez Alvarez, B. Structural dynamics of the cereblon ligand binding domain. PLoS One 2015, 10 (5), e0128342 DOI: 10.1371/journal.pone.0128342Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGru77F&md5=525f48f454c54c466360231d1ab3330aStructural dynamics of the cereblon ligand binding domainHartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Alvarez, Birte HernandezPLoS One (2015), 10 (5), e0128342/1-e0128342/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cereblon, a primary target of thalidomide and its derivs., has been characterized structurally from both bacteria and animals. Esp. well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the cryst. state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resoln., our data provide a view of thalidomide binding at at. resoln.
- 7Mayor-Ruiz, C.; Bauer, S.; Brand, M.; Kozicka, Z.; Siklos, M.; Imrichova, H.; Kaltheuner, I. H.; Hahn, E.; Seiler, K.; Koren, A.; Petzold, G.; Fellner, M.; Bock, C.; Muller, A. C.; Zuber, J.; Geyer, M.; Thoma, N. H.; Kubicek, S.; Winter, G. E. Rational discovery of molecular glue degraders via scalable chemical profiling. Nat. Chem. Biol. 2020, 16 (11), 1199– 1207, DOI: 10.1038/s41589-020-0594-xGoogle Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1OisLnE&md5=5291629b94fd659ae090316f96272b33Rational discovery of molecular glue degraders via scalable chemical profilingMayor-Ruiz, Cristina; Bauer, Sophie; Brand, Matthias; Kozicka, Zuzanna; Siklos, Marton; Imrichova, Hana; Kaltheuner, Ines H.; Hahn, Elisa; Seiler, Kristina; Koren, Anna; Petzold, Georg; Fellner, Michaela; Bock, Christoph; Mueller, Andre C.; Zuber, Johannes; Geyer, Matthias; Thomae, Nicolas H.; Kubicek, Stefan; Winter, Georg E.Nature Chemical Biology (2020), 16 (11), 1199-1207CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Targeted protein degrdn. is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are mol. glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, the authors describe a scalable strategy toward glue degrader discovery that is based on chem. screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led the authors to identify compds. that induce ubiquitination and degrdn. of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, the authors' data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.
- 8Ito, T.; Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 2016, 104 (3), 293– 299, DOI: 10.1007/s12185-016-2073-4Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s3kslSntA%253D%253D&md5=4d26b90fbd5bbaed2c2376fc8ce2ee88Cereblon and its downstream substrates as molecular targets of immunomodulatory drugsIto Takumi; Handa Hiroshi; Ito TakumiInternational journal of hematology (2016), 104 (3), 293-9 ISSN:.Thalidomide was first developed as a sedative around 60 years ago, but exhibited teratogenicity, leading to serious defects such as limb deformities. Nevertheless, thalidomide is now recognized as a therapeutic drug for the treatment of Hansen's disease and myeloma. Immunomodulatory drugs (IMiDs), a new class of anti-cancer drug derived from thalidomide, have also been developed and exert potent anti-cancer effects. Although the molecular mechanism of thalidomide and IMiDs remained unclear for a long time, cereblon, a substrate receptor of the CRL4 E3 ubiquitin ligase was identified as a primary direct target by a new affinity technique. A growing body of evidence suggests that the effect of IMiDs on myeloma and other cancer cells is mediated by CRBN. Each IMiD binds to CRBN and alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in breakdown of intrinsic downstream proteins such as Ikaros and Aiolos. Here we give an overview of the current understanding of mechanism of action of IMiDs via CRBN and prospects for the development of new drugs that degrade protein of interest.
- 9Kronke, J.; Udeshi, N. D.; Narla, A.; Grauman, P.; Hurst, S. N.; McConkey, M.; Svinkina, T.; Heckl, D.; Comer, E.; Li, X.; Ciarlo, C.; Hartman, E.; Munshi, N.; Schenone, M.; Schreiber, S. L.; Carr, S. A.; Ebert, B. L. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 2014, 343 (6168), 301– 5, DOI: 10.1126/science.1244851Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c3itFajsQ%253D%253D&md5=8cae3e554399308493aa38f0d05ed610Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cellsKronke Jan; Udeshi Namrata D; Narla Anupama; Grauman Peter; Hurst Slater N; McConkey Marie; Svinkina Tanya; Heckl Dirk; Comer Eamon; Li Xiaoyu; Ciarlo Christie; Hartman Emily; Munshi Nikhil; Schenone Monica; Schreiber Stuart L; Carr Steven A; Ebert Benjamin LScience (New York, N.Y.) (2014), 343 (6168), 301-5 ISSN:.Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.
- 10Petzold, G.; Fischer, E. S.; Thoma, N. H. Structural basis of lenalidomide-induced CK1alpha degradation by the CRL4(CRBN) ubiquitin ligase. Nature 2016, 532 (7597), 127– 30, DOI: 10.1038/nature16979Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtFOis78%253D&md5=320fc6ff811da335fa8e02feab2d702dStructural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligasePetzold, Georg; Fischer, Eric S.; Thoma, Nicolas H.Nature (London, United Kingdom) (2016), 532 (7597), 127-130CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Thalidomide and its derivs., lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of hematol. malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4CRBN) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4CRBN substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degrdn. Lenalidomide induces degrdn. of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clin. efficacy in the treatment of multiple myeloma and 5q-deletion assocd. myelodysplastic syndrome (del(5q) MDS), resp. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α β-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4CRBN is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compd. and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small mols. will provide opportunities for drug development, particularly for targeted protein degrdn.
- 11Kronke, J.; Fink, E. C.; Hollenbach, P. W.; MacBeth, K. J.; Hurst, S. N.; Udeshi, N. D.; Chamberlain, P. P.; Mani, D. R.; Man, H. W.; Gandhi, A. K.; Svinkina, T.; Schneider, R. K.; McConkey, M.; Jaras, M.; Griffiths, E.; Wetzler, M.; Bullinger, L.; Cathers, B. E.; Carr, S. A.; Chopra, R.; Ebert, B. L. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature 2015, 523 (7559), 183– 8, DOI: 10.1038/nature14610Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1entr%252FN&md5=9b20a91c13e888e6569223ffa25af2cdLenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDSKronke, Jan; Fink, Emma C.; Hollenbach, Paul W.; MacBeth, Kyle J.; Hurst, Slater N.; Udeshi, Namrata D.; Chamberlain, Philip P.; Mani, D. R.; Man, Hon Wah; Gandhi, Anita K.; Svinkina, Tanya; Schneider, Rebekka K.; McConkey, Marie; Jaras, Marcus; Griffiths, Elizabeth; Wetzler, Meir; Bullinger, Lars; Cathers, Brian E.; Carr, Steven A.; Chopra, Rajesh; Ebert, Benjamin L.Nature (London, United Kingdom) (2015), 523 (7559), 183-188CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN), resulting in CK1α degrdn. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haplo-insufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. The authors found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degrdn. of CK1α. The authors further demonstrate that minor side chain modifications in thalidomide and a novel analog, CC-122, can modulate the spectrum of substrates targeted by CRL4CRBN. These findings have implications for the clin. activity of lenalidomide and related compds., and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.
- 12Hartmann, M. D.; Boichenko, I.; Coles, M.; Zanini, F.; Lupas, A. N.; Hernandez Alvarez, B. Thalidomide mimics uridine binding to an aromatic cage in cereblon. J. Struct. Biol. 2014, 188 (3), 225– 32, DOI: 10.1016/j.jsb.2014.10.010Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSnu7jI&md5=e601c6e1091deea840f9738fb30fa7bbThalidomide mimics uridine binding to an aromatic cage in cereblonHartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Zanini, Fabio; Lupas, Andrei N.; Hernandez Alvarez, BirteJournal of Structural Biology (2014), 188 (3), 225-232CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)Thalidomide and its derivs. lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resoln. crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivs. mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an arom. cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.
- 13Boichenko, I.; Bar, K.; Deiss, S.; Heim, C.; Albrecht, R.; Lupas, A. N.; Hernandez Alvarez, B.; Hartmann, M. D. Chemical Ligand Space of Cereblon. ACS Omega 2018, 3 (9), 11163– 11171, DOI: 10.1021/acsomega.8b00959Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslSku7bO&md5=b0fee25453d5ec83098ac0cf28575d44Chemical Ligand Space of CereblonBoichenko, Iuliia; Baer, Kerstin; Deiss, Silvia; Heim, Christopher; Albrecht, Reinhard; Lupas, Andrei N.; Hernandez Alvarez, Birte; Hartmann, Marcus D.ACS Omega (2018), 3 (9), 11163-11171CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)The protein, cereblon, serves as a substrate receptor of a ubiquitin ligase complex that can be tuned toward different target proteins by cereblon-binding agents. This approach to targeted protein degrdn. is exploited in different clin. settings and has sparked the development of a growing no. of thalidomide derivs. Here, we probed the chem. space of cereblon binding beyond such derivs. and worked out a simple set of chem. requirements, delineating the metaclass of cereblon effectors. We report co-crystal structures of Magnetospirillum gryphiswaldense cereblon isoform 4 with a diverse set of compds., including commonly used pharmaceuticals, but we also found that already minimalistic cereblon-binding moieties might exert teratogenic effects in zebrafish. These results may guide the design of a post-thalidomide generation of therapeutic cereblon effectors, and provide a framework for the circumvention of unintended cereblon-binding by neg. design for future pharmaceuticals.
- 14Heim, C.; Pliatsika, D.; Mousavizadeh, F.; Bar, K.; Hernandez Alvarez, B.; Giannis, A.; Hartmann, M. D. De-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide Derivatives. J. Med. Chem. 2019, 62 (14), 6615– 6629, DOI: 10.1021/acs.jmedchem.9b00454Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1OqsbrK&md5=b09f48df1a76f1d2a954563f4842d3fcDe-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide DerivativesHeim, Christopher; Pliatsika, Dimanthi; Mousavizadeh, Farnoush; Baer, Kerstin; Hernandez Alvarez, Birte; Giannis, Athanassios; Hartmann, Marcus D.Journal of Medicinal Chemistry (2019), 62 (14), 6615-6629CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Targeted protein degrdn. via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clin. settings, in which the substrate specificity of CRBN is altered via the binding of small-mol. effectors. To date, such effectors are derived from thalidomide and confer a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular approach to design novel and minimalistic CRBN effectors. In this approach, we took advantage of the binding modes of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallog. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chem. moiety that imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to degrade the neo-substrate IKZF3 in the cell culture.
- 15Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R. J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (15), 8554– 8559, DOI: 10.1073/pnas.141230798Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXls1Wisbk%253D&md5=540358035222f745f7b6367b38781a21Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradationSakamoto, Kathleen M.; Kim, Kyung B.; Kumagai, Akiko; Mercurio, Frank; Crews, Craig M.; Deshaies, Raymond J.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (15), 8554-8559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex contg. Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degrdn. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compd., protein-targeting chimeric mol. 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IκBα phosphopeptide that is recognized by the F-box protein β-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCFβ-TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.
- 16Min, J. H.; Yang, H.; Ivan, M.; Gertler, F.; Kaelin, W. G., Jr.; Pavletich, N. P. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 2002, 296 (5574), 1886– 9, DOI: 10.1126/science.1073440Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XksVWmsr8%253D&md5=c3ccb5f0a383257ae465cc1c5f7a189fStructure of an HIF-1α-pVHL complex: hydroxyproline recognition in signalingMin, Jung-Hyun; Yang, Haifeng; Ivan, Mircea; Gertler, Frank; Kaelin, William G., Jr.; Pavletich, Nikola P.Science (Washington, DC, United States) (2002), 296 (5574), 1886-1889CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) plays a central role in the cellular response to changes in oxygen availability. PVHL binds to HIF only when a conserved proline in HIF is hydroxylated, a modification that is oxygen-dependent. The 1.85 Å structure of a 20-residue HIF-1α peptide-pVHL-Elongin B-Elongin C complex shows that HIF-1α binds to pVHL in an extended β strand-like conformation. The hydroxyproline inserts into a gap in the pVHL hydrophobic core, at a site that is a hotspot for tumorigenic mutations, with its 4-hydroxyl group recognized by buried serine and histidine residues. Although the β sheet-like interactions contribute to the stability of the complex, the hydroxyproline contacts are central to the strict specificity characteristic signaling.
- 17Fischer, E. S.; Bohm, K.; Lydeard, J. R.; Yang, H.; Stadler, M. B.; Cavadini, S.; Nagel, J.; Serluca, F.; Acker, V.; Lingaraju, G. M.; Tichkule, R. B.; Schebesta, M.; Forrester, W. C.; Schirle, M.; Hassiepen, U.; Ottl, J.; Hild, M.; Beckwith, R. E.; Harper, J. W.; Jenkins, J. L.; Thoma, N. H. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 2014, 512 (7512), 49– 53, DOI: 10.1038/nature13527Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht12ms7%252FP&md5=1c1eaa48bef87463cdd3f2b1e1bd400dStructure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomideFischer, Eric S.; Bohm, Kerstin; Lydeard, John R.; Yang, Haidi; Stadler, Michael B.; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M.; Tichkule, Ritesh B.; Schebesta, Michael; Forrester, William C.; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E. J.; Harper, J. Wade; Jenkins, Jeremy L.; Thoma, Nicolas H.Nature (London, United Kingdom) (2014), 512 (7512), 49-53CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivs. lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-assocd. dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN. Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degrdn. This dual activity implies that small mols. can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.
- 18Steinebach, C.; Sosic, I.; Lindner, S.; Bricelj, A.; Kohl, F.; Ng, Y. L. D.; Monschke, M.; Wagner, K. G.; Kronke, J.; Gutschow, M. A MedChem toolbox for cereblon-directed PROTACs. MedChemComm 2019, 10 (6), 1037– 1041, DOI: 10.1039/C9MD00185AGoogle Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVCrtr3L&md5=865c40183453c6473ef7f037aa566ecaA MedChem toolbox for cereblon-directed PROTACsSteinebach, Christian; Sosic, Izidor; Lindner, Stefanie; Bricelj, Alesa; Kohl, Franziska; Ng, Yuen Lam Dora; Monschke, Marius; Wagner, Karl G.; Kroenke, Jan; Guetschow, MichaelMedChemComm (2019), 10 (6), 1037-1041CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)A modular chem. toolbox was developed for cereblon-directed PROTACs. A variety of linkers was attached to a CRBN ligand via the 4-amino position of pomalidomide. We used linkers of different constitution to modulate physicochem. properties. We equipped one terminus of the linker with a set of functional groups, e.g. protected amines, protected carboxylic acids, alkynes, chloroalkanes, and protected alcs., all of which are considered to be attractive for PROTAC design. We also highlight different opportunities for the expansion of the medicinal chemists' PROTAC toolbox towards heterobifunctional mols., e.g. with biotin, fluorescent, hydrophobic and peptide tags.
- 19Buckley, D. L.; Van Molle, I.; Gareiss, P. C.; Tae, H. S.; Michel, J.; Noblin, D. J.; Jorgensen, W. L.; Ciulli, A.; Crews, C. M. Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1alpha interaction. J. Am. Chem. Soc. 2012, 134 (10), 4465– 8, DOI: 10.1021/ja209924vGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivVOgurw%253D&md5=60f7384b6bc932c9828dc12999283a1eTargeting the von Hippel-Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1α InteractionBuckley, Dennis L.; Van Molle, Inge; Gareiss, Peter C.; Tae, Hyun Seop; Michel, Julien; Noblin, Devin J.; Jorgensen, William L.; Ciulli, Alessio; Crews, Craig M.Journal of the American Chemical Society (2012), 134 (10), 4465-4468CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degrdn., are attractive drug targets due to their exquisite substrate specificity. However, the development of small-mol. inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein-protein interactions. Using rational design, we have generated the first small mol. targeting the von Hippel-Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compd. mimics the binding mode of the transcription factor HIF-1α, a substrate of VHL. These results have the potential to guide future development of improved lead compds. as therapeutics for the treatment of chronic anemia and ischemia.
- 20Boichenko, I.; Deiss, S.; Bar, K.; Hartmann, M. D.; Hernandez Alvarez, B. A FRET-Based Assay for the Identification and Characterization of Cereblon Ligands. J. Med. Chem. 2016, 59 (2), 770– 4, DOI: 10.1021/acs.jmedchem.5b01735Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1yktg%253D%253D&md5=f0400506428f439e81f579204e0bd39dA FRET-Based Assay for the Identification and Characterization of Cereblon LigandsBoichenko, Iuliia; Deiss, Silvia; Baer, Kerstin; Hartmann, Marcus D.; Hernandez Alvarez, BirteJournal of Medicinal Chemistry (2016), 59 (2), 770-774CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Cereblon serves as an ubiquitin ligase substrate receptor that can be tuned toward different target proteins by various cereblon-binding agents. This offers one of the most promising avenues for targeted protein degrdn. in cancer therapy, but cereblon binding can also mediate teratogenic effects. We present an effective assay that is suited for high-throughput screening of compd. libraries for off-target cereblon interactions but also can guide lead optimization and rational design of novel cereblon effector mols.
- 21Duhr, S.; Braun, D. Why molecules move along a temperature gradient. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (52), 19678– 82, DOI: 10.1073/pnas.0603873103Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjtVSltQ%253D%253D&md5=91255864cb4828be93545c71d96f30fdWhy molecules move along a temperature gradientDuhr, Stefan; Braun, DieterProceedings of the National Academy of Sciences of the United States of America (2006), 103 (52), 19678-19682CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Mols. drift along temp. gradients, an effect called thermophoresis, the Soret effect, or thermodiffusion. In liqs., its theor. foundation is the subject of a long-standing debate. By using an all-optical microfluidic fluorescence method, we present exptl. results for DNA and polystyrene beads over a large range of particle sizes, salt concns., and temps. The data support a unifying theory based on solvation entropy. Stated in simple terms, the Soret coeff. is given by the neg. solvation entropy, divided by kT. The theory predicts the thermodiffusion of polystyrene beads and DNA without any free parameters. We assume a local thermodn. equil. of the solvent mols. around the mol. This assumption is fulfilled for moderate temp. gradients below a fluctuation criterion. For both DNA and polystyrene beads, thermophoretic motion changes sign at lower temps. This thermophilicity toward lower temps. is attributed to an increasing pos. entropy of hydration, whereas the generally dominating thermophobicity is explained by the neg. entropy of ionic shielding. The understanding of thermodiffusion sets the stage for detailed probing of solvation properties of colloids and biomols. For example, we successfully det. the effective charge of DNA and beads over a size range that is not accessible with electrophoresis.
- 22Gupta, A. J.; Duhr, S.; Baaske, P. Microscale Thermophoresis (MST). In Encyclopedia of Biophysics; Roberts, G., Watts, A., Eds.; Springer: Berlin, Heidelberg, 2018; pp 1– 5.Google ScholarThere is no corresponding record for this reference.
- 23Seidel, S. A.; Dijkman, P. M.; Lea, W. A.; van den Bogaart, G.; Jerabek-Willemsen, M.; Lazic, A.; Joseph, J. S.; Srinivasan, P.; Baaske, P.; Simeonov, A.; Katritch, I.; Melo, F. A.; Ladbury, J. E.; Schreiber, G.; Watts, A.; Braun, D.; Duhr, S. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 2013, 59 (3), 301– 15, DOI: 10.1016/j.ymeth.2012.12.005Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFClsbs%253D&md5=4122dd561a2bd3187b0d9100ed57d4c0Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditionsSeidel, Susanne A. I.; Dijkman, Patricia M.; Lea, Wendy A.; van den Bogaart, Geert; Jerabek-Willemsen, Moran; Lazic, Ana; Joseph, Jeremiah S.; Srinivasan, Prakash; Baaske, Philipp; Simeonov, Anton; Katritch, Ilia; Melo, Fernando A.; Ladbury, John E.; Schreiber, Gideon; Watts, Anthony; Braun, Dieter; Duhr, StefanMethods (Amsterdam, Netherlands) (2013), 59 (3), 301-315CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Microscale thermophoresis (MST) allows for quant. anal. of protein interactions in free soln. and with low sample consumption. The technique is based on thermophoresis, the directed motion of mols. in temp. gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of mol. properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an IR laser is used for local heating, and mol. mobility in the temp. gradient is analyzed via fluorescence. In std. MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence.Despite the high mol. wt. ratio, the interaction of small mols. and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomols., such as membrane proteins to be stabilized in soln. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purifn. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for anal. of biomol. interactions in complex exptl. settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.
- 24Akuffo, A. A.; Alontaga, A. Y.; Metcalf, R.; Beatty, M. S.; Becker, A.; McDaniel, J. M.; Hesterberg, R. S.; Goodheart, W. E.; Gunawan, S.; Ayaz, M.; Yang, Y.; Karim, M. R.; Orobello, M. E.; Daniel, K.; Guida, W.; Yoder, J. A.; Rajadhyaksha, A. M.; Schonbrunn, E.; Lawrence, H. R.; Lawrence, N. J.; Epling-Burnette, P. K. Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon. J. Biol. Chem. 2018, 293 (16), 6187– 6200, DOI: 10.1074/jbc.M117.816868Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFSqtb8%253D&md5=16bc835472884d8b173e96fccdc3019aLigand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblonAkuffo, Afua A.; Alontaga, Aileen Y.; Metcalf, Rainer; Beatty, Matthew S.; Becker, Andreas; McDaniel, Jessica M.; Hesterberg, Rebecca S.; Goodheart, William E.; Gunawan, Steven; Ayaz, Muhammad; Yang, Yan; Karim, Md. Rezaul; Orobello, Morgan E.; Daniel, Kenyon; Guida, Wayne; Yoder, Jeffrey A.; Rajadhyaksha, Anjali M.; Schonbrunn, Ernst; Lawrence, Harshani R.; Lawrence, Nicholas J.; Epling-Burnette, Pearlie K.Journal of Biological Chemistry (2018), 293 (16), 6187-6200CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degrdn. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theor., biophys., and biol. assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chem. compd. and targeting ligand designed to degrade bromodomain-contg. 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting mols. in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.
- 25Hughes, J. P.; Rees, S.; Kalindjian, S. B.; Philpott, K. L. Principles of early drug discovery. Br. J. Pharmacol. 2011, 162 (6), 1239– 49, DOI: 10.1111/j.1476-5381.2010.01127.xGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXivF2lt7o%253D&md5=05a0016f45b155f9fb5822f5c7bcc35dPrinciples of early drug discoveryHughes, J. P.; Rees, S.; Kalindjian, S. B.; Philpott, K. L.British Journal of Pharmacology (2011), 162 (6), 1239-1249CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)A review. Developing a new drug from original idea to the launch of a finished product is a complex process which can take 12-15 years and cost in excess of $1 billion. The idea for a target can come from a variety of sources including academic and clin. research and from the com. sector. It may take many years to build up a body of supporting evidence before selecting a target for a costly drug discovery program. Once a target was chosen, the pharmaceutical industry and more recently some academic centers have streamlined a no. of early processes to identify mols. which possess suitable characteristics to make acceptable drugs. This review will look at key preclin. stages of the drug discovery process, from initial target identification and validation, through assay development, high throughput screening, hit identification, lead optimization and finally the selection of a candidate mol. for clin. development.
- 26Steinebach, C.; Ng, Y. L. D.; Sosic, I.; Lee, C. S.; Chen, S. R.; Lindner, S.; Vu, L. P.; Bricelj, A.; Haschemi, R.; Monschke, M.; Steinwarz, E.; Wagner, K. G.; Bendas, G.; Luo, J.; Gutschow, M.; Kronke, J. Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders. Chem. Sci. 2020, 11 (13), 3474– 3486, DOI: 10.1039/D0SC00167HGoogle Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkt1eqt7k%253D&md5=6f1f042714f46883e8ab7a0b4ae3ae96Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degradersSteinebach, Christian; Ng, Yuen Lam Dora; Sosic, Izidor; Lee, Chih-Shia; Chen, Sirui; Lindner, Stefanie; Vu, Lan Phuong; Bricelj, Alesa; Haschemi, Reza; Monschke, Marius; Steinwarz, Elisabeth; Wagner, Karl G.; Bendas, Gerd; Luo, Ji; Guetschow, Michael; Kroenke, JanChemical Science (2020), 11 (13), 3474-3486CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Cyclin-dependent kinase 6 (CDK6) is an important regulator of the cell cycle. Together with CDK4, it phosphorylates and inactivates retinoblastoma (Rb) protein. In tumor cells, CDK6 is frequently upregulated and CDK4/6 kinase inhibitors like palbociclib possess high activity in breast cancer and other malignancies. Besides its crucial catalytic function, kinase-independent roles of CDK6 have been described. Therefore, targeted degrdn. of CDK6 may be advantageous over kinase inhibition. Proteolysis targeting chimeras (PROTACs) structurally based on the cereblon (CRBN) ligand thalidomide have recently been described to degrade the targets CDK4/6. However, CRBN-based PROTACs have several limitations including the remaining activity of immunomodulatory drugs (IMiDs) on Ikaros transcription factors as well as CRBN inactivation as a resistance mechanism in cancer. Here, we systematically explored the chem. space of CDK4/6 PROTACs by addressing different E3 ligases and connecting their resp. small-mol. binders via various linkers to palbociclib. The spectrum of CDK6-specific PROTACs was extended to von Hippel Lindau (VHL) and cellular inhibitor of apoptosis protein 1 (cIAP1) that are essential for most cancer cells and therefore less likely to be inactivated. Our VHL-based PROTAC series included compds. that were either specific for CDK6 or exhibited dual activity against CDK4 and CDK6. IAP-based PROTACs caused a combined degrdn. of CDK4/6 and IAPs resulting in synergistic effects on cancer cell growth. Our new degraders showed potent and long-lasting degrading activity in human and mouse cells and inhibited proliferation of several leukemia, myeloma and breast cancer cell lines. In conclusion, we show that VHL- and IAP-based PROTACs are an attractive approach for targeted degrdn. of CDK4/6 in cancer.
- 27Matyskiela, M. E.; Zhang, W.; Man, H. W.; Muller, G.; Khambatta, G.; Baculi, F.; Hickman, M.; LeBrun, L.; Pagarigan, B.; Carmel, G.; Lu, C. C.; Lu, G.; Riley, M.; Satoh, Y.; Schafer, P.; Daniel, T. O.; Carmichael, J.; Cathers, B. E.; Chamberlain, P. P. A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos. J. Med. Chem. 2018, 61 (2), 535– 542, DOI: 10.1021/acs.jmedchem.6b01921Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1KqsLs%253D&md5=8358ae8563f8f2c09dbf68683e5e4008A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and AiolosMatyskiela, Mary E.; Zhang, Weihong; Man, Hon-Wah; Muller, George; Khambatta, Godrej; Baculi, Frans; Hickman, Matthew; LeBrun, Laurie; Pagarigan, Barbra; Carmel, Gilles; Lu, Chin-Chun; Lu, Gang; Riley, Mariko; Satoh, Yoshitaka; Schafer, Peter; Daniel, Thomas O.; Carmichael, James; Cathers, Brian E.; Chamberlain, Philip P.Journal of Medicinal Chemistry (2018), 61 (2), 535-542CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degrdn. Here we describe CC-220 (compd. 6), a cereblon modulator in clin. development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compd. 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degrdn. of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compd. 6 reveals that the increase in potency correlates with increased contacts between compd. 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degrdn. via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degrdn.
- 28Gentile, I.; Buonomo, A. R.; Borgia, G. Dasabuvir: A Non-Nucleoside Inhibitor of NS5B for the Treatment of Hepatitis C Virus Infection. Rev. Recent Clin. Trials 2014, 9 (2), 115– 23, DOI: 10.2174/1574887109666140529222602Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslGqu7vJ&md5=75f136bd5bccdf288697d8aba73ba037Dasabuvir: A Non-Nucleoside Inhibitor of NS5B for the Treatment of Hepatitis C Virus InfectionGentile, Ivan; Buonomo, Antonio R.; Borgia, GuglielmoReviews on Recent Clinical Trials (2014), 9 (2), 115-123CODEN: RRCTB2; ISSN:1574-8871. (Bentham Science Publishers Ltd.)Hepatitis C virus (HCV) chronically infects about 2% of the world's population. Approx. a quarter of these patients will develop, during their life, liver cirrhosis, which entails a high risk of complications and death. Successful antiviral therapy can reduce the risk of disease progression, but it is feasible only in a minority of patients because it includes interferon which is contraindicated in the most advanced stages of the disease and in patients with severe impairment of other organs. Consequent to the launch of the first direct antiviral agents (DAA), namely the protease inhibitors telaprevir and boceprevir, several mols. are in an advanced phase of clin. development to be used in assocn. with interferon or with other DAA (in interferon-free combinations). This review focuses on the mechanism of action, pharmacokinetics, efficacy, safety and resistance of dasabuvir, a non-nucleoside inhibitor of NS5B viral RNA-dependent RNA polymerase. Thanks to its pharmacokinetics, dasabuvir can be administered twice daily. In combinations with other oral DAAs, dasabuvir results in very high rates of SVR (about 95%) in patients with HCV genotype 1 infection with a good tolerability and safety. In conclusion, dasabuvir is a good agent to be used in interferon-free combinations for the treatment of chronic hepatitis C.
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, 747-749. https://doi.org/10.1016/j.mencom.2022.11.013
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- Christopher Heim, Marcus D. Hartmann. High-resolution structures of the bound effectors avadomide (CC-122) and iberdomide (CC-220) highlight advantages and limitations of the MsCI4 soaking system. Acta Crystallographica Section D Structural Biology 2022, 78
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- Mikhail Krasavin, Maria Adamchik, Andrey Bubyrev, Christopher Heim, Samuel Maiwald, Daniil Zhukovsky, Petr Zhmurov, Alexander Bunev, Marcus D. Hartmann. Synthesis of Novel Glutarimide Ligands for the E3 Ligase Substrate Receptor Cereblon (CRBN): Investigation of Their Binding Mode and Antiproliferative Effects Against Myeloma Cell Lines. SSRN Electronic Journal 2022, 1 https://doi.org/10.2139/ssrn.4191806
- Christopher Heim, Samuel Maiwald, Christian Steinebach, Matthew K. Collins, Jonathan Strope, Cindy H. Chau, William D. Figg, Michael Gütschow, Marcus D. Hartmann. On the correlation of cereblon binding, fluorination and antiangiogenic properties of immunomodulatory drugs. Biochemical and Biophysical Research Communications 2021, 534 , 67-72. https://doi.org/10.1016/j.bbrc.2020.11.117
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Abstract
Figure 1
Figure 1. Overall structure and ligand binding modes for E3 ligase substrate receptors VHL and CRBN. (A) Structure of VHL-Elongin B-Elongin C (VBC) complex with bound HIF1-α peptide (PDB: 1LM8). (B) Illustrative surface representation of the VHL binding groove with bound 11mer reporter, based on the same structure, highlighting the position of the fluorophore and hydroxyproline (Hyp). (C) Structure of the bacterial CRBN homologue MsCI4 bound to thalidomide (PDB: 4V2Y). (D) Illustrative surface representation of MsCI4 bound to BODIPY-uracil reporter. (E) Proposed clash of nitrofurantoin in the binding pocket of CRBN. Nitrofurantoin (green) was aligned with a structure of hydantoin in complex with MsCI4 (PDB: 5OH7) and shown in comparison to thalidomide (red). The protruding moiety of nitrofurantoin comes in close proximity to W99 of MsCI4. (F) Depiction of the pharmacophore-based nomenclature for 5- and 6-membered rings. R refers to the protruding moiety with the canonical exit vector.
Figure 2
Figure 2. MST traces and dose–response curves for Kd determination of short (FAM-11mer) and long (FAM-19mer) reporter peptide to VHL, and competition experiments with the short (11mer) and long (19mer) peptides. IC50 and derived Ki values are shown in blue and black, respectively, together with their confidence intervals. All values are in nM. FAM, fluorescein amidite; Hyp, hydroxyproline.
Figure 3
Figure 3. MST traces and affinities of the BODIPY-uracil reporter and reference compounds to the bacterial MsCI4 and the thalidomide binding domain of human CRBN (hTBD). The affinity of BODIPY-uracil was determined via initial fluorescence measurements. The MST traces show that binding or unbinding of the reporter yields an inversion of the thermophoretic behavior. The affinities of DMSO, succinimide (without DMSO), and thalidomide (with 0.5% DMSO) were determined via out-competition of the reporter in MST measurements, for which the MST traces and derived dose–response curves are shown. IC50 and derived Ki values are shown in blue and black, respectively, together with their confidence intervals. All values are in μM.
Figure 4
Figure 4. Determination of Z′ factor for hTBD. MST traces of solvent (0.5% DMSO) and positive control thalidomide (top panel) and scatter plot and resulting Z′ values at on-time 5s and on-time 20s (bottom panel). Means of positive and negative controls are shown by solid lines; dashed lines indicate 3-fold standard deviations.
Figure 5
Figure 5. Chemical structures, dose–response curves and affinity values for (A) IMiDs and rolipram, (B) small hydantoins and hydantoins branched via hydrazo groups, and (C) uracils to hTBD. IC50 and derived Ki values are shown in blue and black, respectively, together with their confidence intervals. All values are in μM. n.b., no binding; 2-NP-AHD, 1-(2-nitrobenzylideneamino)hydantoin; NF-DB, 1-(3-(5-nitrofuran-2-yl)allylidene)amino)hydantoin.
References
This article references 28 other publications.
- 1Lai, A. C.; Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discovery 2017, 16 (2), 101– 114, DOI: 10.1038/nrd.2016.2111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFCit7rP&md5=a01ff43e9b696fe4dc03f0815158a98fInduced protein degradation: an emerging drug discovery paradigmLai, Ashton C.; Crews, Craig M.Nature Reviews Drug Discovery (2017), 16 (2), 101-114CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)Small-mol. drug discovery has traditionally focused on occupancy of a binding site that directly affects protein function, and this approach typically precludes targeting proteins that lack such amenable sites. Furthermore, high systemic drug exposures may be needed to maintain sufficient target inhibition in vivo, increasing the risk of undesirable off-target effects. Induced protein degrdn. is an alternative approach that is event-driven: upon drug binding, the target protein is tagged for elimination. Emerging technologies based on proteolysis-targeting chimaeras (PROTACs) that exploit cellular quality control machinery to selectively degrade target proteins are attracting considerable attention in the pharmaceutical industry owing to the advantages they could offer over traditional small-mol. strategies. These advantages include the potential to reduce systemic drug exposure, the ability to counteract increased target protein expression that often accompanies inhibition of protein function and the potential ability to target proteins that are not currently therapeutically tractable, such as transcription factors, scaffolding and regulatory proteins.
- 2Chamberlain, P. P.; Hamann, L. G. Development of targeted protein degradation therapeutics. Nat. Chem. Biol. 2019, 15 (10), 937– 944, DOI: 10.1038/s41589-019-0362-y2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslygur%252FF&md5=468e3fd87ba2b21607dac8b495e5ecf6Development of targeted protein degradation therapeuticsChamberlain, Philip P.; Hamann, Lawrence G.Nature Chemical Biology (2019), 15 (10), 937-944CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Targeted protein degrdn. as a therapeutic modality has seen dramatic progress and massive investment in recent years because of the convergence of two key scientific breakthroughs: optimization of first-generation peptidic proteolysis-targeted chimeras (PROTACs) into more drug-like mols. able to support in vivo proof of concept and the discovery that clin. mols. function as degraders by binding and repurposing the proteins cereblon and DCAF15. This provided clin. validation for the general approach through the cereblon modulator class of drugs and provided highly drug-like and ligand-efficient E3 ligase binders upon which to tether target-binding moieties. Increasingly rational and systematic approaches including biophys. and structural studies on ternary complexes are being leveraged as the field advances. In this Perspective we summarize the discoveries that have laid the foundation for future degrdn. therapeutics, focusing on those classes of small mols. that redirect E3 ubiquitin ligases to non-native substrates.
- 3Schapira, M.; Calabrese, M. F.; Bullock, A. N.; Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discovery 2019, 18 (12), 949– 963, DOI: 10.1038/s41573-019-0047-y3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitVKrtLrM&md5=9bfd48c6c47fc1cce15129495fea7079Targeted protein degradation: expanding the toolboxSchapira, Matthieu; Calabrese, Matthew F.; Bullock, Alex N.; Crews, Craig M.Nature Reviews Drug Discovery (2019), 18 (12), 949-963CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. Proteolysis-targeting chimeras (PROTACs) and related mols. that induce targeted protein degrdn. by the ubiquitin-proteasome system represent a new therapeutic modality and are the focus of great interest, owing to potential advantages over traditional occupancy-based inhibitors with respect to dosing, side effects, drug resistance and modulating 'undruggable' targets. However, the technol. is still maturing, and the design elements for successful PROTAC-based drugs are currently being elucidated. Importantly, fewer than 10 of the more than 600 E3 ubiquitin ligases have so far been exploited for targeted protein degrdn., and expansion of knowledge in this area is a key opportunity. Here, we briefly discuss lessons learned about targeted protein degrdn. in chem. biol. and drug discovery and systematically review the expression profile, domain architecture and chem. tractability of human E3 ligases that could expand the toolbox for PROTAC discovery.
- 4Steinebach, C.; Kehm, H.; Lindner, S.; Vu, L. P.; Kopff, S.; Marmol, A. L.; Weiler, C.; Wagner, K. G.; Reichenzeller, M.; Kronke, J.; Gutschow, M. PROTAC-mediated crosstalk between E3 ligases. Chem. Commun. 2019, 55 (12), 1821– 1824, DOI: 10.1039/C8CC09541H4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFSmtLY%253D&md5=17ed8abc73d45081c73f1995205713cfPROTAC-mediated crosstalk between E3 ligasesSteinebach, Christian; Kehm, Hannes; Lindner, Stefanie; Vu, Lan Phuong; Koepff, Simon; Lopez Marmol, Alvaro; Weiler, Corinna; Wagner, Karl G.; Reichenzeller, Michaela; Kroenke, Jan; Guetschow, MichaelChemical Communications (Cambridge, United Kingdom) (2019), 55 (12), 1821-1824CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Small-mol. heterobifunctional degraders can effectively control protein levels and are useful research tools. We assembled proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN) and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a PROTAC-induced heterodimerization of the two E3 ligases leading to unidirectional and efficient degrdn. of CRBN.
- 5Girardini, M.; Maniaci, C.; Hughes, S. J.; Testa, A.; Ciulli, A. Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs. Bioorg. Med. Chem. 2019, 27 (12), 2466– 2479, DOI: 10.1016/j.bmc.2019.02.0485https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktV2rsb8%253D&md5=ef00788b6428899cf4dfe1c8d357dd64Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACsGirardini, Miriam; Maniaci, Chiara; Hughes, Scott J.; Testa, Andrea; Ciulli, AlessioBioorganic & Medicinal Chemistry (2019), 27 (12), 2466-2479CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biol. important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degrdn. of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degrdn. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compd. 14a induced potent, rapid and profound preferential degrdn. of CRBN over VHL in cancer cell lines. At lower concns., weaker degrdn. of VHL was instead obsd. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.
- 6Hartmann, M. D.; Boichenko, I.; Coles, M.; Lupas, A. N.; Hernandez Alvarez, B. Structural dynamics of the cereblon ligand binding domain. PLoS One 2015, 10 (5), e0128342 DOI: 10.1371/journal.pone.01283426https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGru77F&md5=525f48f454c54c466360231d1ab3330aStructural dynamics of the cereblon ligand binding domainHartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Alvarez, Birte HernandezPLoS One (2015), 10 (5), e0128342/1-e0128342/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cereblon, a primary target of thalidomide and its derivs., has been characterized structurally from both bacteria and animals. Esp. well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the cryst. state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resoln., our data provide a view of thalidomide binding at at. resoln.
- 7Mayor-Ruiz, C.; Bauer, S.; Brand, M.; Kozicka, Z.; Siklos, M.; Imrichova, H.; Kaltheuner, I. H.; Hahn, E.; Seiler, K.; Koren, A.; Petzold, G.; Fellner, M.; Bock, C.; Muller, A. C.; Zuber, J.; Geyer, M.; Thoma, N. H.; Kubicek, S.; Winter, G. E. Rational discovery of molecular glue degraders via scalable chemical profiling. Nat. Chem. Biol. 2020, 16 (11), 1199– 1207, DOI: 10.1038/s41589-020-0594-x7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1OisLnE&md5=5291629b94fd659ae090316f96272b33Rational discovery of molecular glue degraders via scalable chemical profilingMayor-Ruiz, Cristina; Bauer, Sophie; Brand, Matthias; Kozicka, Zuzanna; Siklos, Marton; Imrichova, Hana; Kaltheuner, Ines H.; Hahn, Elisa; Seiler, Kristina; Koren, Anna; Petzold, Georg; Fellner, Michaela; Bock, Christoph; Mueller, Andre C.; Zuber, Johannes; Geyer, Matthias; Thomae, Nicolas H.; Kubicek, Stefan; Winter, Georg E.Nature Chemical Biology (2020), 16 (11), 1199-1207CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Targeted protein degrdn. is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are mol. glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, the authors describe a scalable strategy toward glue degrader discovery that is based on chem. screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led the authors to identify compds. that induce ubiquitination and degrdn. of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, the authors' data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.
- 8Ito, T.; Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 2016, 104 (3), 293– 299, DOI: 10.1007/s12185-016-2073-48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s3kslSntA%253D%253D&md5=4d26b90fbd5bbaed2c2376fc8ce2ee88Cereblon and its downstream substrates as molecular targets of immunomodulatory drugsIto Takumi; Handa Hiroshi; Ito TakumiInternational journal of hematology (2016), 104 (3), 293-9 ISSN:.Thalidomide was first developed as a sedative around 60 years ago, but exhibited teratogenicity, leading to serious defects such as limb deformities. Nevertheless, thalidomide is now recognized as a therapeutic drug for the treatment of Hansen's disease and myeloma. Immunomodulatory drugs (IMiDs), a new class of anti-cancer drug derived from thalidomide, have also been developed and exert potent anti-cancer effects. Although the molecular mechanism of thalidomide and IMiDs remained unclear for a long time, cereblon, a substrate receptor of the CRL4 E3 ubiquitin ligase was identified as a primary direct target by a new affinity technique. A growing body of evidence suggests that the effect of IMiDs on myeloma and other cancer cells is mediated by CRBN. Each IMiD binds to CRBN and alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in breakdown of intrinsic downstream proteins such as Ikaros and Aiolos. Here we give an overview of the current understanding of mechanism of action of IMiDs via CRBN and prospects for the development of new drugs that degrade protein of interest.
- 9Kronke, J.; Udeshi, N. D.; Narla, A.; Grauman, P.; Hurst, S. N.; McConkey, M.; Svinkina, T.; Heckl, D.; Comer, E.; Li, X.; Ciarlo, C.; Hartman, E.; Munshi, N.; Schenone, M.; Schreiber, S. L.; Carr, S. A.; Ebert, B. L. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 2014, 343 (6168), 301– 5, DOI: 10.1126/science.12448519https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c3itFajsQ%253D%253D&md5=8cae3e554399308493aa38f0d05ed610Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cellsKronke Jan; Udeshi Namrata D; Narla Anupama; Grauman Peter; Hurst Slater N; McConkey Marie; Svinkina Tanya; Heckl Dirk; Comer Eamon; Li Xiaoyu; Ciarlo Christie; Hartman Emily; Munshi Nikhil; Schenone Monica; Schreiber Stuart L; Carr Steven A; Ebert Benjamin LScience (New York, N.Y.) (2014), 343 (6168), 301-5 ISSN:.Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.
- 10Petzold, G.; Fischer, E. S.; Thoma, N. H. Structural basis of lenalidomide-induced CK1alpha degradation by the CRL4(CRBN) ubiquitin ligase. Nature 2016, 532 (7597), 127– 30, DOI: 10.1038/nature1697910https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtFOis78%253D&md5=320fc6ff811da335fa8e02feab2d702dStructural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligasePetzold, Georg; Fischer, Eric S.; Thoma, Nicolas H.Nature (London, United Kingdom) (2016), 532 (7597), 127-130CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Thalidomide and its derivs., lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of hematol. malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4CRBN) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4CRBN substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degrdn. Lenalidomide induces degrdn. of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clin. efficacy in the treatment of multiple myeloma and 5q-deletion assocd. myelodysplastic syndrome (del(5q) MDS), resp. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α β-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4CRBN is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compd. and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small mols. will provide opportunities for drug development, particularly for targeted protein degrdn.
- 11Kronke, J.; Fink, E. C.; Hollenbach, P. W.; MacBeth, K. J.; Hurst, S. N.; Udeshi, N. D.; Chamberlain, P. P.; Mani, D. R.; Man, H. W.; Gandhi, A. K.; Svinkina, T.; Schneider, R. K.; McConkey, M.; Jaras, M.; Griffiths, E.; Wetzler, M.; Bullinger, L.; Cathers, B. E.; Carr, S. A.; Chopra, R.; Ebert, B. L. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature 2015, 523 (7559), 183– 8, DOI: 10.1038/nature1461011https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1entr%252FN&md5=9b20a91c13e888e6569223ffa25af2cdLenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDSKronke, Jan; Fink, Emma C.; Hollenbach, Paul W.; MacBeth, Kyle J.; Hurst, Slater N.; Udeshi, Namrata D.; Chamberlain, Philip P.; Mani, D. R.; Man, Hon Wah; Gandhi, Anita K.; Svinkina, Tanya; Schneider, Rebekka K.; McConkey, Marie; Jaras, Marcus; Griffiths, Elizabeth; Wetzler, Meir; Bullinger, Lars; Cathers, Brian E.; Carr, Steven A.; Chopra, Rajesh; Ebert, Benjamin L.Nature (London, United Kingdom) (2015), 523 (7559), 183-188CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN), resulting in CK1α degrdn. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haplo-insufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. The authors found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degrdn. of CK1α. The authors further demonstrate that minor side chain modifications in thalidomide and a novel analog, CC-122, can modulate the spectrum of substrates targeted by CRL4CRBN. These findings have implications for the clin. activity of lenalidomide and related compds., and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.
- 12Hartmann, M. D.; Boichenko, I.; Coles, M.; Zanini, F.; Lupas, A. N.; Hernandez Alvarez, B. Thalidomide mimics uridine binding to an aromatic cage in cereblon. J. Struct. Biol. 2014, 188 (3), 225– 32, DOI: 10.1016/j.jsb.2014.10.01012https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSnu7jI&md5=e601c6e1091deea840f9738fb30fa7bbThalidomide mimics uridine binding to an aromatic cage in cereblonHartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Zanini, Fabio; Lupas, Andrei N.; Hernandez Alvarez, BirteJournal of Structural Biology (2014), 188 (3), 225-232CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)Thalidomide and its derivs. lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resoln. crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivs. mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an arom. cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.
- 13Boichenko, I.; Bar, K.; Deiss, S.; Heim, C.; Albrecht, R.; Lupas, A. N.; Hernandez Alvarez, B.; Hartmann, M. D. Chemical Ligand Space of Cereblon. ACS Omega 2018, 3 (9), 11163– 11171, DOI: 10.1021/acsomega.8b0095913https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslSku7bO&md5=b0fee25453d5ec83098ac0cf28575d44Chemical Ligand Space of CereblonBoichenko, Iuliia; Baer, Kerstin; Deiss, Silvia; Heim, Christopher; Albrecht, Reinhard; Lupas, Andrei N.; Hernandez Alvarez, Birte; Hartmann, Marcus D.ACS Omega (2018), 3 (9), 11163-11171CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)The protein, cereblon, serves as a substrate receptor of a ubiquitin ligase complex that can be tuned toward different target proteins by cereblon-binding agents. This approach to targeted protein degrdn. is exploited in different clin. settings and has sparked the development of a growing no. of thalidomide derivs. Here, we probed the chem. space of cereblon binding beyond such derivs. and worked out a simple set of chem. requirements, delineating the metaclass of cereblon effectors. We report co-crystal structures of Magnetospirillum gryphiswaldense cereblon isoform 4 with a diverse set of compds., including commonly used pharmaceuticals, but we also found that already minimalistic cereblon-binding moieties might exert teratogenic effects in zebrafish. These results may guide the design of a post-thalidomide generation of therapeutic cereblon effectors, and provide a framework for the circumvention of unintended cereblon-binding by neg. design for future pharmaceuticals.
- 14Heim, C.; Pliatsika, D.; Mousavizadeh, F.; Bar, K.; Hernandez Alvarez, B.; Giannis, A.; Hartmann, M. D. De-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide Derivatives. J. Med. Chem. 2019, 62 (14), 6615– 6629, DOI: 10.1021/acs.jmedchem.9b0045414https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1OqsbrK&md5=b09f48df1a76f1d2a954563f4842d3fcDe-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide DerivativesHeim, Christopher; Pliatsika, Dimanthi; Mousavizadeh, Farnoush; Baer, Kerstin; Hernandez Alvarez, Birte; Giannis, Athanassios; Hartmann, Marcus D.Journal of Medicinal Chemistry (2019), 62 (14), 6615-6629CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Targeted protein degrdn. via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clin. settings, in which the substrate specificity of CRBN is altered via the binding of small-mol. effectors. To date, such effectors are derived from thalidomide and confer a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular approach to design novel and minimalistic CRBN effectors. In this approach, we took advantage of the binding modes of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallog. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chem. moiety that imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to degrade the neo-substrate IKZF3 in the cell culture.
- 15Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R. J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (15), 8554– 8559, DOI: 10.1073/pnas.14123079815https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXls1Wisbk%253D&md5=540358035222f745f7b6367b38781a21Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradationSakamoto, Kathleen M.; Kim, Kyung B.; Kumagai, Akiko; Mercurio, Frank; Crews, Craig M.; Deshaies, Raymond J.Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (15), 8554-8559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex contg. Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degrdn. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compd., protein-targeting chimeric mol. 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IκBα phosphopeptide that is recognized by the F-box protein β-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCFβ-TRCP, ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.
- 16Min, J. H.; Yang, H.; Ivan, M.; Gertler, F.; Kaelin, W. G., Jr.; Pavletich, N. P. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 2002, 296 (5574), 1886– 9, DOI: 10.1126/science.107344016https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XksVWmsr8%253D&md5=c3ccb5f0a383257ae465cc1c5f7a189fStructure of an HIF-1α-pVHL complex: hydroxyproline recognition in signalingMin, Jung-Hyun; Yang, Haifeng; Ivan, Mircea; Gertler, Frank; Kaelin, William G., Jr.; Pavletich, Nikola P.Science (Washington, DC, United States) (2002), 296 (5574), 1886-1889CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) plays a central role in the cellular response to changes in oxygen availability. PVHL binds to HIF only when a conserved proline in HIF is hydroxylated, a modification that is oxygen-dependent. The 1.85 Å structure of a 20-residue HIF-1α peptide-pVHL-Elongin B-Elongin C complex shows that HIF-1α binds to pVHL in an extended β strand-like conformation. The hydroxyproline inserts into a gap in the pVHL hydrophobic core, at a site that is a hotspot for tumorigenic mutations, with its 4-hydroxyl group recognized by buried serine and histidine residues. Although the β sheet-like interactions contribute to the stability of the complex, the hydroxyproline contacts are central to the strict specificity characteristic signaling.
- 17Fischer, E. S.; Bohm, K.; Lydeard, J. R.; Yang, H.; Stadler, M. B.; Cavadini, S.; Nagel, J.; Serluca, F.; Acker, V.; Lingaraju, G. M.; Tichkule, R. B.; Schebesta, M.; Forrester, W. C.; Schirle, M.; Hassiepen, U.; Ottl, J.; Hild, M.; Beckwith, R. E.; Harper, J. W.; Jenkins, J. L.; Thoma, N. H. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 2014, 512 (7512), 49– 53, DOI: 10.1038/nature1352717https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht12ms7%252FP&md5=1c1eaa48bef87463cdd3f2b1e1bd400dStructure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomideFischer, Eric S.; Bohm, Kerstin; Lydeard, John R.; Yang, Haidi; Stadler, Michael B.; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M.; Tichkule, Ritesh B.; Schebesta, Michael; Forrester, William C.; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E. J.; Harper, J. Wade; Jenkins, Jeremy L.; Thoma, Nicolas H.Nature (London, United Kingdom) (2014), 512 (7512), 49-53CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivs. lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-assocd. dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN. Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degrdn. This dual activity implies that small mols. can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.
- 18Steinebach, C.; Sosic, I.; Lindner, S.; Bricelj, A.; Kohl, F.; Ng, Y. L. D.; Monschke, M.; Wagner, K. G.; Kronke, J.; Gutschow, M. A MedChem toolbox for cereblon-directed PROTACs. MedChemComm 2019, 10 (6), 1037– 1041, DOI: 10.1039/C9MD00185A18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVCrtr3L&md5=865c40183453c6473ef7f037aa566ecaA MedChem toolbox for cereblon-directed PROTACsSteinebach, Christian; Sosic, Izidor; Lindner, Stefanie; Bricelj, Alesa; Kohl, Franziska; Ng, Yuen Lam Dora; Monschke, Marius; Wagner, Karl G.; Kroenke, Jan; Guetschow, MichaelMedChemComm (2019), 10 (6), 1037-1041CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)A modular chem. toolbox was developed for cereblon-directed PROTACs. A variety of linkers was attached to a CRBN ligand via the 4-amino position of pomalidomide. We used linkers of different constitution to modulate physicochem. properties. We equipped one terminus of the linker with a set of functional groups, e.g. protected amines, protected carboxylic acids, alkynes, chloroalkanes, and protected alcs., all of which are considered to be attractive for PROTAC design. We also highlight different opportunities for the expansion of the medicinal chemists' PROTAC toolbox towards heterobifunctional mols., e.g. with biotin, fluorescent, hydrophobic and peptide tags.
- 19Buckley, D. L.; Van Molle, I.; Gareiss, P. C.; Tae, H. S.; Michel, J.; Noblin, D. J.; Jorgensen, W. L.; Ciulli, A.; Crews, C. M. Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1alpha interaction. J. Am. Chem. Soc. 2012, 134 (10), 4465– 8, DOI: 10.1021/ja209924v19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivVOgurw%253D&md5=60f7384b6bc932c9828dc12999283a1eTargeting the von Hippel-Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1α InteractionBuckley, Dennis L.; Van Molle, Inge; Gareiss, Peter C.; Tae, Hyun Seop; Michel, Julien; Noblin, Devin J.; Jorgensen, William L.; Ciulli, Alessio; Crews, Craig M.Journal of the American Chemical Society (2012), 134 (10), 4465-4468CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degrdn., are attractive drug targets due to their exquisite substrate specificity. However, the development of small-mol. inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein-protein interactions. Using rational design, we have generated the first small mol. targeting the von Hippel-Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compd. mimics the binding mode of the transcription factor HIF-1α, a substrate of VHL. These results have the potential to guide future development of improved lead compds. as therapeutics for the treatment of chronic anemia and ischemia.
- 20Boichenko, I.; Deiss, S.; Bar, K.; Hartmann, M. D.; Hernandez Alvarez, B. A FRET-Based Assay for the Identification and Characterization of Cereblon Ligands. J. Med. Chem. 2016, 59 (2), 770– 4, DOI: 10.1021/acs.jmedchem.5b0173520https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1yktg%253D%253D&md5=f0400506428f439e81f579204e0bd39dA FRET-Based Assay for the Identification and Characterization of Cereblon LigandsBoichenko, Iuliia; Deiss, Silvia; Baer, Kerstin; Hartmann, Marcus D.; Hernandez Alvarez, BirteJournal of Medicinal Chemistry (2016), 59 (2), 770-774CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Cereblon serves as an ubiquitin ligase substrate receptor that can be tuned toward different target proteins by various cereblon-binding agents. This offers one of the most promising avenues for targeted protein degrdn. in cancer therapy, but cereblon binding can also mediate teratogenic effects. We present an effective assay that is suited for high-throughput screening of compd. libraries for off-target cereblon interactions but also can guide lead optimization and rational design of novel cereblon effector mols.
- 21Duhr, S.; Braun, D. Why molecules move along a temperature gradient. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (52), 19678– 82, DOI: 10.1073/pnas.060387310321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjtVSltQ%253D%253D&md5=91255864cb4828be93545c71d96f30fdWhy molecules move along a temperature gradientDuhr, Stefan; Braun, DieterProceedings of the National Academy of Sciences of the United States of America (2006), 103 (52), 19678-19682CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Mols. drift along temp. gradients, an effect called thermophoresis, the Soret effect, or thermodiffusion. In liqs., its theor. foundation is the subject of a long-standing debate. By using an all-optical microfluidic fluorescence method, we present exptl. results for DNA and polystyrene beads over a large range of particle sizes, salt concns., and temps. The data support a unifying theory based on solvation entropy. Stated in simple terms, the Soret coeff. is given by the neg. solvation entropy, divided by kT. The theory predicts the thermodiffusion of polystyrene beads and DNA without any free parameters. We assume a local thermodn. equil. of the solvent mols. around the mol. This assumption is fulfilled for moderate temp. gradients below a fluctuation criterion. For both DNA and polystyrene beads, thermophoretic motion changes sign at lower temps. This thermophilicity toward lower temps. is attributed to an increasing pos. entropy of hydration, whereas the generally dominating thermophobicity is explained by the neg. entropy of ionic shielding. The understanding of thermodiffusion sets the stage for detailed probing of solvation properties of colloids and biomols. For example, we successfully det. the effective charge of DNA and beads over a size range that is not accessible with electrophoresis.
- 22Gupta, A. J.; Duhr, S.; Baaske, P. Microscale Thermophoresis (MST). In Encyclopedia of Biophysics; Roberts, G., Watts, A., Eds.; Springer: Berlin, Heidelberg, 2018; pp 1– 5.There is no corresponding record for this reference.
- 23Seidel, S. A.; Dijkman, P. M.; Lea, W. A.; van den Bogaart, G.; Jerabek-Willemsen, M.; Lazic, A.; Joseph, J. S.; Srinivasan, P.; Baaske, P.; Simeonov, A.; Katritch, I.; Melo, F. A.; Ladbury, J. E.; Schreiber, G.; Watts, A.; Braun, D.; Duhr, S. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 2013, 59 (3), 301– 15, DOI: 10.1016/j.ymeth.2012.12.00523https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFClsbs%253D&md5=4122dd561a2bd3187b0d9100ed57d4c0Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditionsSeidel, Susanne A. I.; Dijkman, Patricia M.; Lea, Wendy A.; van den Bogaart, Geert; Jerabek-Willemsen, Moran; Lazic, Ana; Joseph, Jeremiah S.; Srinivasan, Prakash; Baaske, Philipp; Simeonov, Anton; Katritch, Ilia; Melo, Fernando A.; Ladbury, John E.; Schreiber, Gideon; Watts, Anthony; Braun, Dieter; Duhr, StefanMethods (Amsterdam, Netherlands) (2013), 59 (3), 301-315CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Microscale thermophoresis (MST) allows for quant. anal. of protein interactions in free soln. and with low sample consumption. The technique is based on thermophoresis, the directed motion of mols. in temp. gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of mol. properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an IR laser is used for local heating, and mol. mobility in the temp. gradient is analyzed via fluorescence. In std. MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence.Despite the high mol. wt. ratio, the interaction of small mols. and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomols., such as membrane proteins to be stabilized in soln. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purifn. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for anal. of biomol. interactions in complex exptl. settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.
- 24Akuffo, A. A.; Alontaga, A. Y.; Metcalf, R.; Beatty, M. S.; Becker, A.; McDaniel, J. M.; Hesterberg, R. S.; Goodheart, W. E.; Gunawan, S.; Ayaz, M.; Yang, Y.; Karim, M. R.; Orobello, M. E.; Daniel, K.; Guida, W.; Yoder, J. A.; Rajadhyaksha, A. M.; Schonbrunn, E.; Lawrence, H. R.; Lawrence, N. J.; Epling-Burnette, P. K. Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon. J. Biol. Chem. 2018, 293 (16), 6187– 6200, DOI: 10.1074/jbc.M117.81686824https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFSqtb8%253D&md5=16bc835472884d8b173e96fccdc3019aLigand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblonAkuffo, Afua A.; Alontaga, Aileen Y.; Metcalf, Rainer; Beatty, Matthew S.; Becker, Andreas; McDaniel, Jessica M.; Hesterberg, Rebecca S.; Goodheart, William E.; Gunawan, Steven; Ayaz, Muhammad; Yang, Yan; Karim, Md. Rezaul; Orobello, Morgan E.; Daniel, Kenyon; Guida, Wayne; Yoder, Jeffrey A.; Rajadhyaksha, Anjali M.; Schonbrunn, Ernst; Lawrence, Harshani R.; Lawrence, Nicholas J.; Epling-Burnette, Pearlie K.Journal of Biological Chemistry (2018), 293 (16), 6187-6200CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degrdn. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theor., biophys., and biol. assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chem. compd. and targeting ligand designed to degrade bromodomain-contg. 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting mols. in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.
- 25Hughes, J. P.; Rees, S.; Kalindjian, S. B.; Philpott, K. L. Principles of early drug discovery. Br. J. Pharmacol. 2011, 162 (6), 1239– 49, DOI: 10.1111/j.1476-5381.2010.01127.x25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXivF2lt7o%253D&md5=05a0016f45b155f9fb5822f5c7bcc35dPrinciples of early drug discoveryHughes, J. P.; Rees, S.; Kalindjian, S. B.; Philpott, K. L.British Journal of Pharmacology (2011), 162 (6), 1239-1249CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)A review. Developing a new drug from original idea to the launch of a finished product is a complex process which can take 12-15 years and cost in excess of $1 billion. The idea for a target can come from a variety of sources including academic and clin. research and from the com. sector. It may take many years to build up a body of supporting evidence before selecting a target for a costly drug discovery program. Once a target was chosen, the pharmaceutical industry and more recently some academic centers have streamlined a no. of early processes to identify mols. which possess suitable characteristics to make acceptable drugs. This review will look at key preclin. stages of the drug discovery process, from initial target identification and validation, through assay development, high throughput screening, hit identification, lead optimization and finally the selection of a candidate mol. for clin. development.
- 26Steinebach, C.; Ng, Y. L. D.; Sosic, I.; Lee, C. S.; Chen, S. R.; Lindner, S.; Vu, L. P.; Bricelj, A.; Haschemi, R.; Monschke, M.; Steinwarz, E.; Wagner, K. G.; Bendas, G.; Luo, J.; Gutschow, M.; Kronke, J. Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders. Chem. Sci. 2020, 11 (13), 3474– 3486, DOI: 10.1039/D0SC00167H26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkt1eqt7k%253D&md5=6f1f042714f46883e8ab7a0b4ae3ae96Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degradersSteinebach, Christian; Ng, Yuen Lam Dora; Sosic, Izidor; Lee, Chih-Shia; Chen, Sirui; Lindner, Stefanie; Vu, Lan Phuong; Bricelj, Alesa; Haschemi, Reza; Monschke, Marius; Steinwarz, Elisabeth; Wagner, Karl G.; Bendas, Gerd; Luo, Ji; Guetschow, Michael; Kroenke, JanChemical Science (2020), 11 (13), 3474-3486CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Cyclin-dependent kinase 6 (CDK6) is an important regulator of the cell cycle. Together with CDK4, it phosphorylates and inactivates retinoblastoma (Rb) protein. In tumor cells, CDK6 is frequently upregulated and CDK4/6 kinase inhibitors like palbociclib possess high activity in breast cancer and other malignancies. Besides its crucial catalytic function, kinase-independent roles of CDK6 have been described. Therefore, targeted degrdn. of CDK6 may be advantageous over kinase inhibition. Proteolysis targeting chimeras (PROTACs) structurally based on the cereblon (CRBN) ligand thalidomide have recently been described to degrade the targets CDK4/6. However, CRBN-based PROTACs have several limitations including the remaining activity of immunomodulatory drugs (IMiDs) on Ikaros transcription factors as well as CRBN inactivation as a resistance mechanism in cancer. Here, we systematically explored the chem. space of CDK4/6 PROTACs by addressing different E3 ligases and connecting their resp. small-mol. binders via various linkers to palbociclib. The spectrum of CDK6-specific PROTACs was extended to von Hippel Lindau (VHL) and cellular inhibitor of apoptosis protein 1 (cIAP1) that are essential for most cancer cells and therefore less likely to be inactivated. Our VHL-based PROTAC series included compds. that were either specific for CDK6 or exhibited dual activity against CDK4 and CDK6. IAP-based PROTACs caused a combined degrdn. of CDK4/6 and IAPs resulting in synergistic effects on cancer cell growth. Our new degraders showed potent and long-lasting degrading activity in human and mouse cells and inhibited proliferation of several leukemia, myeloma and breast cancer cell lines. In conclusion, we show that VHL- and IAP-based PROTACs are an attractive approach for targeted degrdn. of CDK4/6 in cancer.
- 27Matyskiela, M. E.; Zhang, W.; Man, H. W.; Muller, G.; Khambatta, G.; Baculi, F.; Hickman, M.; LeBrun, L.; Pagarigan, B.; Carmel, G.; Lu, C. C.; Lu, G.; Riley, M.; Satoh, Y.; Schafer, P.; Daniel, T. O.; Carmichael, J.; Cathers, B. E.; Chamberlain, P. P. A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos. J. Med. Chem. 2018, 61 (2), 535– 542, DOI: 10.1021/acs.jmedchem.6b0192127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1KqsLs%253D&md5=8358ae8563f8f2c09dbf68683e5e4008A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and AiolosMatyskiela, Mary E.; Zhang, Weihong; Man, Hon-Wah; Muller, George; Khambatta, Godrej; Baculi, Frans; Hickman, Matthew; LeBrun, Laurie; Pagarigan, Barbra; Carmel, Gilles; Lu, Chin-Chun; Lu, Gang; Riley, Mariko; Satoh, Yoshitaka; Schafer, Peter; Daniel, Thomas O.; Carmichael, James; Cathers, Brian E.; Chamberlain, Philip P.Journal of Medicinal Chemistry (2018), 61 (2), 535-542CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degrdn. Here we describe CC-220 (compd. 6), a cereblon modulator in clin. development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compd. 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degrdn. of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compd. 6 reveals that the increase in potency correlates with increased contacts between compd. 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degrdn. via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degrdn.
- 28Gentile, I.; Buonomo, A. R.; Borgia, G. Dasabuvir: A Non-Nucleoside Inhibitor of NS5B for the Treatment of Hepatitis C Virus Infection. Rev. Recent Clin. Trials 2014, 9 (2), 115– 23, DOI: 10.2174/157488710966614052922260228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslGqu7vJ&md5=75f136bd5bccdf288697d8aba73ba037Dasabuvir: A Non-Nucleoside Inhibitor of NS5B for the Treatment of Hepatitis C Virus InfectionGentile, Ivan; Buonomo, Antonio R.; Borgia, GuglielmoReviews on Recent Clinical Trials (2014), 9 (2), 115-123CODEN: RRCTB2; ISSN:1574-8871. (Bentham Science Publishers Ltd.)Hepatitis C virus (HCV) chronically infects about 2% of the world's population. Approx. a quarter of these patients will develop, during their life, liver cirrhosis, which entails a high risk of complications and death. Successful antiviral therapy can reduce the risk of disease progression, but it is feasible only in a minority of patients because it includes interferon which is contraindicated in the most advanced stages of the disease and in patients with severe impairment of other organs. Consequent to the launch of the first direct antiviral agents (DAA), namely the protease inhibitors telaprevir and boceprevir, several mols. are in an advanced phase of clin. development to be used in assocn. with interferon or with other DAA (in interferon-free combinations). This review focuses on the mechanism of action, pharmacokinetics, efficacy, safety and resistance of dasabuvir, a non-nucleoside inhibitor of NS5B viral RNA-dependent RNA polymerase. Thanks to its pharmacokinetics, dasabuvir can be administered twice daily. In combinations with other oral DAAs, dasabuvir results in very high rates of SVR (about 95%) in patients with HCV genotype 1 infection with a good tolerability and safety. In conclusion, dasabuvir is a good agent to be used in interferon-free combinations for the treatment of chronic hepatitis C.
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00440.
Analysis and discussion of the impact of DMSO competition on the characterization of CRBN ligands; analysis and discussion of the influence of fluorescence quenching and autofluorescent test compounds; experimental procedures; FRET assay data for avadomide, nitrofurantoin and UMP; influence of DMSO; influence of initial fluorescence on MST behavior (PDF)
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