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De-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide Derivatives
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  • Christopher Heim
    Christopher Heim
    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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  • Dimanthi Pliatsika
    Dimanthi Pliatsika
    Faculty for Chemistry und Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany
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  • Farnoush Mousavizadeh
    Farnoush Mousavizadeh
    Faculty for Chemistry und Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany
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  • Kerstin Bär
    Kerstin Bär
    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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  • Birte Hernandez Alvarez
    Birte Hernandez Alvarez
    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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  • Athanassios Giannis*
    Athanassios Giannis
    Faculty for Chemistry und Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany
    *E-mail: [email protected] (A.G.).
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    Orcidhttp://orcid.org/0000-0003-2203-0959
  • Marcus D. Hartmann*
    Marcus D. Hartmann
    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
    *E-mail: [email protected] (M.D.H.).
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    Orcidhttp://orcid.org/0000-0001-6937-5677
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2019, 62, 14, 6615–6629
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https://doi.org/10.1021/acs.jmedchem.9b00454
Published June 28, 2019

Copyright © 2019 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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Targeted protein degradation via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clinical settings, in which the substrate specificity of CRBN is altered via the binding of small-molecule 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 crystallography. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chemical 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.

Copyright © 2019 American Chemical Society

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Introduction

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Classical immunomodulatory drugs (IMiDs) like thalidomide and its second- and third-generation analogues lenalidomide, pomalidomide, avadomide (CC-122), and iberdomide (CC-220) have constantly emerged to new therapeutic areas. Originally developed as a sedative (1) and banned in 1961 for its teratogenic effects when used during pregnancy, (2,3) thalidomide and a number of newly developed analogues are approved for the treatment of multiple myeloma (MM), (4) erythema nodosum (5) and myelodysplastic syndrome (MDS). (6,7) Because of their pleiotropic and especially anti-angiogenic properties, IMiDs have further been reported effective in many off-label indications as for Hodgkin’s lymphoma, (8−10) light chain-associated (AL) amyloidosis, (11) and acute myeloid leukemia (AML). (12,13)
Currently, IMiDs share a common glutarimide moiety (Figure 1), which is connected to a second moiety that is typically derived from phthaloyl. Via the glutarimide moiety, they are able to bind to a tri-tryptophan pocket within the thalidomide-binding domain (14−17) of cereblon (CRBN). CRBN is the substrate receptor of the Cullin RING E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN) (17) and responsible for the recognition of endogenous substrates such as glutamine synthetase, (18) MEIS2, (17) and amyloid precursor protein (APP). (19) In the presence of IMiDs, however, its substrate specificity is changed. The solvent-exposed second moiety, the protruding moiety that is unique to each IMiD, recruits novel substrates to the CRBN surface for ubiquitination. The degradation of these neo-substrates accounts for most of the efficacy of IMiDs in MM (IKZF1 and IKZF3), (20) 5q-deletion-associated MDS (CK1α), (21) and AML (GSPT1). (22) Recently, it has also been shown that the degradation of the neo-substrate SALL4 is linked to developmental malformations caused by thalidomide. (23,24)

Figure 1

Figure 1. Chemical structures of thalidomide, lenalidomide, pomalidomide, avadomide, and iberdomide with their glutarimide moiety shown in red.

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Crystal structures of the neo-substrate complexes CRBN·lenalidomide·CK1α, CRBN·pomalidomide·IKZF1, and CRBN·pomalidomide·ZNF692 provided first insights into the binding mode of neo-substrates. (25,26) Many of the identified neo-substrates possess no obvious sequence homology, but they all exhibit a structurally analogous β-hairpin, which binds to the surface around the IMiD-binding site, involving interactions with both surface residues and the IMiD itself. A potential prerequisite for the recruitment is the presence of a glycine at the tip of this β-hairpin; this glycine was found to be conserved for many neo-substrates, like CK1α (25) and GSPT1, (22) and appears in the common C-X(2)-C-G motif (24) in many transcription factors belonging to the C2H2 zinc finger class, including IKZF3, (25) IKZF1, (25) ZFP91, (24) and SALL4. (24) While IMiDs seem to generally recruit several members of the C2H2 zinc finger family, only lenalidomide was proven to recruit CK1α, indicating that the protruding moiety imparts substrate specificity.
The CRBN-binding ability of IMiDs has further been exploited for targeted protein degradation in an approach called proteolysis targeting chimera (PROTAC), coined in 2001 by Craig Crews and co-workers. (27,28) PROTACs are bifunctional small molecules with a binding moiety for a target protein linked to a binding moiety for an E3 ubiquitin ligase, thus inducing ubiquitination and proteasomal degradation of the target protein. Recent examples of successful PROTACs target the estrogen (29) and the androgen receptor (30) via a von-Hippel-Lindau (VHL) E3 ligase ligand but also BET bromodomains via linkage to thalidomide as a CRBN ligand. (31) Notably, also PROTACs with a VHL ligand on one and thalidomide as the CRBN ligand on the other end have been tested, leading to unidirectional degradation of CRBN. (32) In general, also the PROTAC approach has high potential to target the undruggable; a particularly illustrative example for this potential is exemplified by the recent discovery of PROTACs targeting the tau protein. (33−35)
To date, essentially all CRBN effectors—IMiDs and CRBN-based PROTACs—are derived from thalidomide and its derivatives. However, the chemical space of CRBN ligands ranges far beyond thalidomide: in first systematic characterization, we have previously revealed that a large variety of lactams and cyclic imides are potent binders, including several marketed drugs. (36) Specifically, we have shown succinimide to exhibit higher affinity than glutarimide, using the single-domain bacterial CRBN homologue from Magnetospirillum gryphiswaldense (MsCI4) in a FRET assay. (14) In this study, we set out to further explore and exploit the chemical space of CRBN binding by designing effectors based on succinimide and glutarimide, which we characterize with regard to their affinity, their structural binding mode, and their ability to induce proteasomal degradation of neo-substrates. Guided by hydrolyzed metabolites of thalidomide and of three of our designs, we present novel minimalistic motifs that are able to recruit and degrade neo-substrates and may serve as E3-recruiting ligands for future PROTACs.

Results and Discussion

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Biophysical and Structural Characterization of IMiD Analogues and Their Hydrolysis Products

As a starting point for new effectors, we chose the classical IMiD scaffold. Based on the finding that succinimide is able to bind to CRBN with a higher affinity than glutarimide (Ki values of 4.3 μM vs 28 μM for MsCI4 (36)), we designed a first panel of derivatives based on glutarimide and succinimide, in which we probed the effect of different substitutions in the phthaloyl moiety (Figure 2). The respective compounds 2a–5b were prepared by the synthetic route shown in Scheme 1. The imides 3a and 3b were synthesized from the commercially available Nα-(tert-butoxycarbonyl)-l-asparagine (1a) and Nα-(tert-butoxycarbonyl)-l-glutamine (1b), respectively, via an imide formation using N,N-carbodiimidazole (CDI) and 4-dimethylaminopyridine (4-DMAP) in tetrahydrofuran (THF) as well as a deprotection reaction in the presence of trifluoracetic acid (TFA). Compounds 4a–4d were obtained through coupling between the imides 3a or 3b and the commercially available 3-nitrophthalic anhydride or 4-nitrophthalic anhydride. Catalytic hydrogenation of the molecules 4a and 4b using 10 wt % Pd/C in EtOAc produced the derivatives 5a and 5b.

Figure 2

Figure 2. The first panel of chemical structures, five- and six-ring based thalidomide analogues and their affinity to MsCI4 determined as Ki values in the FRET assay.

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Scheme 1

Scheme 1. a

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aReagents and conditions: (a) CDI, 4-DMAP, THF, reflux, 17–48 h; (b) TFA, RT, 30 min; (c) CDI, 4-DMAP, THF, reflux, 5–20 h; and (d) 10 wt % Pd/C, EtOAc, RT, 20 h.

The binding affinities of these compounds for MsCI4 were assessed in the FRET assay, starting with the glutarimide-based compounds 4c and 4d in comparison to the commercial thalidomide derivatives, lenalidomide and pomalidomide. Lenalidomide (Ki = 3.1 μM) and pomalidomide (Ki = 0.8 μM), both carrying an additional amino group in the R2 position, show improved affinity to MsCI4 as compared to thalidomide (Ki = 4.4 μM). When we exchanged this amino group for a nitro group (4c), we saw a significant drop in affinity into a range in which a precise value could not be obtained (Ki > 40 μM); (36) moving this nitro group to the R1 position (4d) had less impact on the affinity (Ki = 8.9 μM, Figure 2A). Similar effects were observed in a recent study that was published during the preparation of this manuscript. (37) As both compounds, 4c and 4d, retained affinity to MsCI4, their overall binding mode is supposedly conserved and comparable to thalidomide, which is consistent with previous studies, showing that small modifications on the protruding moiety have little influence on the overall affinity to CRBN. (17,37) The binding modes of lenalidomide and pomalidomide have previously been reported to be virtually identical to thalidomide. (14,17)
For the succinimide-based compounds 4a, 4b, 5a, and 5b, we obtained a similar picture as for the glutarimide-based compounds. Although they showed overall weaker binding in the FRET assay (Figure 2B), substitutions in the R2 position are less favorable for the affinity as substitutions in R1. For 4a and 5a, which have a nitro or an amino group in R2, respectively, we obtained Ki values of >40 μM. For 4b and 5b, which have the respective groups in the R1 position, the obtained Ki values are 11 and 12 μM, respectively.
In a next step, we determined the molecular-binding determinants of the succinimide-based compounds via X-ray crystallography. To this end, crystals of the MsCI4·thalidomide complex were reproduced (36) and subsequently used for soaking experiments, in which the thalidomide molecules bound to MsCI4 may be displaced by the compound of interest. There are three chains, that is, three MsCI4·thalidomide complexes, in the asymmetric unit (ASU) of these crystals, in which the bound thalidomide molecules can potentially be displaced. However, as the three chains form different crystal contacts and differ slightly in their conformation, it is possible that thalidomide is only replaced in one or two chains of the ASU, depending on the particular compound. (36) These experiments yielded crystal structures for the four compounds tested, 4a, 4b, 5a, and 5b, all showing the classical IMiD binding mode with the basal main-chain interactions of the succinimide amino group with F77 and the carbonyl group with W79 of MsCI4. Also, the orientation of the protruding moieties of 4a, 5a, and 5b is very similar to that of thalidomide, despite the different ring size of glutarimide and succinimide. As a result, the hydrogen bond that is typically observed between the conserved N50 and a carbonyl group of the phthaloyl moieties for the classical glutarimide-based compounds is also found for 5b.
However, the crystal structures also held surprises. They did not only reveal the binding modes of the pure compounds but also that of hydrolysis products of 4a, 4b, and 5a with unambiguous electron density; only 5b was exclusively observed in the nonhydrolyzed form (Figures 3 and 4). For 4a and 5a, hydrolyzed metabolites were only observed in one chain of the ASU, with the second chain occupied with a nonhydrolyzed version and the third chain with thalidomide. For 4b, which showed the highest affinity with a Ki of 11 μM, all three binding pockets in the ASU were occupied by a hydrolyzed metabolite, so the binding mode of nonhydrolyzed 4b could not be studied. In all cases, for the hydrolysis products of 4a, 4b, and 5a, ring opening of the phthaloyl group had led to the formation of a secondary amide and a carboxyl group, which are clearly resolved in FO–FC omit maps (Figure 3). Both of these form important interactions with the binding pocket: first, the secondary amide rescues the hydrogen bond to the conserved N50, which is typically formed by a carbonyl of the phthaloyl moiety. Second, the additional carboxyl group replaces a conserved water molecule previously coordinated by W99 and engages in direct hydrogen bonding with the W99 side chain. In contrary, the primary amino group of hydrolyzed 5a and the solvent-exposed nitro groups of hydrolyzed 4a and 4b do not show additional interactions.

Figure 3

Figure 3. Overview of the thalidomide-binding mode and electron densities of thalidomide analogues and their hydrolysis products bound to MsCI4. Left: Cartoon representation of thalidomide-bound MsCI4 with key residues of the binding pocket shown as sticks. Right: FO–FC maps of CBG (PDB 6R0Q), 4a and hydrolyzed 4a (PDB 6R0S), hydrolyzed 4b (PDB 6R0V), 5a, and hydrolyzed 5a (PDB 6R0U) and 5b (PDB 6R11) in the MsCI4-binding pocket, contoured at 2σ.

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Figure 4

Figure 4. Binding mode of the initial compounds and their hydrolysis products inside the binding pocket. Thalidomide, 4a, and 5a are shown with their respective hydrolysis products. Ring opening of the phtaloyl moiety that leads to the observed hydrolysis product is indicated in red in the chemical drawings. 5b was exclusively found in the nonhydrolyzed form, whereas 4b was exclusively found as a hydrolysis product. PDB codes are 4V2Y (thalidomide), 6R0Q (CBG), 6R0S (4a), 6R0U (5a), 6R11 (5b), and 6R0V (4b).

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Binding Mode of CBG, a Major Hydrolysis Product of Thalidomide

Unintentionally, in addition to the hydrolysis products of 4a, 4b, and 5a, we were able to characterize the binding mode of a major hydrolyzed thalidomide metabolite. It is known that IMiDs can rapidly racemize in bodily fluids and water (38) and spontaneously hydrolyze under physiological conditions. (39−41) For thalidomide, being eliminated mainly by spontaneous hydrolysis in blood and tissues, a half-life of about 5.5–7.3 h at the physiological pH of 7.4 was reported. (42) Among several proposed hydrolysis products, the two main urinary metabolites are 2-phthalimidoglutaramic acid (∼50%) and α-(2-carboxybenzamido)glutarimide (CBG) (∼30%). (43) Of these, CBG has an unmodified glutarimide moiety and is also one of three major metabolites in human plasma. (44) As CBG was also reported to possess a higher TNF-α production-inhibitory activity (80%) than thalidomide (32%) at concentrations of 3 μM, (44,45) it is of great pharmacological interest, but its mode of interaction remained elusive so far. (46) During the course of this study, we obtained a crystal structure of CBG in the complex with a humanized mutant form of MsCI4 (Figures 3 and 4). This structure was the result of a cocrystallization trial of this mutant with thalidomide, which aimed at the characterization of the mutant protein. However, the mutant selectively bound CGB that was presumably present in traces in our crystallization setup with thalidomide. This mutant has a number of nonconserved residues in the direct vicinity of the thalidomide-binding pocket mutated to the residues in the human protein, including the substitution F56H. While this residue is not directly involved in classical IMiD binding in MsCI4 or the human protein, it plays an important role in the binding of CBG: together with N50, it coordinates one oxygen of the carboxyl group in the CBG-protruding moiety; the other oxygen of this carboxyl coordinates the conserved water molecule bound to W99. However, in contrast to the hydrolysis products of the succinimide-based compounds 4a, 4b, and 5a, the amide moiety resulting from ring opening is not found to be involved in defined hydrogen bonding—although superposing approximately with the phthaloyl moiety of thalidomide, it does not form the canonical hydrogen bond with N50, as the latter is engaged in the hydrogen bond to the carboxyl group.

Rational Design of Novel Succinimide Effectors Guided by Hydrolyzed Metabolites

In a next step, we aimed to exploit our knowledge on the binding mode of hydrolyzed metabolites for the design of novel effectors. The fact that the hydrolysis products were selected against their parent compounds in several cocrystallization or soaking experiments suggested that they pose binders of similar, if not superior affinity. Comparing the binding modes of CBG and hydrolyzed 4a, 4b, and 5a, we further hypothesized that the difference seen for the hydrogen bonding of their amide linkers should yield increased affinity for succinimide-based effectors. Consequently, we used this amide linker to connect different functional groups as protruding moieties to succinimide as the binding moiety. To this aim, we prepared derivatives 7a–7h, as shown in Scheme 2, by treating imide 3a with the corresponding acyl chlorides 6a–6h and N,N-diisopropylethylamine (DIPEA) in THF.

Scheme 2

Scheme 2. a

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aReagents and conditions: (a) CDI, 4-DMAP, THF, reflux, 48 h; (b) TFA, RT, 30 min; (c) 6a–h, DIPEA, THF, 0 oC to reflux, and 2 h.

All derivatives of this second panel were tested in the FRET assay. Only for compound 7e, with a 3,5-dinitrobenzol group, the affinity was decreased under the detectable levels of the FRET assay. For the compounds with an isobutylene (7a), benzyl (7b), or chlorobenzothiophene group (7g) as the protruding moiety, binding was well detectable but could not be quantified (Ki > 40 μM). Better binding was observed for compounds with a styryl (7f) and dichloro-benzothiophene (7h) group, both showing Ki values of 20 μM (Figure 5A). Finally, the highest affinities were achieved with a 2,4,6-trichlorobenzol moiety (7c, Ki = 9 μM) and benzyloxy group (7d, Ki = 4 μM), rendering 7d, the highest-affinity binder in this study, with a Ki value comparable to an unmodified succinimide (36) (Figure 5A).

Figure 5

Figure 5. Compounds inspired by hydrolysis products of thalidomide analogues and their affinity data. (A) Second panel of compounds, based on the 3-amidosuccinimide scaffold. (B) Third panel of compounds, inspired by 7d. * 20a could not be purified to satisfactory levels for affinity testing.

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We continued with characterizing the binding modes of compounds 7a–c and 7f via crystallography. For the best binder 7d, we performed cocrystallization screening, which yielded a new crystal form diffracting to 1.1 Å resolution; the other compounds were successfully evaluated in soaking experiments. All compounds revealed the expected binding mode as observed for the hydrolyzed metabolites of 4a, 4b, and 5a, with the succinimide moiety forming the canonical interaction within the binding pocket, and the amide linker forming the hydrogen bond with N50. Besides these hydrogen bonds of the binding and linking moiety, no defined interactions with the protein were found for the protruding moieties of any of the five compounds, including the best binder 7d. Figure 6 shows a superposition of the compounds of this panel and their conserved binding mode in the aromatic cage.

Figure 6

Figure 6. Binding modes of compounds from the second and third panel. (A) Superposition of all compounds bound to MsCI4. (B) Detailed side and top view of bound effectors based on the 3-amidosuccinimide scaffold: 7a (yellow, PDB 6R1X), 7b (sand, PDB 6R12), 7c (pink, PDB 6R1K), 7d (green, PDB 6R1D), 7f (blue, PDB 6R13), and the water soluble 11a (cyan, PDB 6R18), and 12a (light blue, PDB 6R1C), indicating interactions with N50. (C) Side and top view of compounds based on 3-amidoglutarimide, 16b (brown, PDB 6R1W) and 20b (dark green, PDB 6R1A). Although the depicted instances for this scaffold do not show the interaction with N50, this interaction was observed in one other instance for 16b.

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Based on these data, we hypothesized that the planar benzene connected via an oxygen as in 7d is favorable for the affinity. Consequently, we designed a third panel of derivatives carrying this feature based on succinimide and glutarimide (Figure 5B). In this panel, in addition to affinity improvement, we also sought to increase the solubility in water via additional polar or charged groups. As described in Scheme 3, reaction of the intermediates 8, 13, and 17 with phosgene solution (15 wt % in toluene) in THF yielded to the corresponding chloroformates 9, 14, and 18. Derivatives 11a, 11b, 16a, 16b, 20a, and 20b were prepared by a coupling reaction between the imides 3a or 3b and the aforementioned chloroformates 9, 14, and 18 using DIPEA in THF followed by deprotection using TFA. Treatment of the derivatives 11a and 11b with succinic anhydride and triethylamine (Et3N) in DMF produced the molecules 12a and 12b, respectively.

Scheme 3

Scheme 3. a

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aReagents and conditions: (a) Phosgene solution 15 wt % in toluene, THF, 0 oC to RT, 20 h; (b) 3a or 3b, DIPEA, THF, 0 oC to reflux, 20 h; (c) TFA, DCM, 0 oC to RT, 2 h; (d) Et3N, DMF, 0 oC to RT, 20 h. Note that 13 and thereby also 14–16 are in cis conformation.

Indeed, the final compounds with terminal amino groups were water-soluble (11a, 11b, 16a, 16b), while 12a and 12b, with a terminal succinyl group, were highly soluble in bicarbonate buffer (>200 mM). The compounds were subsequently tested in the FRET assay, apart from 20a, which still contained impurities (see Methods). The assay indicated that all derivatives retained high affinity for CRBN independent of the binding moiety and planarity of the substituent; a clear preference for either binding moiety was not recognizable.
In soaking experiments, we obtained crystal structures with the 5-ring members 11a, 12a, and 20a, and the 6-ring members 16b, and 20b, all forming the canonical interactions within the aromatic cage. As expected, the 5-ring effectors form the interaction of the amide linker with N50, which is also observed in one instance for the 6-ring effector 16b. Further interactions of the protruding moieties are not observed for any of the compounds. Consequently, the prolonged extensions are less resolved in the electron density map, which is especially evident for 12a (Figure 7). As these prolonged compounds still retain high affinity, this confirms that the amidosuccinimide scaffold can serve as the universal CRBN-binding moiety, allowing great chemical variability on the protruding moiety.

Figure 7

Figure 7. FO–FC maps of bound compounds shown in Figure 6. All compounds are clearly defined by their electron density maps with the exception of the prolonged extension in 12a. Crystallographic structures were refined to resolutions between 1.1 and 1.8 Å, and the maps are contoured at 2σ. PDB accession codes are 6R1X (7a), 6R12 (7b), 6R1K (7c), 6R1D (7d), 6R13 (7f), 6R18 (11a), 6R1C (12a), 6R1W (16b), 6R19 (20a), and 6R1A (20b).

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Degradation of Neo-Substrates

After assessing the biophysical and structural parameters of our designs, we tested their potential for the degradation of neo-substrates in the MM-derived human cell line OPM-2. To this end, we have selected the established neo-substrates IKZF3, which is targeted via a variety of IMiDs (20,47−49) and CK1α, which is so far only targeted via lenalidomide, (21) as two complementary targets. We treated OPM-2 cells for 24 h with the different compounds and assayed for the endogenous levels of both neo-substrates; for comparison, the classical IMiDs, thalidomide, lenalidomide, and pomalidomide were included in the test set. As anticipated, the results for the two neo-substrates were very different. None of the marketed IMiDs apart from lenalidomide was able to reduce the levels of CK1α significantly, (21) but also none of our compounds showed any effect on CK1α. This indicates that our compounds could neither supersede nor sufficiently mimic the interface for CK1α recruitment formed by lenalidomide, substantiating the notion of a very narrow specificity window for this substrate. (21)
However, the situation was completely different for IKZF3. Here, in addition to all classical IMiDs, multiple of our designs were successful: significant effects were observed for 5a, 7d, and 7f, which are all compounds with a rather compact structure from our first and second panel (Figure 8). The only successful compound from the first, phthaloyl-based panel, 5a, represents a direct succinimide analogue of pomalidomide. It reduced IKZF3 levels by almost 40%. Comparison of its structural binding mode to that of thalidomide or pomalidomide already suggested that it may be a possible functional substitute for pomalidomide. The fact that the other compounds of that panel did not show similar effects indicate that substitutions larger than an amino group in the R2 position of the phthaloyl moiety, as for 4a and 4c, or any substitution in R1, as for 4b, 4d, and 5b, abolish IKZF3 recruitment.

Figure 8

Figure 8. Compound-mediated IKFZ3 degradation in OPM-2 cells. (A) Immunoblot analysis of IKZF3 levels after treatment with compounds 7d, 5a, and 7f (100 μM) for 24 h, compared to DMSO (control) and lenalidomide (20 μM) (n = 3). Lenalidomide at 100 μM reduced IKFZ3 levels effectively by 100% (not shown). (B) Averaged IKZF3 levels from the three independent experiments, normalized against total protein loading control (Figure S2). The significance of the data was tested comparing IKZF3 levels in the presence of the compounds and DMSO (p > 0.05).

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Most interesting is the analysis of the successful compounds of the second panel, 7d and 7f, which are not derived from classical IMiDs but from the hydrolysis products of the first panel. 7d, which is also the highest affinity binder in this study, reduced IKZF3 levels by about 20%. Moreover, the analogous 7f, which is a weaker binder that only differs from 7d in one heavy atom in the linker, reduced IKZF3 levels by even 40%. Both compounds are more flexible and elongated than 5a, so it appears impossible that their protruding moieties adopt a conformation mimicking the classical IMiDs at the interface between CRBN and IKZF3. Their terminal benzyl groups inevitably project further away from the thalidomide-binding pocket, requiring another mode of interaction with the zinc finger motif than characterized for classical IMiDs. (26) This interaction mode is also very sensitive to chemical changes: shortening of the linker by only one atom, as for 7b, further modifications on the benzyl group, as for 11a or 12a, or any other variant we designed in the second or third panel abolished IKZF3 recruitment, which is indicative for a highly specific interaction. Therefore, 7d and 7f are the first representatives of a novel type of CRBN effectors with a recognition mode that is clearly distinct from that of the classical thalidomide-based IMiDs.

Conclusions

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The development of CRBN effectors is a rapidly growing field, and novel IMiDs and PROTACS are reported frequently. To date, essentially all of these compounds are based on the classical thalidomide scaffold, which significantly restrains the chemical space available for the recruitment of neo-substrates. Here, following on from our previous characterization of CRBN-binding moieties, we have probed the chemical space for the linking and protruding moiety, taking advantage of the structural binding modes of hydrolyzed metabolites. Although not in the focus of this work, the apparent specificity of CRBN for these hydrolysis products, especially for the major thalidomide metabolite CBG, may inspire further research toward the understanding of their pharmacological relevance.
As a consensus from the binding modes of our initial designs and their hydrolysis products, we derived an amidosuccinimide scaffold as a minimal binding and linking moiety. 3-Amidosuccinimide can be used to mount almost arbitrary chemistry as the protruding moiety, while retaining affinities to CRBN in the range of classical IMiDs, rendering it an attractive CRBN-binding moiety for future PROTACS.
However, the most relevant aspect of our study concerns the versatility of the 3-amidosuccinimide scaffold for the design of IMiD-like CRBN effectors. As a minimal binding and linking moiety, it does not restrain the chemical space of protruding moieties, allowing for the design of substrate-recruiting motifs that cannot be realized on a classical IMiD scaffold. In our attempt to probe the effect of different protruding moieties mounted on the amidosuccinimide scaffold, two out of twelve compounds were able to recruit the neo-substrate IKZF3 without further optimization. Although we do not have structural insight into the IKZF3-recognition mode of these effectors, it is clearly incompatible with that of the classical IMiDs, which points at a large unexplored chemical space for the recruitment of new therapeutic targets. For the ongoing characterization of the proteome druggable via the IMiD approach, novel CRBN effectors are needed both for probing the space of neo-substrates, as well as for future pharmacological exploitation. With this work, we contribute first steps toward the rational design of a post-thalidomide generation of such effectors, toward unlocking the full potential of the IMiD approach.

Experimental Section

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Chemistry

General Procedures

Room temperature (RT) refers to 22 °C. Reagents and anhydrous solvents were transferred via an oven-dried syringe or a cannula. Flasks were flame-dried under vacuum and cooled under a constant stream of argon. Anhydrous solvents (toluene, dioxane, and DMF) were purchased from Sigma-Aldrich (anhydrous over molecular sieves). THF was dried over potassium. All other chemicals were purchased from ABCR, Acros, Alfa Aesar, Fluorochem, Merck, Sigma-Aldrich, and TCI Europe at highest commercially available purity and used without further purification. Compound 2b and 3b were prepared according to the procedures reported by Capitosti et al. (50) Compounds 8, 13, and 17 were prepared according to the procedures previously reported. (51−54) Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F 254 TLC aluminium sheets and visualized by ultraviolet light (254 nm) and/or with ceric ammonium molybdate, potassium permanganate, or ninhydrine staining solution. Flash column chromatography was performed on Acros silica gel 35–70, 60 Å, using a forced flow of eluent (method of Still). Yields refer to chromatographically purified and spectroscopically pure compounds. NMR spectra were recorded on Varian Mercury plus 400 (operating at 400 MHz for 1H and 100 MHz for 13C acquisitions), and Varian Mercury plus 300 (operating at 300 MHz for 1H and 75 MHz for 13C acquisitions). Chemical shifts are reported in ppm with the solvent resonance as the internal standard (d1-chloroform: 7.26 (1H NMR), 77.16 (13C NMR); d6-dimethylsulfoxide: 2.50 (1H NMR), 39.52 (13C NMR); (d4-methanol: 3.31 (1H NMR), 49.00 (13C NMR); [d6-acetone: 2.05 (1H NMR), and 29.84, 206.26 (13C NMR)]. Coupling constants J are reported in Hertz (Hz). Multiplicities are indicated by s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, dd = doublet of doublet, dt = doublet of triplet, m = multiplet, and br = broad resonance. High-resolution mass spectra were obtained on Bruker Daltonics ESI-FT-ICR-MS APEX II. IR spectra were measured on ATI/Mattson Genesis FT-IR as thin film (in CCl4) or KBr-disk. Absorbance frequencies are reported in reciprocal centimeters (cm–1). Melting points were measured on a Boetius-micro hot stage and are uncorrected. The purity of compounds was analyzed by detecting UV absorbance at 254 nm using a Poroshell 120 EC-C18 column on a 1260 Infinity II system (Agilent Technologies, Inc.) (Figure S1). All compounds showed >95% purity with the exception of 7a (87.6%), 7b (91.5%), 7c (88.2%). 12a (89.4%), 12b (88.2%), and 20a (<50%).
General Procedure A
Preparation of the compounds 4a–d: to a solution of 3a or 3b (1.0 equiv) in anhydrous THF (4.60 mL/mmol), 4-nitrophthalic anhydride (1.5 equiv) in anhydrous THF (0.5 mL/mmol) was added. CDI (1.7 equiv), Et3N (2.9 equiv), and catalytic amounts of 4-DMAP were added, and the reaction mixture was heated to reflux for 5 h. The solvent was evaporated, and the resulting crude oil was purified by column chromatography to give 4a–d.
General Procedure B
Reduction of the nitro group: to a solution of 4a or 4b (1.0 equiv) in EtOAc (100 mL/mmol), 10 wt % Pd/C (27 mol %) was added. The flask was degassed and let to stir under a hydrogen atmosphere for 20 h at RT. After completion of the reaction (TLC control), the mixture was filtered over Celite and washed with copious amounts of acetone. Afterward, the solvent was evaporated and 5a or 5b was obtained.
General Procedure C
Preparation of the compounds 7a–h (amide bond formation): to a solution of the TFA-salt 3a (1.0 equiv) in THF (5 mL/mmol), DIPEA (2.0 equiv) was added. The mixture was cooled down to 0 °C, the acyl chloride 6a–h (1.0 equiv) was added, and the reaction mixture was left to stir under reflux for 2 h. Upon completion, the reaction was diluted with EtOAc and washed with 3 M HCl and brine. The organic layer was dried over Na2SO4, and the solvent was evaporated in vacuo. The resulting crude product was purified by flash column chromatography to give 7a–h.
General Procedure D
Phosgenation, carbamate formation, and Boc deprotection: to a 15% wt solution of COCl2 in toluene (1.3 equiv) was added dry THF (0.58 mL/mmol) under argon. The solution was cooled to 0 °C, and a solution of 8, 13, or 17 (1.0 equiv) in dry THF (0.6 mL/mmol) was added over 10 min. The reaction mixture was stirred at 0 °C for 15 min and then warmed up to RT and stirred for two more hours. After completion of the reaction, the volatiles were completely evaporated under reduced pressure to give the chloroformates 9, 14, or 18, respectively, which were used for the next step without further purification. To a stirred suspension of the TFA-salt 3a or 3b (1.03 equiv) in dry THF (2 mL/mmol), DIPEA (0.35 mL/mmol) was added at 0 °C. Then, a solution of the chloroformate 9, 14, or 18 (1.0 equiv) in dry THF (0.35 mL/mmol) was added gradually to the suspension. The reaction was warmed up to RT, stirred for 30 min at RT, and then refluxed overnight. After completion of the reaction, the solvent was evaporated, and the crude product was purified by column chromatography giving the carbamates 10a–b, 15a–b, or 19 respectively. To a solution of 10a–b, 15a–b, or 19 (1.0 equiv) in DCM (4.5 mL/mmol), TFA (30.0 equiv) was added at 0 °C. The reaction solution was warmed up to RT and stirred for 2 h. After completion of the reaction, the volatiles were completely evaporated to give 11a–b, 16a–b, or 20 respectively.
General Procedure E
Preparation of 12a–b (amide bond formation): derivatives 11a–b (1.0 equiv) were dissolved in DMF (2.8 mL/mmol), and Et3N (2.2 equiv) was added at 0 °C. After 20 min of stirring, succinic anhydride (1.0 equiv) was added, and the reaction mixture was stirred at RT overnight. After the end of the reaction, the solvent was evaporated, and the corresponding acid 12a–b was afforded via flash column chromatography.

tert-Butyl (S)-(2,5-Dioxopyrrolidin-3-yl)carbamate (2a)

A mixture of N-Boc-L-asparagin (2 g, 8.61 mmol, 1 equiv), CDI (1.39 g, 8.61 mmol, 1 equiv), and catalytic amounts 4-DMAP in anhydrous THF (21.5 mL) was stirred and heated to reflux for 48 h. After the end of the reaction, the solvent was evaporated, and the crude product was purified by column chromatography (n-hexane/ethyl acetate, 1:1 v/v) to give 2a (1.54 g, 6.75 mmol, 78%) as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.48; mp 160–162 °C; IR (KBr) ν̃max: 3376.75, 3361.32, 1709.59, 1521.56, 1171.54 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 11.18 (s, 1H), 7.42 (d, J = 8.1 Hz, 1H), 4.33–4.24 (m, 1H), 2.85 (dd, J = 17.5, 9.4 Hz, 1H), 2.44 (dd, J = 17.5, 5.7 Hz, 1H), 1.37 (s, 9H); 13C NMR (101 MHz, DMSO): δ 178.2, 176.5, 155.2, 78.8, 50.3, 36.4, 28.2; HRMS ESI (m/z): [M + Na]+ calcd for C9H14N2O4Na+, 237.08513; found, 237.08513.

2,5-Dioxopyrrolidin-3-aminium 2,2,2-Trifluoroacetate (3a)

Compound 3a (1.54 g, 6.75 mmol, 1 equiv) was dissolved in TFA (7.8 mL, 101.25 mmol, 15 equiv) and stirred for 30 min. The excess of the acid was removed in vacuo, and the resulting product was dried under vacuum to give 3a (1.54 g, 6.75 mmol, quant) as an off-white solid. TLC (EtOAc): Rf = 0.0; mp 109–114 °C; IR (KBr) ν̃max: 3446.17, 1665.23, 1617.02, 1593.88, 1207.22, 1181.19, 1149.37, 1137.8, 723.175 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 11.71 (s, 1H), 8.61 (s, 3H), 4.39–4.26 (m, 1H), 2.95 (d, J = 26.9 Hz, 1H), 2.61 (dd, J = 17.7, 5.5 Hz, 1H); 13C NMR (101 MHz, DMSO-d6): δ 175.29, 175.14, 158.69 (q, J = 31.8 Hz), 117.08 (q, J = 298.4 Hz), 48.53, 34.73; 19F NMR (377 MHz, DMSO-d6): δ −73.84; HRMS ESI (m/z): [M + H]+ calcd for C4H7N2O2+, 115.05075; found, 115.05142.

2-(2,5-Dioxopyrrolidin-3-yl)-4-nitroisoindoline-1,3-dione (4a)

Compound 4a was synthesized according to general procedure A using 3b (0.50 g, 2.19 mmol), 3-nitrophthalic anhydride (655.7 mg, 3.29 mmol), CDI (603.2 mg, 3.72 mmol), Et3N (0.89 mL, 6.35 mmol), and catalytic amounts of 4-DMAP. After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v), compound 4a (412 mg, 1.43 mmol 65%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 1:3 v/v): RF = 0.72; mp 129–131 °C; IR (KBr) ν̃max: 3442.31, 1722.12, 1541.81, 1396.21 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.55 (br s, 1H), 8.35–8.28 (m, 1H), 8.28–8.21 (m, 1H), 8.22–8.15 (m, 1H), 5.47 (dd, J = 9.7, 5.9 Hz, 1H), 3.21 (dd, J = 18.0, 9.7 Hz, 1H), 3.05 (dd, J = 18.0, 5.9 Hz, 1H); 13C NMR (101 MHz, acetone-d6): δ 175.2, 174.9, 165.9, 163.3, 146.0, 137.4, 134.6, 129.7, 128.0, 124.1, 49.5, 35.2; HRMS ESI (m/z): [M + Na]+ calcd for C12H7N3O6Na+, 312.02325; found, 312.04880.

2-(2,5-Dioxopyrrolidin-3-yl)-5-nitroisoindoline-1,3-dione (4b)

Compound 4b was synthesized according to general procedure A using 3a (0.5 g, 2.19 mmol), 4-nitrophthalic anhydride (656 mg, 3.29 mmol), CDI (603 mg, 3.72 mmol), Et3N (0.89 mL, 6.35 mmol), and catalytic amounts of 4-DMAP. After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v) compound 4b (483 mg, 1.67 mmol 76%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 1:3 v/v): RF = 0.61; mp 203–204 °C; IR (KBr) ν̃max: 3489.56, 1787.69, 1538.92, 1399.1, 1349.93, 722.211 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.55 (br s, 1H), 8.75 (dd, J = 8.2, 2.0 Hz, 1H), 8.62 (dd, J = 2.0, 0.5 Hz, 1H), 8.21 (dd, J = 8.2, 0.5 Hz, 1H), 5.50 (dd, J = 9.7, 5.9 Hz, 1H), 3.23 (dd, J = 18.0, 9.7 Hz, 1H), 3.06 (dd, J = 18.0, 5.9 Hz, 1H); 13C NMR (101 MHz, acetone-d6): δ 175.2, 174.9, 166.3, 166.0, 153.1, 137.2, 134.1, 130.8, 125.8, 119.3, 49.5, 35.3; HRMS ESI (m/z): [M + Na + MeOH]+ calcd for C13H11N3O7Na+, 344.04947; found, 344.04985.

2-(2,6-Dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (4c)

Compound 4c was synthesized according to the general procedure A using 3b (3.11 g, 12.9 mmol), 3-nitrophthalic anhydride (3.73 g, 19.3 mmol), CDI (3.55 g, 21.9 mmol) Et3N (5.2 mL, 37.3 mmol), and catalytic amounts of 4-DMAP. After purification via flash column chromatography (n-hexane/EtOAc, 1:1–1:4 v/v), compound 4c (3.48 g, 11.5 mmol, 89%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): RF = 0.40; 1H NMR (400 MHz, DMSO-d6): δ 11.17 (s, 1H), 8.34 (dd, J = 8.1, 0.7 Hz, 1H), 8.23 (dd, J = 7.5, 0.7 Hz, 1H), 8.15–8.08 (m, 1H), 5.19 (dd, J = 12.9, 5.4 Hz, 1H), 2.88 (ddd, J = 17.3, 13.9, 5.4 Hz, 1H), 2.61 (dt, J = 17.0, 2.7 Hz, 1H), 2.56–2.44 (m, 1H), 2.08 (dtd, J = 12.9, 5.3, 2.1 Hz, 1H); 13C NMR (101 MHz, DMSO): δ 172.8, 169.6, 165.3, 162.6, 144.5, 136.9, 133.1, 129.0, 127.4, 122.6, 49.5, 30.9, 21.8; HRMS ESI (m/z): [M + Na + MeOH]+ calcd for C14H13N3O7Na+, 358.06512; found, 358.06703; spectral data were consistent with the literature. (55)

2-(2,6-Dioxopiperidin-3-yl)-5-nitroisoindoline-1,3-dione (4d)

Compound 4d was synthesized according to the general procedure A using 3b (4.17 g, 17.2 mmol), 4-nitrophthalic anhydride (4.99 g, 25.9 mmol), CDI (4.73 g, 29.1 mmol) Et3N (6.97 mL, 50.00 mmol), and catalytic amounts of 4-DMAP. After purification via flash column chromatography (n-hexane/EtOAc, 1:1–1:4 v/v), compound 4d (2.35 g, 7.8 mmol, 45%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.50; 1H NMR (400 MHz, DMSO-d6): δ 11.18 (s, 1H), 8.67 (dd, J = 8.2, 2.0 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 5.23 (dd, J = 12.9, 5.4 Hz, 1H), 2.90 (ddd, J = 17.1, 13.9, 5.4 Hz, 1H), 2.63 (ddd, J = 17.1, 4.3, 2.3 Hz, 1H), 2.58–2.47 (m, 1H), 2.10 (dtd, J = 12.9, 5.2, 2.2 Hz, 1H); 13C NMR (101 MHz, DMSO): δ 172.82, 169.61, 165.63, 165.36, 151.79, 135.80, 132.60, 130.17, 125.09, 118.46, 49.57, 30.95, 21.91; HRMS ESI (m/z): [M + Na + MeOH]+ calcd for C14H13N3O7Na+, 358.06512; found, 358.06642; spectral data were consistent with the literature. (50)

4-Amino-2-(2,5-dioxopyrrolidin-3-yl)isoindoline-1,3-dione (5a)

Compound 5a was synthesized according to the general procedure B using 4a (0.30 g, 1.04 mmol) and 10 wt % Pd/C (0.30 g, 0.28 mmol). Compound 5a (0.25 g, 0.95 mmol, 92%) was obtained as a yellow-green solid. TLC (n-hexane/EtOAc, 1:3 v/v): Rf = 0.61; mp 292–295 °C; IR (KBr) ν̃max: 3474.13, 1727.91, 1702.84, 1632.45 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.45 (br s, 1H), 7.50 (dd, J = 8.4, 7.1 Hz, 1H), 7.07 (dd, J = 10.7, 7.7 Hz, 2H), 6.17 (s, 1H), 5.34 (dd, J = 9.7, 5.9 Hz, 1H), 3.18 (dd, J = 17.9, 9.7 Hz, 1H), 3.02 (dd, J = 17.9, 5.9 Hz, 1H); 13C NMR (101 MHz, acetone-d6): δ 175.7, 175.5, 170.9, 170.9, 147.8, 136.4, 133.4, 122.5, 122.4, 112.4, 48.7, 35.6; HRMS ESI (m/z): [M + Na]+ calcd for C12H9N3O4Na+, 282.04908; found, 282.04853.

5-Amino-2-(2,5-dioxopyrrolidin-3-yl)isoindoline-1,3-dione (5b)

Compound 5b was synthesized according to the general procedure B using 4a (0.30 g, 1.0 mmol) and 10 wt % Pd/C (0.30 g, 0.3 mmol). Compound 5b (0.26 g, 1.0 mmol, 97%) was obtained as a yellow-green solid. TLC (n-hexane/EtOAc, 1:3 v/v): RF = 0.68; mp 259–260 °C; IR (KBr) ν̃max: 3473.17, 3354.57, 1701.87, 1628.59, 1613.16, 1405.85 cm–1; 1H NMR (300 MHz, acetone-d6): δ 10.40 (br s, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 1.8 Hz, 1H), 6.97 (dd, J = 8.2, 2.1 Hz, 1H), 6.00 (br s, 2H), 5.32 (dd, J = 9.7, 6.0 Hz, 1H), 3.16 (dd, J = 17.9, 9.7 Hz, 1H), 2.98 (dd, J = 17.9, 6.0 Hz, 1H); 13C NMR (75 MHz, acetone-d6): δ 175.8, 175.6, 155.9, 135.6, 126.0, 118.8, 118.4, 108.5, 48.8, 35.6; HRMS ESI (m/z): [M + Na]+ calcd for C12H9N3O4Na+, 282.04908; found, 282.04853.

N-(2,5-Dioxopyrrolidin-3-yl)-3-methylbut-2-enamide (7a)

Compound 7a was synthesized according to the general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 mL, 0.88 mmol), and 3, 3-dimethylacryloyl chloride 6a (51.9 mg, 0.44 mmol). After purification via flash column chromatography, (n-hexane/EtOAc, 5:5 v/v) compound 7a (56.8 mg, 0.29 mmol, 66%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.18; mp 181–188 °C; IR (KBr) ν̃max: 3354.57, 1729.83, 1715.37, 1624.73, 1530.24, 1363.43, 1261.22, 1193.72, 1177.33 cm–1; 1H NMR (300 MHz, acetone-d6): δ 10.04 (s, 1H), 7.63 (s, 1H), 5.72 (s, 1H), 4.54 (ddd, J = 9.3, 7.5, 5.8 Hz, 1H), 2.97 (dd, J = 17.6, 9.3 Hz, 1H), 2.71 (dd, J = 17.6, 5.8 Hz, 1H), 2.11 (d, J = 1.2 Hz, 3H), 1.82 (d, J = 1.2 Hz, 3H); 13C NMR (75 MHz, acetone-d6): δ 176.0, 172.1, 167.3, 152.3, 118.6, 51.1, 37.2, 27.1, 19.7; HRMS ESI (m/z): [M + Na]+ calcd for C9H12N2O3Na+, 219.07456; found, 219.07401.

N-(2,5-Dioxopyrrolidin-3-yl)-2-phenylacetamide (7b)

Compound 7b was synthesized according to the general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 mL, 0.876 mmol), and phenylacetyl chloride 6b (67.7 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v), compound 7b (75.9 mg, 0.33 mmol, 75%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.15; mp 181–184 °C; IR (KBr) ν̃max: 3502.1, 3259.11, 1731.76, 1715.35, 1654.62, 1542.77, 1358.6, 1196.61, 1172.51 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.02 (br s, 1H), 7.81 (br s, 1H), 7.35–7.18 (m, 5H), 4.57 (ddd, J = 9.3, 7.5, 5.8 Hz, 1H), 3.56 (s, 2H), 2.96 (dd, J = 17.6, 9.4 Hz, 1H), 2.66 (dd, J = 17.6, 5.8 Hz, 1H); 13C NMR (101 MHz, acetone-d6): δ 176.3, 175.0, 170.7, 135.7, 129.2, 128.3, 126.6, 50.4, 42.2, 36.2; HRMS ESI (m/z): [M + Na]+ calcd for C12H12N3O6Na+, 255.07456; found, 255.07401.

2,4,6-Trichloro-N-(2,5-dioxopyrrolidin-3-yl)benzamide (7c)

Compound 7c was synthesized according to the general procedure C using 3a (0.10 g, 0.438 mmol), DIPEA (0.15 mL, 0.88 mmol), and 2,4,6-trichlorobenzoyl chloride 6c (106.8 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 5:5 v/v), compound 7c (81.7 mg, 0.25 mmol, 58%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): RF = 0.47; mp 210–212 °C; IR (KBr) ν̃max: 3450.03, 3249.47, 1717.3, 1640.16, 1582.31, 1547.59 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 11.35 (s, 1H), 9.37 (d, J = 7.5 Hz, 1H), 7.75 (s, 1H), 4.59 (ddd, J = 9.3, 7.6, 5.3 Hz, 1H), 3.01 (dd, J = 17.6, 9.4 Hz, 1H), 2.59 (dd, J = 17.6, 5.3 Hz, 1H); 13C NMR (101 MHz, DMSO): δ 176.5, 176.3, 163.3, 134.9, 134.3, 132.3, 128.1, 50.0, 35.9; HRMS ESI (m/z): [M + Na]+ calcd for C11H7Cl3N2O3Na+, 342.94199; found, 342.94145.

Benzyl (2,5-Dioxopyrrolidin-3-yl)carbamate (7d)

Compound 7d was synthesized according to the general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 mL, 0.876 mmol), and benzyl chloroformate 6d (74.7 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 5:5 v/v), compound 7d (105.9 mg, 0.43 mmol, 98%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.44; mp 93–96 °C; IR (KBr) ν̃max: 3363.25, 1718.26, 1532.17, 1267.97, 1203.36, 1177.33 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.09 (br s, 1H), 7.41–7.27 (m, 5H), 7.08 (br s, 1H), 5.10 (s, 2H), 4.63–4.53 (m, 1H), 3.05 (dd, J = 17.6, 9.4 Hz, 1H), 2.74 (dd, J = 17.6, 5.5 Hz, 1H); 13C NMR (101 MHz, acetone-d6): δ 177.5, 175.8, 156.9, 137.8, 129.2, 128.7, 67.1, 52.0, 37.1; HRMS ESI (m/z): [M + Na]+ calcd for C12H12N2O4Na+, 271.06948; found, 271.06893.

N-(2,5-Dioxopyrrolidin-3-yl)-3,5-dinitrobenzamide (7e)

Compound 7e was synthesized according to the general procedure C using 3a (0.1 g, 0.44 mmol), DIPEA (0.15 mL, 0.88 mmol), and 3,5-dinitrobenzoyl chloride 6e (0.10 g, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 5:5 v/v), compound 7e (0.10 g, 0.34 mmol, 76%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.28; mp 238–239 °C; IR (KBr) ν̃max: 3447.16, 3389.28, 1717.3, 1659.45, 1540.85, 1348, 1188.9 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.24 (br s, 1H), 9.15 (d, J = 7.1 Hz, 1H), 9.12–9.06 (m, 3H), 4.99–4.91 (m, 1H), 3.20–3.11 (m, 1H), 2.93 (dd, J = 17.8, 5.8 Hz, 1H); 13C NMR (101 MHz, acetone): δ 176.7, 175.7, 163.7, 149.7, 137.6, 128.3, 122.1, 51.8, 36.8; HRMS ESI (m/z): [M + Na]+ calcd for C11H8N4O7Na+, 331.02907; found, 331.02842.

N-(2,5-Dioxopyrrolidin-3-yl)cinnamamide (7f)

Compound 7f was synthesized according to the general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 mL, 0.88 mmol), and cinnamoyl chloride 6f (73.0 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v), compound 7f (102 mg, 0.42 mmol, 96%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.19; mp 213–214 °C; IR (KBr) ν̃max: 3431.71, 1718.26, 2654.62, 1617.02, 1522.52, 1191.79 cm–1; 1H NMR (300 MHz, acetone-d6): δ 10.11 (s, 1H), 8.01 (d, J = 6.2 Hz, 1H), 7.71–7.33 (m, 6H), 6.71 (d, J = 15.8 Hz, 1H), 4.76–4.63 (m, 1H), 3.10–2.97 (m, 1H), 2.80 (dd, J = 17.6, 5.9 Hz, 1H); 13C NMR (75 MHz, acetone-d6): δ 177.3, 175.9, 166.4, 141.5, 136.0, 130.5, 129.7, 128.6, 121.5, 51.5, 37.2; HRMS ESI (m/z): [M + Na]+ calcd for C13H12N2O3Na+, 267.07456; found, 267.07401.

3-Chloro-N-(2,5-dioxopyrrolidin-3-yl)benzo[b]thiophene-2-carboxamide (7g)

Compound 7g was synthesized according to the general procedure C using 3a (100 mg, 0.44 mmol), DIPEA (0.15 mL, 0.88 mmol), and 3-chlorobenzo[b]thiophen-2-carbonyl chloride 6g (101 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v), compound 7g (128 mg, 0.41 mmol, 94%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.46; mp 215–218 °C; IR (KBr) ν̃max: 3445.21, 2932.23, 1729.83, 1636.3, 1534.1 cm–1; 1H NMR (400 MHz, acetone-d6): δ 10.18 (br s, 1H), 8.47 (br d, J = 6.6 Hz, 1H), 8.10–7.98 (m, 1H), 8.00–7.90 (m, 1H), 7.66–7.55 (m, 2H), 4.99 (ddt, J = 10.4, 7.2, 5.2 Hz, 1H), 3.14 (ddd, J = 17.7, 9.5, 3.5 Hz, 1H), 2.92 (dd, J = 17.7, 6.0 Hz, 1H); 13C NMR (101 MHz, acetone-d6): δ 176.8, 175.8, 161.4, 138.7, 137.7, 133.0, 128.7, 126.7, 124.0, 123.8, 120.6, 51.8, 37.1; HRMS ESI (m/z): [M + Na]+ calcd for C13H9ClN2O3SNa, 303.991201; found, 303.99146; [2M + Na]+ calcd for C26H18Cl2N4O6S2Na+, 638.99425; found, 638.99370.

3,4-Dichloro-N-(2,5-dioxopyrrolidin-3-yl)benzo[b]thiophene-2-carboxamide (7h)

Compound 7h was synthesized according to the general procedure C using 3a (100 mg, 0.44 mmol), DIPEA (0.15 mL, 0.88 mmol), and 3,4-dichlorobenzo[b]thiophene-2-carbonyl chloride 6h (116 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v), compound 7h (63.6 mg, 0.27 mmol, 62%) was isolated as a light-yellow solid. TLC (n-hexane/EtOAc, 3:7 v/v): RF = 0.46; mp 159–162 °C; IR (KBr) ν̃max: 3396.03, 3192.58, 1135.62, 1620.88, 1532.17, 1193.72.755.244 cm–1; 1H NMR (300 MHz, DMSO-d6): δ 11.35 (s, 1H), 9.10 (d, J = 7.8 Hz, 1H), 8.10 (dd, J = 7.9, 1.2 Hz, 1H), 7.62–7.48 (m, 2H), 4.83–4.73 (m, 1H), 2.99 (dd, J = 17.6, 9.3 Hz, 1H), 2.67 (dd, J = 17.5, 5.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): δ 177.1, 176.4, 160.3, 139.6, 133.7, 130.6, 128.4, 128.1, 123.1, 118.5, 50.4, 36.1, 30.8; HRMS ESI (m/z): [M + Na]+ calcd for C13H8Cl2N2O3SNa+, 364.95304; found, 364.95249.

tert-Butyl (4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)benzyl)carbamate (10a)

Compound 10a was synthesized according to the general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 mL, 17.29 mmol), a solution of 8 (3.15 g, 13.3 mmol) in THF was added. After evaporation of the volatiles, compound 9 was produced. By using 3a (2.8 g, 12.4 mmol), DIPEA (4.0 mL, 23.0 mmol), and the chloroformate 9 (3.6 g, 12.0 mmol), compound 10a was synthesized. After purification via flash column chromatography (DCM/MeOH 0–2% v/v), compound 10a (4.3 g, 11.4 mmol, 95%) was isolated as a white solid. TLC (DCM/MeOH, 5:0.3 v/v): Rf = 0.81; mp 80–82 °C; IR (KBr) ν̃max: 3412, 1718, 1528, 1267, 1170 cm–1; 1H NMR: (300 MHz, CDCl3): δ 9.32 (s, 1H), 7.21 (q, J = 8.0 Hz, 4H), 6.20 (s, 1H), 5.14 (s, 1H), 5.02 (s, 2H), 4.24 (s, 3H), 2.90 (d, J = 8.8 Hz, 1H), 2.71 (d, J = 17.6 Hz, 1H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 28.5, 36.7, 44.3, 51.3, 67.3, 79.9, 127.5, 128.6, 134.9, 139.4, 156.3, 175.2, 177.0; HRMS ESI (m/z): [M + Na]+ calcd for C18H23N3O6Na+, 400.14850; found, 400.14782.

tert-Butyl (4-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)benzyl)carbamate (10b)

Compound 10b was synthesized according to the general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 mL, 17.3 mmol), a solution of 8 (3.15 g, 13.3 mmol) in THF was added. After evaporation of the volatiles, compound 9 was produced. By using 3b (3.0 mg, 12.4 mmol), DIPEA (4.0 mL, 23 mmol), and the chloroformate 9 (3.6 g, 12.0 mmol) compound 10b was synthesized. After purification via flash column chromatography (DCM/MeOH 0–2% v/v), compound 10b (4.23 g, 10.8 mmol, 90%) was isolated as a white solid. TLC (n-hexane/EtOAc, 3:7 v/v): RF = 0.16; mp 154.4 °C; IR (KBr) ν̃max: 3412.42, 3100.01, 2977.55, 2927.41, 2853.17, 1698.98, 1524.45 cm–1; 1H NMR: (300 MHz, CDCl3): δ 8.19 (s, 1H), 7.35–7.27 (m, 4H), 5.65 (s, 1H), 5.11 (s, 2H), 4.90 (s, 1H), 4.44–4.20 (m, 3H), 2.90–2.47 (m, 3H), 1.96–1.83 (m, 1H), 1.45 (s, 9H); 13C NMR: (75 MHz, CDCl3): δ 171.1, 171.0, 156.1, 155.9, 139.2, 135.0, 128.5, 127.6, 77.2, 67.0, 52.2, 44.4, 31.2, 28.4, 25.3; HRMS ESI (m/z): [M + Na]+ calcd for C19H25N3O6Na+, 414.16410; found, 414.16326.

(4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)phenyl)methanaminium 2,2,2-Trifluoroacetate (11a)

Compound 11a was synthesized according to the general procedure D using 10a (3.8 g, 10 mmol) and TFA (23 mL, 300 mmol). Upon completion, the volatiles were completely evaporated to give 11a (3.91 g, 10 mmol, quant) as a white solid. TLC (DCM/MeOH, 5:2 v/v): Rf = 0.26; mp 197–200 °C; IR (KBr) ν̃max: 3450, 1780, 1719, 1526, 1203, 1180 cm–1; 1H NMR: (400 MHz, DMSO-d6): δ 7.47 (s, 4H), 5.15 (s, 2H), 4.46 (dd, J = 9.3, 5.9 Hz, 1H), 4.13 (s, 2H), 3.02 (dd, J = 17.8, 9.3 Hz, 1H), 2.68 (dd, J = 17.8, 5.9 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 179.5, 177.9, 139.3, 134.2, 130.1, 129.5, 67.2, 52.4, 44.0, 37.3, 27.72; 19F NMR (282 MHz, DMSO-d6): δ −73.61.; HRMS ESI (m/z): [M + Na]+ calcd for C13H15N3O4Na+, 278.11353; found, 278.11345.

(4-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)phenyl)methanaminium 2,2,2-Trifluoroacetate (11b)

Compound 11b was synthesized according to the general procedure D using (3.91 g, 10 mmol) and TFA (23 mg, 300 mmol). Upon completion, the volatiles were completely evaporated to give 11b (4.05 g, 10 mmol, quant) as a white solid. TLC (DCM/MeOH, 5:2 v/v): RF = 0.3; mp 58–60 °C; IR (KBr) ν̃max: 3419.17, 3240.79, 3094.23, 1698.98, 1532.17, 1251.58, 1204.33, 1136.83 cm–1; 1H NMR: (300 MHz, DMSO): δ 7.54–7.38 (m, 4H), 5.24–5.11 (m, 2H), 4.45–4.35 (m, 1H), 4.12 (s, 2H), 2.87–2.61 (m, 2H), 2.24–1.94 (m, 2H); 13C NMR: (100 MHz, DMSO): δ 173.4, 172.4, 157.1, 138.1, 132.6, 128.7, 128.0, 65.6, 51.3, 42.6, 30.7, 24.4; 19F NMR (282 MHz, DMSO-d6): δ −73.70; HRMS (m/z): [M + H]+ calcd for C14H18N3O4+, 292.12973; found, 292.12885.

4-((4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)benzyl)amino)-4-oxobutanoic Acid (12a)

Compound 12a was synthesized according to the general procedure E using 11a (175 mg, 0.45 mmol), succinic anhydride (44.7 mg, 0.45 mmol), and Et3N (0.14 mL, 0.98 mmol). After purification via flash column chromatography, (EtOAc/IPA/H2O 4:2:1) compound 12a (128 mg, 0.34 mmol, 76%) was isolated as a white solid. TLC (EtOAc/iPA/H2O, 4:3:2 v/v): Rf = 0.45; mp 110 °C; IR (KBr) ν̃max: 3747.98, 3739.3, 2922.59, 1649.8, 1555.31, 1539.88, 1520.6, 1511.92 cm–1; 1H NMR (300 MHz, DMSO-d6): δ 8.53 (t, J = 5.8 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.25 (q, J = 8.1 Hz, 4H), 4.99 (s, 2H), 4.42–4.32 (m, 1H), 4.23 (d, J = 5.9 Hz, 2H), 2.94–2.83 (m, 1H), 2.53–2.43 (m, 1H), 2.37–2.25 (m, 4H); 13C NMR (101 MHz, DMSO-d6): δ 178.1, 176.5, 176.0, 172.6, 155.9, 139.7, 135.2, 128.1, 127.3, 65.8, 50.7, 41.9, 36.3, 32.1, 32.0; HRMS ESI (m/z): [M – H]− calcd for C17H18N3O7–, 376.11447; found, 376.11646.

4-((4-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)benzyl)amino)-4-oxobutanoic Acid (12b)

Compound 12b was synthesized according to the general procedure E using 11b (121 mg, 0.3 mmol), succinic anhydride (30.0 mg, 0.3 mmol), and Et3N (0.092 mL, 0.66 mmol). After purification via flash column chromatography, (EtOAc/IPA/H2O 4:2:1) compound 12a (117 mg, 0.3 mmol, quant) was isolated as a white solid. TLC (EtOAc/iPA/H2O, 4:3:2 v/v): Rf = 0.60; mp 129 °C; IR (KBr) ν̃max: 3443.28, 1712.48, 1700.91, 1555.31, 1539.88, 1522.52, 1202.4 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 10.80 (s, 1H), 8.49 (s, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.38–7.16 (m, 4H), 5.02 (s, 2H), 4.36–4.17 (m, 3H), 2.73 (ddd, J = 18.1, 11.8, 7.1 Hz, 1H), 2.47 (d, J = 3.7 Hz, 1H), 2.32 (dq, J = 12.3, 7.2, 6.5 Hz, 3H), 1.95 (ddt, J = 17.6, 13.4, 5.2 Hz, 2H); 13C NMR: (101 MHz, DMSO-d6): δ 173.1, 172.5, 172.3, 156.2, 139.5, 135.4, 127.9, 127.3, 65.5, 50.9, 41.9, 31.9, 31.8, 31.7, 31.0, 24.4; HRMS ESI (m/z): [M – H]− calcd for C18H20N3O7–, 390.13013; found, 390.13109.

((1s,4s)-4-((tert-Butoxycarbonyl)amino)cyclohexyl)methyl (2,5-Dioxopyrrolidin-3-yl)carbamate (15a)

Compound 15a was synthesized according to the general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 mL, 17.3 mmol), a solution of 13 (3.05 g, 13.3 mmol), in THF was added. After evaporation of the volatiles, compound 14 was produced. By using 3a (2.8 mg, 12.4 mmol), DIPEA (4 mL, 23 mmol), and the chloroformate 14 (3.5 g, 12 mmol), compound 15a was synthesized. After purification via flash column chromatography (DCM/MeOH 0–2% v/v), compound 15a (3.9 mg, 10.44 mmol, 87%) was isolated as a white solid. TLC (DCM/MeOH, 5:1 v/v): Rf = 0.71; mp 95–97 °C; IR (KBr) ν̃max: 3381, 1715, 1523, 1366, 1257, 1172 cm–1; 1H NMR: (400 MHz, CDCl3): δ 8.97 (s, 1H), 5.77 (s, 1H), 4.76 (s, 1H), 4.40 (s, 1H), 4.00 (s, 2H), 3.74 (s, 1H), 3.12 (dd, J = 18.8, 10.0 Hz, 1H), 2.88 (d, J = 18.0 Hz, 1H), 1.82–1.56 (m, 6H), 1.47 (s, 9H), 1.31 (s, 2H); 13C NMR (101 MHz, CDCl3): δ 177.0, 175.2, 156.5, 155.3, 79.2, 69.1, 51.3, 46.3, 36.7, 35.2, 29.2, 28.4, 24.3; HRMS ESI (m/z): [M + Na]+ calcd for C17H27N3O6Na+, 392.17976; found, 392.18342.

((1s,4s)-4-((tert-Butoxycarbonyl)amino)cyclohexyl)methyl (2,6-Dioxopiperidin-3-yl)carbamate (15b)

Compound 15b was synthesized according to the general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 mL, 17.3 mmol), a solution of 13 (3.05 g, 13.3 mmol) in THF was added. After evaporation of the volatiles, compound 14 was produced. By using 3b (3.0 mg, 12.4 mmol), DIPEA (4 mL, 23 mmol), and the chloroformate 14 (3.5 mg, 12 mmol), compound 15b was synthesized. After purification via flash column chromatography (DCM/MeOH 0–2% v/v), compound 15b (2.99 g, 7.8 mmol, 65%) was isolated as a white solid. TLC (DCM/MeOH, 5:2 v/v): Rf = 0.76; mp 133–137 °C; IR (KBr) ν̃max: 3379, 1703, 1523, 1247, 1198, 1171 cm–1; 1H NMR: (400 MHz, CDCl3): δ 8.33 (s, 1H), 5.57 (s, 1H), 4.50 (d, J = 130.1 Hz, 2H), 3.85 (d, J = 97.0 Hz, 3H), 2.91–2.32 (m, 3H), 1.91–1.13 (m, 18H); 13C NMR (101 MHz, CDCl3): δ 171.4, 171.3, 156.5, 155.4, 79.5, 69.0, 53.4, 46.4, 35.5, 31.3, 29.3, 28.5, 25.3, 24.3; HRMS ESI (m/z): [M + Na]+ calcd for C18H29N3O6Na+, 406.19541; found, 406.19381.

((1s,4s)-4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)cyclohexyl)methanaminium 2,2,2-Trifluoroacetate (16a)

Compound 16a was synthesized according to the general procedure D using 15a (3.7 g, 10 mmol) and TFA (23 mL, 300 mmol). Upon completion, the volatiles were completely evaporated to give 16a (3.83 g, 10 mmol, quant) as a white solid. TLC (DCM/MeOH, 5:1 v/v): Rf = 0.21; mp 74–76 °C; IR (KBr) ν̃max: 3447, 1783, 1717, 1629, 1529, 1267, 1203, 1182, 1138 cm–1; 1H NMR: (400 MHz, DMSO-d6): δ 11.23 (s, 1H), 7.90–7.71 (m, 4H), 4.53–4.21 (m, 1H), 3.98–3.86 (m, 3H), 3.16 (s, 1H), 2.88 (dd, J = 17.5, 9.4 Hz, 1H), 2.45 (d, J = 5.7 Hz, 1H), 1.87–1.35 (m, 9H); 13C NMR (75 MHz, DMSO): δ 178.3, 176.6, 156.4, 66.6, 50.9, 48.1, 36.6, 33.5, 26.4, 23.6; 19F NMR (282 MHz, DMSO-d6): δ −73.69; HRMS (m/z): [M + 2H]+ calcd for C12H20N3O4+, 270.14483; found, 270.14076.

((1s,4s)-4-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)cyclohexyl)methanaminium 2,2,2-Trifluoroacetate (16b)

Compound 16b was synthesized according to the general procedure D using 15b (3.8 g, 10 mmol) and TFA (23 mL, 300 mmol). Upon completion, the volatiles were completely evaporated to give 16b (3.97 g, 10.0 mmol, quant) as a white solid. TLC (DCM/MeOH, 5:1 v/v): RF = 0.20; mp 189–191 °C; IR (KBr) ν̃max: 3454, 1747, 1724, 1677, 1622, 1546, 1248, 1201, 1188, 1137 cm–1; 1H NMR: (400 MHz, DMSO-d6): δ 10.78 (s, 1H), 7.80 (s, 3H), 7.44 (s, 1H), 4.28 (s, 1H), 3.94 (dd, J = 16.4, 5.5 Hz, 3H), 3.19 (s, 1H), 2.73 (s, 1H), 2.00–1.40 (m, 11H); 13C NMR (101 MHz, DMSO): 173.4, 172.9, 156.8, 66.4, 51.2, 48.2, 33.6, 31.4, 26.4, 24.8, 23.6; 19F NMR (282 MHz, DMSO-d6): δ −73.96; HRMS (m/z): [M + 2H]+ calcd for C13H22N3O4+, 284.16048; found, 284.15961.

tert-Butyl (2S)-2-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)pyrrolidine-1-carboxylate (19b)

Compound 19b was synthesized according to the general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 mL, 17.3 mmol), a solution of 17 (2.68 g, 13.3 mmol) in THF was added. After evaporation of the volatiles, compound 18 was produced. By using 3b (3.0 g, 12.4 mmol), DIPEA (4 mL, 23 mmol), and the chloroformate 18 (3.16 g, 12 mmol), compound 19b was synthesized. After purification via flash column chromatography (DCM/MeOH 0–2% v/v), compound 19b (3.07 g, 8.64 mmol, 72%) was isolated as a white solid. TLC (DCM/MeOH, 5:0.5 v/v): Rf = 0.42; mp 81–83 °C; IR (KBr) ν̃max: 3437, 1698, 1540, 1403, 1249, 1201, 1171 cm–1; 1H NMR: (400 MHz, CDCl3): δ 8.30 (s, 1H), 5.59 (s, 1H), 4.46–3.82 (m, 4H), 3.52–3.10 (m, 2H), 2.94–2.33 (m, 3H), 2.03–1.77 (m, 5H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 171.2, 156.1, 154.5, 65.7, 55.7, 52.2, 46.7, 46.5, 31.2, 28.5, 27.8, 25.3, 23.7, 22.9; HRMS ESI (m/z): [M + Na]+ calcd for C16H25N3O6Na+, 378.16411; found, 378.16862.

(2S)-2-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)pyrrolidin-1-ium 2,2,2-Trifluoroacetate (20b)

Compound 20b was synthesized according to the general procedure D using 19b (3.55 g, 10 mmol) and TFA (23 mL, 300 mmol). Upon completion, the volatiles were completely evaporated to give 20b (3.69 g, 10 mmol, quant) as a white solid. TLC (DCM/MeOH, 5:0.5 v/v): RF = 0.19; mp 149–151 °C; IR (KBr) ν̃max: 3479, 1705, 1541, 1253, 1204, 1135 cm–1; 1H NMR: (400 MHz, DMSO): δ 10.82 (s, 1H), 9.24 (s, 1H), 8.74 (s, 1H), 7.62 (d, J = 8.5 Hz, 1H), 4.48–4.04 (m, 3H), 3.76 (s, 1H), 3.19 (s, 2H), 2.74 (ddd, J = 18.2, 12.7, 6.1 Hz, 1H), 2.12–1.81 (m, 5H), 1.79–1.57 (m, 1H); 13C NMR (101 MHz, DMSO): δ 173.4, 172.7, 156.0, 63.8, 58.2, 51.3, 45.8, 31.4, 26.9, 24.7, 23.7; 19F NMR (282 MHz, DMSO-d6): δ −74.36; HRMS (m/z): [M + H]+ calcd for C11H18N3O4+, 256.12918; found, 256.12905.

Cloning, Expression and Protein Purification

WT MsCI4 was cloned, expressed, and purified as previously described. (56) In an additional construct (humanized MsCI4), surface exposed residues were mutated to mimic hCRBN. The gene carrying the mutations A52H, M54Y, F56H, R68N, A72R, I87V, and L89Q was codon optimized for expression in E. coli and synthesized (eurofins). Humanized MsCI4 was cloned via BamHI and XhoI restriction sites into pETHis_1a; expression and purification were performed as described for WT MsCI4.

FRET Assay

FRET-based binding assay was performed using WT MsCI4 as described previously. (56) Compounds were dissolved in DMSO with the exception of 11a/b, 16a/b, and 20a/b, which were water-soluble, and compounds 12a/b that were dissolved in bicarbonate buffer. Because of remaining impurities, 20a was not tested in this assay and only used for structural studies. For compounds 4a, 4c, 5a, 7a, 7b, 7e, and 7g, binding was observed but could not be quantified, as saturation at higher concentrations was not appreciable in their binding curves. Ki values are summarized in Table S1.

Crystallography

For crystallization, MsCI4 and humanized MsCI4 protein solution were concentrated to 17 mg/mL and mixed with either 3 mM thalidomide (MsCI4 and humanized MsCI4) or 7d (MsCI4). Crystallization trials for humanized MsCI4·thalidomide, which turned out to be humanized MsCI4·CBG, and MsCI4·7d were performed via the vapor diffusion technique at 294 K in sitting-drops. 400 nL of protein solution was mixed with 400 nL of reservoir solution with the Honeybee 963 robot (Genomic Solutions Ltd.). Diffraction-quality crystals of humanized MsCI4·CBG were obtained in a condition containing 20% PEG 3350 and 0.2 M ammonium acetate, and crystals of MsCI4·7d were obtained using 15% PEG 6 K and 5% glycerol. For all other compounds, crystals were prepared in hanging drop experiments by mixing 1 μL 0.4 M (NH4)2HPO4 ground solution with 1 μL MsCI4·thalidomide solution. Crystals grown in this setup were transferred to 3 μL ground solution spiked with individual compounds for soaking. After 36 h, crystals were cryoprotected by streaking through a 50% PEG 3350 solution. Crystals were flash-cooled in liquid nitrogen, and diffraction data were collected at 100 K and a wavelength of 1 Å on beamline X10SA at the Swiss Light Source using a PILATUS 6M-F hybrid pixel detector (Dectris Ltd.). Data were processed and scaled using XDS. (57) Structures of soaking experiments were solved based on the MsCI4·thalidomide coordinates (PDB 4v2y). The cocrystal structures of the complexes with CBG and 7d were of other crystal forms and were solved using molrep (58) with 4v2y as a search model, locating four and two chains in the ASU, respectively. All structures were rebuilt using Coot (59) and the integrated suite Lidia for chemical structures and generation of restraints. The models were finalized via cyclic modeling in Coot and refinement using REFMAC5. Molecular figures were generated using PyMOL. (60) Data collection and refinement statistics are summarized in Table 1.
Table 1. Data Collection and Refinement Statistics
 MsCI4·4aMsCI4·4bMsCI4·5aMsCI4·5bMsCI4·7aMsCI4·7bMsCI4·7cMsCI4·7dMsCI4·7f
Data Collection
space groupP212121P212121P212121P212121P212121P212121P212121P21P212121
Unit Cell
a, b, c (Å)56.46, 58.82, 88.2356.75, 58.82, 88.5756.95, 59.58, 89.0556.66, 58.71, 88.0156.33, 58.68, 89.2756.42, 58.80, 88.2356.33, 58.68, 89.2731.63, 52.39, 59.2956.87, 58.55, 88.28
α, β, γ (deg)90, 90, 9090, 90, 9090, 90, 9090, 90, 9090, 90, 9090, 90, 9090, 90, 9090.0, 95.8, 90.090, 90, 90
resolution range, Å44.1–1.55 (1.64–1.55)44.28–1.6 (1.7–1.6)44.53–1.7 (1.8–1.7)47.64–1.75 (1.85–1.75)49.03–1.8 (1.86–1.8)48.93–1.73 (1.84–1.73)49.03–1.85 (1.96–1.85)39.2–1.1 (1.16–1.1)44.14–1.65 (1.75–1.65)
redundancy12.7 (11.8)12.9 (12.9)12.4 (12.8)12.8 (12.8)12.86 (12)12.5 (12.0)12.9 (13.0)6.3 (5.3)12.9 (13.2)
completeness %99.8 (98.9)99.9 (99.4)99.0 (98.2)99.6 (97.7)99.6 (97.6)98.8 (92.4)99.8 (98.6)99.1 (95.1)99.9 (99.6)
R merge %9.0 (98.2)5.3 (82.8)7.3 (94.6)6.6 (98.5)7.2 (89.0)14.2 (88.8)6.1 (102.8)5.3 (39.4)6.9 (81.5)
CC (1/2)99.9 (85.4)100 (90.4)99.9 (87.8)100 (87.6)100 (84.6)99.6 (85.7)99.9 (78.1)99.8 (92.4)99.9 (86.6)
I/s(I)15.09 (1.78)15.09 (1.78)20.22 (2.02)20.47 (2.27)22.12 (2.39)10.55 (1.73)21.46 (2.25)17.67 (3.64)20.72 (2.35)
Refinement
number of reflections (total/test)43 370 (4254)39 841 (3904)33 803 (3354)30 301 (2985)28 122 (2771)30 582 (2948)28 122 (2771)77 822 (7458)36 182 (3558)
no. of atoms287127872767270925442778267520822464
protein257225192504252323382521246817682229
solvent223173185134175187123276166
ligand769578523170843869
R work %0.170.190.180.190.170.170.180.120.20
R free %0.200.220.230.230.220.230.220.150.22
Ligand in Chain
Athalidomidehydrolyzed 4bthalidomide5b7athalidomide7c thalidomide
B4ahydrolyzed 4bhydrolyzed 5a5b7a7b7cco-crystal with 7d7f
Chydrolyzed 4ahydrolyzed 4b5a  thalidomide7c  
PDB ID6R0S6R0V6R0U6R116R1X6R126R1K6R1D6R13
 MsCI4·11aMsCI4·12aMsCI4·16bMsCI4·20aMsCI4·20bMsCI4·CBG   
Data Collection   
space groupP212121P212121P212121P212121P212121P21   
Unit Cell   
a, b, c (Å)56.63, 59.59, 88.9056.66, 59.02, 88.5956.53, 59.44, 88.3656.56, 59.23, 88.4356.56, 59.3, 88.061.7, 59.1, 61.7   
α, β, γ (deg)90, 90, 9090, 90, 9090, 90, 9090, 90, 9090, 90, 9090, 105.6, 90   
resolution range, Å44.45–1.35 (1.43–1.35)49.12–1.5 (1.59–1.5)47.62–1.35 (1.43–1.35)49.21–1.45 (1.50–1.45)49.18–1.54 (1.64–1.54)49.17–1.50 (1.59–1.50)   
redundancy12.7 (12.4)12.6 (12.6)12.6 (12.7)12.8 (12.9)12.6 (12.1)3.4 (3.1)   
completeness %99.9 (99.3)99.5 (97.2)99.8 (99.1)99.8 (98.5)99.5 (96.7)99.0 (97.5)   
R merge %7.4 (96.3)11.4 (78.6)7.9 (98.8)9.5 (89.4)13.1 (81.5)8.0 (88.0)   
CC (1/2)99.9 (83.2)99.8 (86.5)99.9 (84.0)99.8 (86.2)99.7 (83.8)99.7 (83.8)   
I/s(I)17.62 (1.97)14.29 (2.32)16.63 (1.85)14.83 (2.11)10.28 (1.71)9.70 (1.15)   
Refinement   
number of reflections (total/test)66 738 (6544)48 282 (4739)66 083 (6516)53 344 (5211)44 026 (4112)68 399 (6707)   
no. of atoms297729953057307429283985   
protein261926652638263426173480   
solvent281252338339232421   
ligand7778811017984   
R work %0.160.210.160.170.180.16   
R free %0.190.240.200.200.210.20   
Ligand in Chain   
Athalidomidethalidomidethalidomidethalidomidethalidomide    
B10a12a16b20athalidomideco-crystal with CBG   
Cthalidomidethalidomide16bthalidomide20b    
PDB ID6R186R1C6R1W6R196R1A6R0Q   

Cell Culture and Western Blot

OPM-2 cells were routinely cultivated in 90% RPMI 1640 + 10% FBS and 1% penicillin/streptomycin. Cells were split to 0.3 × 106 cells/mL 2 days prior to experiments. 4 mL of culture per well was pipetted in a sterile environment. Stock solutions of lenalidomide were prepared at concentrations of 80 and 400 mM and stock solutions of test compounds were prepared at a concentration of 400 mM. For the assay, 10 μL of stock solutions was added to the 4 mL cell culture. DMSO controls were prepared in the same manner, leading to a final DMSO concentration of 0.25%. After 24 h of incubation, cell solution was centrifugated at 500g. After one washing step with ice cold PBS, cell pellets were resuspended in 35 μL of lysis buffer (20 mM Hepes, 175 mM NaCl, 1% NP40, 2 mM MgCl2) on ice and supplemented with 0.5 μL Benzonase. Samples were resolved by Mini-PROTEAN TGX gels (Bio-Rad) and activated under UV light, before transfer to low-fluorescence PVDF membranes, according to manufacturer’s protocol. Membranes were imaged before blocking in 5% milk in PBS-T and incubating with primary antibodies anti-IKZF3 (15103S, Cell Signaling Technology, Inc.) and anti-CK1α (ab108296, Abcam plc). After overnight incubation, horseradish peroxidase conjugated secondary antibodies goat anti-rabbit (111-035-144, Jackson Immuno Research) and goat anti-mouse (115-035-003, Jackson Immuno Research) were used at 1:20 000 dilutions for detection of bands by chemiluminescence (ECL Vilbert). Protein bands were detected and integrated using the Bio-Rad analysis suite. Endogenous protein levels were normalized using Stain-Free Technology (Figure S2) and analyzed by one-way ANOVA, according to the Holm-Sidak method integrated into SigmaPlot. The linearity of anti-IKZF3 mAb was tested via serial dilutions of cell extracts (Figure S3).

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00454.

  • HPLC traces of tested compounds, western blot quantification and normalization IKZF3, concentration response curve for IKZF3 mAb, and Ki values of tested compounds (PDF)

  • Molecular formula strings (CSV)

Accession Codes

Crystal structures have been deposited in the protein data bank (PDB) under the accession codes 6R0S (4a), 6R0V (4b), 6R0U (5a), 6R11 (5b), 6R1X (7a), 6R12 (7b), 6R1K (7c), 6R1D (7d), 6R13 (7f), 6R18 (11a), 6R1C (12a), 6R1W (16b), 6R19 (20a), and 6R1A (20b), 6R0Q (CBG). The authors will release the atomic coordinates and experimental data upon article publication.

Terms & Conditions

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

Author Information

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  • Corresponding Authors
  • Authors
    • Christopher Heim - Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
    • Dimanthi Pliatsika - Faculty for Chemistry und Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany
    • Farnoush Mousavizadeh - Faculty for Chemistry und Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany
    • Kerstin Bär - Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
    • Birte Hernandez Alvarez - Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Andrei Lupas for continuing support, Reinhard Albrecht for assistance with crystallization and crystallographic data collection, and the staff of beamline X10SA of the Swiss Light Source (PSI, Villigen, Switzerland) for excellent technical support. This work was supported by institutional funds of the Max Planck Society.

Abbreviations

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4-DMAP

4-dimethylaminopyridine

AML

acute myeloid leukemia

APP

amyloid precursor protein

ASU

asymmetric unit

BET

bromodomain and extraterminal domain

CBG

α-(2-carboxybenzamido)glutarimide

CDI

N,N-carbodiimidazole

CK1α

casein kinase 1α

CRBN

cereblon

CRL4CRBN

CUL4-RBX1-DDB1-CRBN

DIPEA

N,N-diisopropylethylamine

Et3N

triethylamine

EtOAc

ethyl acetate

FBS

fetal bovine serum

GSPT1

eukaryotic peptide chain release factor GTP-binding subunit ERF3A

IKZF1

IKAROS family zinc finger 1

IKZF3

zinc finger protein Aiolos

IMiD

immunomodulatory drug

MDS

myelodysplastic syndrome

MM

multiple myeloma

MsCI4

CRBN homologue from magnetospirillum gryphiswaldense

PROTAC

proteolysis targeting chimera

SALL4

sal-like protein 4

VHL

von-Hippel-Lindau

References

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This article references 60 other publications.

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    Thalidomide is an effective treatment for relapsed multiple myeloma (MM), but is assocd. with a significant side effect profile at higher doses. In a recent study, only half of the enrolled patients were able to tolerate the max. dose of 800 mg/day [Singhal, S., et al. (1999) "Antitumor activity of thalidomide in refractory multiple myeloma", New Engl. J. Med. 341, 1565-1571]. Moreover, the dose-response relationship has not been defined. We report our use of low dose thalidomide in a small cohort of 12 patients-eight with relapsed or refractory MM and four with plasma cell leukemia (PCL). Five of the 12 (42%) patients had a partial response, showing a median fall in their PP/BJP of 80% (63-90%) at a median dose of 175 mg (100-300 mg) with negligible side effects. Three of four patients with PCL showed an impressive response to treatment with thalidomide as a single agent. No patient who failed to show any evidence of response at low dose (<150 mg/day) responded to higher doses. In this study, thalidomide induces a similar rate of response at a lower and better tolerated dose than previously reported and produced "best ever" responses in patients with resistant PCL.
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    The treatment of T-cell non-Hodgkin's lymphoma (T-NHL) remains challenging. There is currently no standard regimen for the treatment of T-NHL in the first- or second-line setting. Thalidomide was previously shown to exert antitumor effects through inhibiting angiogenesis, promoting apoptosis and immunomodulatory activity. However, all the previous studies on the treatment of lymphoma with thalidomide included patient samples of limited size. In the present study, 46 cases of eligible T-NHL patients were randomized into i) the control group (conventional combined chemotherapy, n=22) and ii) the thalidomide group (thalidomide plus combined chemotherapy, n=24). The median dose of thalidomide was 200 mg (range, 150-400 mg) every night, without reported severe side effects. The clinical response to treatment was as follows: Complete response (CR) in 12 cases, partial response (PR) in 7, stable disease (SD) in 1 and progressive disease (PD) in 4 cases in the thalidomide group; and CR in 8 cases, PR in 6, SD in 3 and PD in 5 cases in the control group. The CR rate was 50.0 and 36.4% in the thalidomide and the control groups, respectively (P<0.05). The median progression-free and overall survival were 12 and undefined months, respectively, in the thalidomide group and 6 and 17 months, respectively, in the control group. The toxicity profile was considered acceptable in both groups. Our results indicated that thalidomide plus combined chemotherapy may exhibit enhanced efficacy in the clinical treatment of T-NHL. In addition, this type of treatment may reduce the frequency of adverse gastrointestinal reactions and help alleviate fear of chemotherapy. Therefore, thalidomide plus combined chemotherapy may be a viable option for the clinical treatment of T-NHL.
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    Few diseases have a prognosis worse than Hodgkin's lymphoma (HL), patients relapsing after autologous or allogeneic stem cell transplantation. Here, we report two highly refractory patients with HL who successfully responded to a combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex). Despite the use of a very large no. of different drugs (>5 different schemes) including high-dose therapy and autologous and allogeneic stem cell transplantation, both patients proved to be suffering from a highly resistant disease. Fortunately, they finally responded to the ThaCyDex combination, achieving sustained complete remission that would support the running of a trial within this setting.
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    Hematology (Abingdon, United Kingdom) (2006), 11 (1), 25-29CODEN: HMATFL; ISSN:1024-5332. (Taylor & Francis Ltd.)
    Introduction: Patients with Hodgkin's Lymphoma (HL) who relapse or progress after primary therapy and subsequent high dose chemotherapy with autologous stem cell transplantation (ASCT) cannot be cured with conventional treatment. We combined thalidomide (THAL), an agent with anti-angiogenic and immunomodulatory properties, with vinblastine, which is active after ASCT, to det. the objective response rate, improvement in B symptoms and toxicity in patients with refractory HD. Methods: Patients were eligible if they HD that progressed after chemotherapy and ASCT or had declined or were ineligible for curative therapy. Treatment consisted of THAL 200 mg orally given daily. After 2 wk, VBL 6 mg IV was given weekly × 6 doses on an eight-week cycle. Response and toxicity assessment occurred following each cycle. Results: Eleven patients were enrolled, 1 progressed within 6 days of study enrollment and was subsequently treated with alternative palliative therapy and thus 11 patients are response evaluable and 10 are evaluable for toxicity. Patient characteristics: relapsed after ASCT: 7; median no. of prior chemotherapy regimens: 3 (range 1-5); median time to progression post-ASCT: 7 mo (range 2-29). Four patients had a partial response to treatment (response rate 36%); two patients had stable disease. B symptoms were present at enrollment in four patients and resolved completely on treatment in two patients. Five had disease progression within 3 mo of starting treatment. The median duration of response was 9 mo (range 0-22 mo). Toxicity was mild and limited to grade 2 neuropathy in 6 patients and grade 2 or 3 neutropenia in 4 patients. Conclusions: In this small study in chemotherapy- refractory HL, THAL and VBL demonstrated encouraging activity with some durable responses and acceptable toxicity. These results suggest that chronic low dose chemotherapy combined with less toxic immunomodulatory or anti-angiogenic drugs warrants further study.
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  11. 11
    Seldin, D. C.; Choufani, E. B.; Dember, L. M.; Wiesman, J. F.; Berk, J. L.; Falk, R. H.; O’Hara, C.; Fennessey, S.; Finn, K. T.; Wright, D. G.; Skinner, M.; Sanchorawala, V. Tolerability and efficacy of thalidomide for the treatment of patients with light chain-associated (AL) amyloidosis. Clin. Lymphoma 2003, 3, 241– 246,  DOI: 10.3816/clm.2003.n.005
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    11
    Tolerability and efficacy of thalidomide for the treatment of patients with light-chain-associated (AL) amyloidosis
    Seldin, David C.; Choufani, Elie B.; Dember, Laura M.; Wiesman, Janice F.; Berk, John L.; Falk, Rodney H.; O'Hara, Carl; Fennessey, Salli; Finn, Kathleen T.; Wright, Daniel G.; Skinner, Martha; Sanchorawala, Vaishali
    Clinical Lymphoma (2003), 3 (4), 241-246CODEN: CLLYAO; ISSN:1526-9655. (Cancer Information Group)
    A phase I/II trial was carried out with patients with AL amyloidosis, most of whom had failed prior therapy with high-dose melphalan and autologous stem cell transplantation. This trial was designed as an individualized 6-mo dose-escalation study with re-evaluation of bone marrow plasmacytosis and serum and urine monoclonal proteins after 3 and 6 mo. Sixteen patients with a median age of 62 yr (range, 37-70 yr) were enrolled. Fourteen of the patients had renal involvement, 4 had cardiac involvement, 4 had liver involvement, and 2 had predominant soft tissue or lymph node involvement. The median max. tolerated dose was 300 mg, with fatigue and other central nervous system side effects being the major dose-limiting toxicities. Side effects not frequently reported for other patient populations included exacerbation of peripheral and pulmonary edema and worsening azotemia. In all, 50% of the patients experienced grade 3/4 toxicity, and 25% had to discontinue the drug. No complete hematol. responses were seen, but 25% of the patients had a significant redn. in Bence-Jones proteinuria. Thus, while thalidomide has activity in AL amyloidosis, it also has significant toxicity in this patient population.
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  12. 12
    Baird, R.; van Zyl-Smit, R. N.; Iveson, A.; Duddy, J.; Rassam, S. M. Thalidomide is highly effective in a patient with meningeal acute myeloid leukaemia. Leuk. Lymphoma 2004, 45, 179– 181,  DOI: 10.1080/1042819031000149412
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    12
    Thalidomide is highly effective in a patient with meningeal acute myeloid leukaemia
    Baird Richard; van Zyl-Smit Richard Nellis; Iveson Anne; Duddy James; Rassam Saad M B
    Leukemia & lymphoma (2004), 45 (1), 179-81 ISSN:1042-8194.
    We report a case of secondary acute myeloid leukaemia (AML) following high dose therapy for diffuse large B-cell non-Hodgkin's lymphoma (NHL) who developed meningeal leukaemia. This was refractory to systemic and intrathecal chemotherapy and cranial irradiation. Thalidomide has been reported to have anti-AML activity and appears to cross the blood brain barrier (BBB). We, therefore, attempted a trial of oral Thalidomide and achieved rapid biochemical and cytological remission with a short course. The patient, however, progressed systemically and succumbed to her illness.
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  13. 13
    Thomas, D. A.; Estey, E.; Giles, F. J.; Faderl, S.; Cortes, J.; Keating, M.; O’Brien, S.; Albitar, M.; Kantarjian, H. Single agent thalidomide in patients with relapsed or refractory acute myeloid leukaemia. Br. J. Haematol. 2003, 123, 436– 441,  DOI: 10.1046/j.1365-2141.2003.04639.x
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    13
    Single agent thalidomide in patients with relapsed or refractory acute myeloid leukaemia
    Thomas, Deborah A.; Estey, Elihu; Giles, Francis J.; Faderi, Stefan; Cortes, Jorge; Keating, Michael; O'Brien, Susan; Albitar, Maher; Kantarjian, Hagop
    British Journal of Haematology (2003), 123 (3), 436-441CODEN: BJHEAL; ISSN:0007-1048. (Blackwell Publishing Ltd.)
    Thalidomide is a putative anti-angiogenesis agent that has significant anti-tumor activity in haematol. malignancies with increased bone marrow angiogenesis, including multiple myeloma (MM) and myelodysplastic syndromes (MDS). Increased levels of the mitogen for angiogenesis, vascular endothelial growth factor (VEGF), correlate with worse survival in acute myeloid leukemia (AML). A phase II trial of thalidomide was conducted in patients with relapsed- or refractory-AML previously treated with cytarabine-contg. regimens. A total of 16 patients with refractory- or relapsed-AML were treated with thalidomide 200-800 mg orally daily (median dose 400 mg daily) for a median of 27 d (range, 3-94 d). Overall, one patient (6%) achieved complete remission (CR) lasting for 36 mo, and two patients had a transient redn. in marrow blasts from 8% and 7% to less than 5% in both cases. There was no correlation between redn. in levels of angiogenesis markers and response. Toxicities related to thalidomide were significant, and precluded dose escalation beyond 400 mg orally daily in most patients. Although there appears to be some evidence of biol. activity, single agent thalidomide is not an optimal choice of therapy for salvaging patients with relapsed- or refractory-AML. Thalidomide analogs with more potent immunomodulatory activities and more favorable toxicity profiles may offer more promise as anti-AML therapy.
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  14. 14
    Hartmann, 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, 225– 232,  DOI: 10.1016/j.jsb.2014.10.010
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    14
    Thalidomide mimics uridine binding to an aromatic cage in cereblon
    Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Zanini, Fabio; Lupas, Andrei N.; Hernandez Alvarez, Birte
    Journal 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.
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  15. 15
    Hartmann, M. D.; Boichenko, I.; Coles, M.; Lupas, A. N.; Hernandez Alvarez, B. Structural dynamics of the cereblon ligand binding domain. PLoS One 2015, 10, e0128342  DOI: 10.1371/journal.pone.0128342
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    15
    Structural dynamics of the cereblon ligand binding domain
    Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Alvarez, Birte Hernandez
    PLoS 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.
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  16. 16
    Chamberlain, P. P.; Lopez-Girona, A.; Miller, K.; Carmel, G.; Pagarigan, B.; Chie-Leon, B.; Rychak, E.; Corral, L. G.; Ren, Y. J.; Wang, M.; Riley, M.; Delker, S. L.; Ito, T.; Ando, H.; Mori, T.; Hirano, Y.; Handa, H.; Hakoshima, T.; Daniel, T. O.; Cathers, B. E. Structure of the human cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 2014, 21, 803– 809,  DOI: 10.1038/nsmb.2874
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    16
    Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs
    Chamberlain, Philip P.; Lopez-Girona, Antonia; Miller, Karen; Carmel, Gilles; Pagarigan, Barbra; Chie-Leon, Barbara; Rychak, Emily; Corral, Laura G.; Ren, Yan J.; Wang, Maria; Riley, Mariko; Delker, Silvia L.; Ito, Takumi; Ando, Hideki; Mori, Tomoyuki; Hirano, Yoshinori; Handa, Hiroshi; Hakoshima, Toshio; Daniel, Thomas O.; Cathers, Brian E.
    Nature Structural & Molecular Biology (2014), 21 (9), 803-809CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degrdn. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are crit. for antiproliferative effects.
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  17. 17
    Fischer, E. S.; Böhm, 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. J.; Harper, J. W.; Jenkins, J. L.; Thomä, N. H. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 2014, 512, 49– 53,  DOI: 10.1038/nature13527
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    17
    Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide
    Fischer, 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.
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  18. 18
    Nguyen, T. V.; Lee, J. E.; Sweredoski, M. J.; Yang, S. J.; Jeon, S. J.; Harrison, J. S.; Yim, J. H.; Lee, S. G.; Handa, H.; Kuhlman, B.; Jeong, J. S.; Reitsma, J. M.; Park, C. S.; Hess, S.; Deshaies, R. J. Glutamine triggers acetylation-dependent degradation of glutamine synthetase via the thalidomide receptor cereblon. Mol. Cell 2016, 61, 809– 820,  DOI: 10.1016/j.molcel.2016.02.032
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    18
    Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon
    Nguyen, T. Van; Lee, J. Eugene; Sweredoski, Michael J.; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S.; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M.; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J.
    Molecular Cell (2016), 61 (6), 809-820CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4CRBN. Upon exposing cells to high glutamine concn., GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4CRBN and degrdn. by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4CRBN that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.
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  19. 19
    Del Prete, D.; Rice, R. C.; Rajadhyaksha, A. M.; D’Adamio, L. Amyloid precursor protein (APP) may act as a substrate and a recognition unit for CRL4(CRBN) and Stub1 E3 ligases facilitating ubiquitination of proteins involved in presynaptic functions and neurodegeneration. J. Biol. Chem. 2016, 291, 17209– 17227,  DOI: 10.1074/jbc.m116.733626
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    19
    Amyloid precursor protein (APP) may act as a substrate and a recognition unit for CRL4CRBN and Stub1 E3 ligases facilitating ubiquitination of proteins involved in presynaptic functions and neurodegeneration
    Del Prete, Dolores; Rice, Richard C.; Rajadhyaksha, Anjali M.; D'Adamio, Luciano
    Journal of Biological Chemistry (2016), 291 (33), 17209-17227CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the N-terminus of the ACR, and CRL4CRBN, which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the C-terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4CRBN, pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome anal. indicates that these E3 ligases are enzymically active and ubiquitinated the ACR residues Lys649/650/651/676/688. Deletion of Crbn reduces ubiquitination of Lys676 suggesting that Lys676 is physiol. ubiquitinated by CRL4CRBN. The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro. This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.
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  20. 20
    Kronke, 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, 301– 305,  DOI: 10.1126/science.1244851
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    20
    Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells
    Kronke 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 L
    Science (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.
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  21. 21
    Krönke, 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.; Järås, 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, 183– 188,  DOI: 10.1038/nature14610
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    21
    Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS
    Kronke, 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.
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  22. 22
    Matyskiela, M. E.; Lu, G.; Ito, T.; Pagarigan, B.; Lu, C.-C.; Miller, K.; Fang, W.; Wang, N.-Y.; Nguyen, D.; Houston, J.; Carmel, G.; Tran, T.; Riley, M.; Nosaka, L. A.; Lander, G. C.; Gaidarova, S.; Xu, S.; Ruchelman, A. L.; Handa, H.; Carmichael, J.; Daniel, T. O.; Cathers, B. E.; Lopez-Girona, A.; Chamberlain, P. P. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase. Nature 2016, 535, 252– 257,  DOI: 10.1038/nature18611
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    22
    A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase
    Matyskiela, Mary E.; Lu, Gang; Ito, Takumi; Pagarigan, Barbra; Lu, Chin-Chun; Miller, Karen; Fang, Wei; Wang, Nai-Yu; Nguyen, Derek; Houston, Jack; Carmel, Gilles; Tran, Tam; Riley, Mariko; Nosaka, Lyn'Al; Lander, Gabriel C.; Gaidarova, Svetlana; Xu, Shuichan; Ruchelman, Alexander L.; Handa, Hiroshi; Carmichael, James; Daniel, Thomas O.; Cathers, Brian E.; Lopez-Girona, Antonia; Chamberlain, Philip P.
    Nature (London, United Kingdom) (2016), 535 (7611), 252-257CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4CRBN E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumor activity. The anti-tumor activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degrdn. of the translation termination factor GSPT1. Patient-derived acute myeloid leukemia tumor cells exhibit high sensitivity to CC-885, indicating the clin. potential of this mechanism. Crystallog. studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn contg. a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homol. to previously known cereblon substrates, mutational anal. and modeling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumor target rendered druggable by cereblon modulation.
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  23. 23
    Matyskiela, M. E.; Couto, S.; Zheng, X.; Lu, G.; Hui, J.; Stamp, K.; Drew, C.; Ren, Y.; Wang, M.; Carpenter, A.; Lee, C.-W.; Clayton, T.; Fang, W.; Lu, C.-C.; Riley, M.; Abdubek, P.; Blease, K.; Hartke, J.; Kumar, G.; Vessey, R.; Rolfe, M.; Hamann, L. G.; Chamberlain, P. P. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 2018, 14, 981– 987,  DOI: 10.1038/s41589-018-0129-x
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    23
    SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate
    Matyskiela, Mary E.; Couto, Suzana; Zheng, Xinde; Lu, Gang; Hui, Julia; Stamp, Katie; Drew, Clifton; Ren, Yan; Wang, Maria; Carpenter, Aaron; Lee, Chung-Wein; Clayton, Thomas; Fang, Wei; Lu, Chin-Chun; Riley, Mariko; Abdubek, Polat; Blease, Kate; Hartke, James; Kumar, Gondi; Vessey, Rupert; Rolfe, Mark; Hamann, Lawrence G.; Chamberlain, Philip P.
    Nature Chemical Biology (2018), 14 (10), 981-987CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    Targeted protein degrdn. via small-mol. modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clin. presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the mol. basis of thalidomide-induced teratogenicity.
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  24. 24
    Donovan, K. A.; An, J.; Nowak, R. P.; Yuan, J. C.; Fink, E. C.; Berry, B. C.; Ebert, B. L.; Fischer, E. S., Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray Syndrome. Elife 2018, 7. DOI: 10.7554/elife.38430
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  25. 25
    Petzold, G.; Fischer, E. S.; Thomä, N. H. Structural basis of lenalidomide-induced CK1alpha degradation by the CRL4(CRBN) ubiquitin ligase. Nature 2016, 532, 127– 130,  DOI: 10.1038/nature16979
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    25
    Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase
    Petzold, 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.
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  26. 26
    Sievers, Q. L.; Petzold, G.; Bunker, R. D.; Renneville, A.; Słabicki, M.; Liddicoat, B. J.; Abdulrahman, W.; Mikkelsen, T.; Ebert, B. L.; Thomä, N. H. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 2018, 362, eaat0572  DOI: 10.1126/science.aat0572
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    There is no corresponding record for this reference.
  27. 27
    Lai, A. C.; Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discovery 2017, 16, 101– 114,  DOI: 10.1038/nrd.2016.211
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    27
    Induced protein degradation: an emerging drug discovery paradigm
    Lai, 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.
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  28. 28
    Sakamoto, 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, 8554– 8559,  DOI: 10.1073/pnas.141230798
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    28
    Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation
    Sakamoto, 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.
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  29. 29
    Hu, J.; Hu, B.; Wang, M.; Xu, F.; Miao, B.; Yang, C.-Y.; Wang, M.; Liu, Z.; Hayes, D. F.; Chinnaswamy, K.; Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Estrogen Receptor (ER). J. Med. Chem. 2019, 62, 1420– 1442,  DOI: 10.1021/acs.jmedchem.8b01572
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    29
    Discovery of ERD-308 as a highly potent proteolysis targeting chimera (PROTAC) degrader of estrogen receptor (ER)
    Hu, Jiantao; Hu, Biao; Wang, Mingliang; Xu, Fuming; Miao, Bukeyan; Yang, Chao-Yie; Wang, Mi; Liu, Zhaomin; Hayes, Daniel F.; Chinnaswamy, Krishnapriya; Delproposto, James; Stuckey, Jeanne; Wang, Shaomeng
    Journal of Medicinal Chemistry (2019), 62 (3), 1420-1442CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The estrogen receptor (ER) is a validated target for the treatment of estrogen receptor-pos. (ER+) breast cancer. Here, we describe the design, synthesis, and extensive structure-activity relationship (SAR) studies of small-mol. ERα degraders based on the proteolysis targeting chimeras (PROTAC) concept. Our efforts have resulted in the discovery of highly potent and effective PROTAC ER degraders, as exemplified by ERD-308 I. I achieves DC50 (concn. causing 50% of protein degrdn.) values of 0.17 and 0.43 nM in MCF-7 and T47D ER+ breast cancer cell lines, resp., and induces >95% of ER degrdn. at concns. as low as 5 nM in both cell lines. Significantly, I induces more complete ER degrdn. than fulvestrant, the only approved selective ER degrader (SERD), and is more effective in inhibition of cell proliferation than fulvestrant in MCF-7 cells. Further optimization of I may lead to a new therapy for advanced ER+ breast cancer.
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  30. 30
    Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.; Yang, C.-Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.; Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ARD-69 as a highly potent proteolysis targeting chimera (PROTAC) degrader of androgen receptor (AR) for the treatment of prostate cancer. J. Med. Chem. 2019, 62, 941– 964,  DOI: 10.1021/acs.jmedchem.8b01631
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    30
    Discovery of ARD-69 as a highly potent proteolysis targeting chimera (PROTAC) degrader of androgen receptor (AR) for the treatment of prostate cancer
    Han, Xin; Wang, Chao; Qin, Chong; Xiang, Weiguo; Fernandez-Salas, Ester; Yang, Chao-Yie; Wang, Mi; Zhao, Lijie; Xu, Tianfeng; Chinnaswamy, Krishnapriya; Delproposto, James; Stuckey, Jeanne; Wang, Shaomeng
    Journal of Medicinal Chemistry (2019), 62 (2), 941-964CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We report herein the discovery of highly potent PROTAC degraders of androgen receptor (AR), as exemplified by IARD-69. I induces degrdn. of AR protein in AR-pos. prostate cancer cell lines in a dose- and time-dependent manner. I achieves DC50 values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1 AR+ prostate cancer cell lines, resp. I is capable of reducing the AR protein level by >95% in these prostate cancer cell lines and effectively suppressing AR-regulated gene expression. I potently inhibits cell growth in these AR-pos. prostate cancer cell lines and is >100 times more potent than AR antagonists. A single dose of I effectively reduces the level of AR protein in xenograft tumor tissue in mice. Further optimization of I may ultimately lead to a new therapy for AR+, castration-resistant prostate cancer.
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  31. 31
    Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza, A.; Dhe-Paganon, S.; Bradner, J. E. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 2015, 348, 1376– 1381,  DOI: 10.1126/science.aab1433
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    31
    Phthalimide conjugation as a strategy for in vivo target protein degradation
    Winter, Georg E.; Buckley, Dennis L.; Paulk, Joshiawa; Roberts, Justin M.; Souza, Amanda; Dhe-Paganon, Sirano; Bradner, James E.
    Science (Washington, DC, United States) (2015), 348 (6241), 1376-1381CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The development of effective pharmacol. inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-mol. antagonists disrupt the activity of only one domain in the target protein. The authors devised a chem. strategy that promotes ligand-dependent target protein degrdn. using as an example the transcriptional coactivator BRD4, a protein crit. for cancer cell growth and survival. The authors appended a competitive antagonist of BET bromodomains to a phthalimide moiety to hijack the cereblon E3 ubiquitin ligase complex. The resultant compd., dBET1, induced highly selective cereblon-dependent BET protein degrdn. in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degrdn. of the cytosolic protein FKBP12. This chem. strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins.
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  32. 32
    Steinebach, C.; Kehm, H.; Lindner, S.; Vu, L. P.; Köpff, S.; López Mármol, Á.; Weiler, C.; Wagner, K. G.; Reichenzeller, M.; Krönke, J.; Gütschow, M. PROTAC-mediated crosstalk between E3 ligases. Chem. Commun. 2019, 55, 1821– 1824,  DOI: 10.1039/c8cc09541h
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    PROTAC-mediated crosstalk between E3 ligases
    Steinebach, Christian; Kehm, Hannes; Lindner, Stefanie; Vu, Lan Phuong; Koepff, Simon; Lopez Marmol, Alvaro; Weiler, Corinna; Wagner, Karl G.; Reichenzeller, Michaela; Kroenke, Jan; Guetschow, Michael
    Chemical 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.
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  33. 33
    Lu, M.; Liu, T.; Jiao, Q.; Ji, J.; Tao, M.; Liu, Y.; You, Q.; Jiang, Z. Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway. Eur. J. Med. Chem. 2018, 146, 251– 259,  DOI: 10.1016/j.ejmech.2018.01.063
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    33
    Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway
    Lu, Mengchen; Liu, Tian; Jiao, Qiong; Ji, Jianai; Tao, Mengmin; Liu, Yijun; You, Qidong; Jiang, Zhengyu
    European Journal of Medicinal Chemistry (2018), 146 (), 251-259CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    Induced protein degrdn. by PROTACs has emerged as a promising strategy to target nonenzymic proteins inside the cell. The aim of this study was to identify Keap1, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degrdn. of the nonenzymic protein Tau. A peptide PROTAC by recruiting Keap1-Cul3 ubiquitin E3 ligase was developed and applied in the degrdn. of intracellular Tau. Peptide 1 showed strong in vitro binding with Keap1 and Tau. With proper cell permeability, peptide 1 was found to colocalize with cellular Keap1 and resulted in the coimmunopptn. of Tau and Keap1. The results of flow cytometry and western blotting assays showed that peptide 1 can downregulate the intracellular Tau level in both time- and concn.-dependent manner. The application of Keap1 siRNA silencing and the proteasome inhibitor MG132 confirmed that peptide 1 could promote the Keap1-dependent poly-ubiquitination and proteasome-dependent degrdn. of Tau. The results suggested that using PROTACs to recruit Keap1 to induce the degrdn. of Tau may show promising character in the treatment of neurodegenerative disease. In addn., our research demonstrated that Keap1 should be a promising E3 ligase adaptor to be used in the design of novel PROTACs.
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    Kargbo, R. B. Treatment of Alzheimer’s by PROTAC-Tau protein degradation. ACS Med. Chem. Lett. 2019, 10, 699– 700,  DOI: 10.1021/acsmedchemlett.9b00083
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    Treatment of Alzheimer's by PROTAC-Tau Protein Degradation
    Kargbo, Robert B.
    ACS Medicinal Chemistry Letters (2019), 10 (5), 699-700CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    There is no expanded citation for this reference.
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  35. 35
    Chu, T.-T.; Gao, N.; Li, Q.-Q.; Chen, P.-G.; Yang, X.-F.; Chen, Y.-X.; Zhao, Y.-F.; Li, Y.-M. Specific knockdown of endogenous Tau protein by peptide-directed ubiquitin-proteasome degradation. Cell Chem. Biol. 2016, 23, 453– 461,  DOI: 10.1016/j.chembiol.2016.02.016
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    Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation
    Chu, Ting-Ting; Gao, Na; Li, Qian-Qian; Chen, Pu-Guang; Yang, Xi-Fei; Chen, Yong-Xiang; Zhao, Yu-Fen; Li, Yan-Mei
    Cell Chemical Biology (2016), 23 (4), 453-461CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
    Tau, an important pathol. protein of Alzheimer's disease (AD), can mediate the toxicity of amyloid β (Aβ). Thus, redn. of Tau with chem. mols. may offer a novel strategy for treating AD. Here, we designed and synthesized a series of multifunctional mols. that contained Tau-recognition moieties and E3 ligase-binding moieties to enhance Tau degrdn. Among these mols., TH006 had the highest activity of inducing Tau degrdn. by increasing its poly-ubiquitination. The decrement in Tau induced by TH006 could decrease the cytotoxicity caused by Aβ. Furthermore, TH006 could regulate the Tau level in the brain of an AD mouse model. Therefore, partial redn. of Tau with such multifunctional peptides may open up a novel therapeutic strategy for AD treatment.
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    Boichenko, I.; Bär, 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, 11163– 11171,  DOI: 10.1021/acsomega.8b00959
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    Chemical Ligand Space of Cereblon
    Boichenko, 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.
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    Burslem, G. M.; Ottis, P.; Jaime-Figueroa, S.; Morgan, A.; Cromm, P. M.; Toure, M.; Crews, C. M. Efficient synthesis of immunomodulatory drug analogues enables exploration of structure-degradation relationships. ChemMedChem 2018, 13, 1508– 1512,  DOI: 10.1002/cmdc.201800271
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    Efficient Synthesis of Immunomodulatory Drug Analogues Enables Exploration of Structure-Degradation Relationships
    Burslem, George M.; Ottis, Philipp; Jaime-Figueroa, Saul; Morgan, Alicia; Cromm, Philipp M.; Toure, Momar; Crews, Craig M.
    ChemMedChem (2018), 13 (15), 1508-1512CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The immunomodulatory drugs (IMiDs) thalidomide, pomalidomide, and lenalidomide have been approved for the treatment of multiple myeloma for many years. Recently, their use as E3 ligase recruiting elements for small-mol.-induced protein degrdn. has led to a resurgence in interest in IMiD synthesis and functionalization. Traditional IMiD synthesis follows a stepwise route with multiple purifn. steps. Herein we describe a novel one-pot synthesis without purifn. that provides rapid access to a multitude of IMiD analogs. Binding studies with the IMiD target protein cereblon (CRBN) reveals a narrow structure-activity relationship with only a few compds. showing sub-micromolar binding affinity in the range of pomalidomide and lenalidomide. However, anti-proliferative activity as well as Aiolos degrdn. could be identified for two IMiD analogs. This study provides useful insight into the structure-degrdn. relationships for mols. of this type as well as a rapid and robust method for IMiD synthesis.
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    Bartlett, J. B.; Dredge, K.; Dalgleish, A. G. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat. Rev. Cancer 2004, 4, 314– 322,  DOI: 10.1038/nrc1323
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    Timeline: The evolution of thalidomide and its IMiD derivatives as anticancer agents
    Bartlett, J. Blake; Dredge, Keith; Dalgleish, Angus G.
    Nature Reviews Cancer (2004), 4 (4), 314-322CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
    A review. Thalidomide was originally used to treat morning sickness, but was banned in the 1960s for causing serious congenital birth defects. Remarkably, thalidomide was subsequently discovered to have anti-inflammatory and anti-angiogenic properties, and was identified as an effective treatment for multiple myeloma. A series of immunomodulatory drugs - created by chem. modification of thalidomide - have been developed to overcome the original devastating side effects. Their powerful anticancer properties mean that these drugs are now emerging from thalidomide's shadow as useful anticancer agents.
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    Reist, M.; Carrupt, P.-A.; Francotte, E.; Testa, B. Chiral inversion and hydrolysis of thalidomide: mechanisms and catalysis by bases and serum albumin, and chiral stability of teratogenic metabolites. Chem. Res. Toxicol. 1998, 11, 1521– 1528,  DOI: 10.1021/tx9801817
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    Chiral Inversion and Hydrolysis of Thalidomide: Mechanisms and Catalysis by Bases and Serum Albumin, and Chiral Stability of Teratogenic Metabolites
    Reist, Marianne; Carrupt, Pierre-Alain; Francotte, Eric; Testa, Bernard
    Chemical Research in Toxicology (1998), 11 (12), 1521-1528CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    The chiral inversion and hydrolysis of thalidomide and the catalysis by bases and human serum albumin were investigated by using a stereoselective HPLC assay. Chiral inversion was catalyzed by albumin, hydroxyl ions, phosphate, and amino acids. Basic amino acids (Arg and Lys) had a superior potency in catalyzing chiral inversion compared to acid and neutral ones. The chiral inversion of thalidomide is thus subject to specific and general base catalysis, and it is suggested that the ability of HSA to catalyze the reaction is due to the basic groups of the amino acids Arg and Lys and not to a single catalytic site on the macromol. The hydrolysis of thalidomide was also base-catalyzed. However, albumin had no effect on hydrolysis, and there was no difference between the catalytic potencies of acidic, neutral, and basic amino acids. This may be explained by different reaction mechanisms of the chiral inversion and hydrolysis of thalidomide. Chiral inversion is deduced to occur by electrophilic substitution involving specific and general base catalysis, whereas hydrolysis is thought to occur by nucleophilic substitution involving specific and general base as well as nucleophilic catalysis. As nucleophilic attack is sensitive to steric properties of the catalyst, steric hindrance might be the reason albumin is not able to catalyze hydrolysis. 1H NMR expts. revealed that the three teratogenic metabolites of thalidomide, in sharp contrast to the drug itself, had complete chiral stability. This leads to the speculation that, were some enantioselectivity to exist in the teratogenicity of thalidomide, it could result from fast hydrolysis to chirally stable teratogenic metabolites.
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  40. 40
    Chen, T. L.; Vogelsang, G. B.; Petty, B. G.; Brundrett, R. B.; Noe, D. A.; Santos, G. W.; Colvin, O. M. Plasma pharmacokinetics and urinary excretion of thalidomide after oral dosing in healthy male volunteers. Drug Metab Dispos 1989, 17, 402– 405
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    Plasma pharmacokinetics and urinary excretion of thalidomide after oral dosing in healthy male volunteers
    Chen, Tian Ling; Vogelsang, Georgia B.; Petty, Brent G.; Brundrett, Robert B.; Noe, Dennis A.; Santos, George W.; Colvin, O. Michael
    Drug Metabolism and Disposition (1989), 17 (4), 402-5CODEN: DMDSAI; ISSN:0090-9556.
    The plasma pharmacokinetics and urinary excretion of thalidomide were evaluated in healthy male volunteers receiving a single oral dose of 200 mg. Concns. of thalidomide were detd. by a new HPLC assay. Plasma concn. vs. time data were well fit by a 1-compartment model. The mean peak concn., 1.15 μg/mL, was achieved at 4.39 h. Absorption and elimination half-lives were 1.70 and 8.70 h, resp., with a lag time of 0.41 h obsd. in 6 of 8 subjects. The apparent vol. of distribution and total body clearance rate, based on assumed complete bioavailability, were 120.69 L and 10.41 L/h. The urinary excretion of thalidomide accounted for only 0.6% of the total dose administered over 24 h, and the renal clearance rate was 0.08 L/h. Thus, the major route of elimination of thalidomide is nonrenal.
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    Schumacher, H.; Smith, R. L.; Williams, R. T. The metabolism of thalidomide: the fate of thalidomide and some of its hydrolysis products in various species. Br. J. Pharmacol. Chemother. 1965, 25, 338– 351,  DOI: 10.1111/j.1476-5381.1965.tb02054.x
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    The metabolism of thalidomide. The fate of thalidomide and some of its hydrolysis products in various species
    Schumacher, H.; Smith, R. L.; Williams, R. T.
    British Journal of Pharmacology and Chemotherapy (1965), 25 (2), 338-51CODEN: BJPCAL; ISSN:0366-0826.
    cf. preceding abstr. The metabolism of thalidomide in the rabbit, rat, mouse, and guinea pig was investigated. The metabolites of thalidomide present in the urine, blood, and tissues of various species dosed with the drug were characterized by comparing their chromatographic mobility and color reactions with those given by authentic samples of the compds. The rabbit urinary metabolites were isolated in cryst. form by solvent extn. and adsorption chromatography, and their identity was established by analysis, m.p. behavior, and comparison of their ir spectra with those of the authentic compds. When thalidomide is fed to rabbits, rats, mice, and guinea pigs a no. of hydrolysis products appear in the urine. These hydrolysis products are formed by the spontaneous hydrolysis of thalidomide. In addn. the urine of rabbits dosed with thalidomide contains derivs. of 3- and 4-hydroxyphthalic acid; these minor metabolites were not identified. The hydrolysis products appear to be derived by spontaneous breakdown of thalidomide in the body, although it is possible that any of the hydrolytic reactions of thalidomide may be assisted by hydrolases in the body. In rats, some breakdown of thalidomide occurs in the gut before absorption; hydrolysis products are present in the gastrointestinal tract following the oral administration of the drug. Thalidomide and some of its hydrolysis products can be detected in the plasma and brain of rats dosed orally with thalidomide.
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  42. 42
    Teo, S. K.; Colburn, W. A.; Tracewell, W. G.; Kook, K. A.; Stirling, D. I.; Jaworsky, M. S.; Scheffler, M. A.; Thomas, S. D.; Laskin, O. L. Clinical pharmacokinetics of thalidomide. Clin. Pharmacokinet. 2004, 43, 311– 327,  DOI: 10.2165/00003088-200443050-00004
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    Clinical pharmacokinetics of thalidomide
    Teo, Steve K.; Colburn, Wayne A.; Tracewell, William G.; Kook, Karin A.; Stirling, David I.; Jaworsky, Markian S.; Scheffler, Michael A.; Thomas, Steve D.; Laskin, Oscar L.
    Clinical Pharmacokinetics (2004), 43 (5), 311-327CODEN: CPKNDH; ISSN:0312-5963. (Adis International Ltd.)
    A review. Thalidomide is a racemic glutamic acid deriv. approved in the US for erythema nodosum leprosum, a complication of leprosy. In addn., its use in various inflammatory and oncol. conditions is being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, resp. More than 90% of the absorbed drug is excreted in the urine and feces within 48 h. Thalidomide is minimally metabolized by the liver, but is spontaneously hydrolyzed into numerous renally excreted products. After a single oral dose of thalidomide 200mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concn. (Cmax) of 1-2 mg/L at 3-4 h after administration, absorption lag time of 30 min, total exposure (AUC∞) of 18 mg • h/L, apparent elimination half-life of 6 h and apparent systemic clearance of 10 L/h. Thalidomide pharmacokinetics are best described by a one-compartment model with first-order absorption and elimination. Because of the low soly. of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacokinetics (the "flip-flop" phenomenon), with its elimination rate being faster than its absorption rate. The apparent elimination half-life of 6 h therefore represents absorption, not elimination. The "true" apparent vol. of distribution was estd. to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state Cmax (Cssmax) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accumulation, with Cssmax of 3.5 and 6.0 mg/L, resp. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400mg. Because of the low soly. of thalidomide, Cmax is less than proportional to dose, and tmax is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolyzed and passively excreted, its pharmacokinetics are not expected to change in patients with impaired liver or kidney function.
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    Beckmann, R. Ueber das Verhalten von Thalidomid im Organismus. Arzneimittelforschung 1963, 13, 185– 191
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    C-14])
    BECKMANN R
    Arzneimittel-Forschung (1963), 13 (), 185-91 ISSN:0004-4172.
    There is no expanded citation for this reference.
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    Chung, F.; Lu, J.; Palmer, B. D.; Kestell, P.; Browett, P.; Baguley, B. C.; Tingle, M.; Ching, L. M. Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients. Clin. Cancer Res. 2004, 10, 5949– 5956,  DOI: 10.1158/1078-0432.ccr-04-0421
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    Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients
    Chung, Francisco; Lu, Jun; Palmer, Brian D.; Kestell, Philip; Browett, Peter; Baguley, Bruce C.; Tingle, Malcolm; Ching, Lai-Ming
    Clinical Cancer Research (2004), 10 (17), 5949-5956CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    Thalidomide has a variety of biol. effects that vary considerably according to the species tested. The authors sought to establish whether differences in pharmacokinetics could form a basis for the species-specific effects of thalidomide. Mice and rabbits were administered thalidomide (2 mg/kg) p.o. or i.v., and plasma concns. of thalidomide were measured after drug administration using high performance liq. chromatog. Plasma samples from five multiple myeloma patients over 24 h after their first dose of thalidomide (200 mg) were similarly analyzed and all data were fitted to a one-compartment model. Metabolites of thalidomide in plasma were identified simultaneously using liq. chromatog.-mass spectrometry. Plasma concn.-time profiles for the individual patients were very similar to each other, but widely different pharmacokinetic properties were found between patients compared with those in mice or rabbits. Area under the concn. curve values for mice, rabbits, and multiple myeloma patients were 4, 8, and 81 μmol/L · hour, resp., and corresponding elimination half-lives were 0.5, 2.2, and 7.3 h, resp. Large differences were also obsd. between the metabolite profiles from the three species. Hydrolysis products were detected for all species, and the proportion of hydroxylated metabolites was higher in mice than in rabbits and undetectable in patients. Our results show major interspecies differences in the pharmacokinetics of thalidomide that are related to the altered degree of metab. The authors suggest that the interspecies differences in biol. effects of thalidomide may be attributable, at least in part, to the differences in its metab. and hence pharmacokinetics.
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    Nakamura, T.; Noguchi, T.; Miyachi, H.; Hashimoto, Y. Hydrolyzed metabolites of thalidomide: synthesis and TNF-alpha production-inhibitory activity. Chem. Pharm. Bull. 2007, 55, 651– 654,  DOI: 10.1248/cpb.55.651
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    Hydrolyzed metabolites of thalidomide: synthesis and TNF-α production-inhibitory activity
    Nakamura, Takanori; Noguchi, Tomomi; Miyachi, Hiroyuki; Hashimoto, Yuichi
    Chemical & Pharmaceutical Bulletin (2007), 55 (4), 651-654CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)
    Putative hydrolyzed metabolites of thalidomide (I) were prepd. and characterized, and their inhibitory activity on tumor necrosis factor (TNF)-α prodn. in the human monocytic leukemia cell line THP-1 was evaluated. α-(2-Carboxybenzamido)glutarimide (II) was a more potent TNF-α prodn. inhibitor than I.
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    Otogawa, K.; Ogino, Y.; Ishikawa, K.; Tanaka, M.; Shiro, M.; Osaka, T.; Asahi, T. Structural and thermal analyses of a hydrolysis compound of thalidomide. Acta Crystallogr., Sect. A: Found. Adv. 2014, 70, C113,  DOI: 10.1107/s2053273314098866
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    Krönke, J.; Hurst, S. N.; Ebert, B. L. Lenalidomide induces degradation of IKZF1 and IKZF3. OncoImmunology 2014, 3, e941742  DOI: 10.4161/21624011.2014.941742
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    Schafer, P. H.; Ye, Y.; Wu, L.; Kosek, J.; Ringheim, G.; Yang, Z.; Liu, L.; Thomas, M.; Palmisano, M.; Chopra, R. Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus. Ann. Rheum. Dis. 2018, 77, 1516– 1523,  DOI: 10.1136/annrheumdis-2017-212916
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    48
    Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus
    Schafer, Peter H.; Ye, Ying; Wu, Lei; Kosek, Jolanta; Ringheim, Garth; Yang, Zhihong; Liu, Liangang; Thomas, Michael; Palmisano, Maria; Chopra, Rajesh
    Annals of the Rheumatic Diseases (2018), 77 (10), 1516-1523CODEN: ARDIAO; ISSN:0003-4967. (BMJ Publishing Group)
    IKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacol. of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos, was studied in SLE patient cells and in a phase 1 healthy volunteer study. CRBN, IKZF1 and IKZF3 gene expression was measured in peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy volunteers. Ikaros and Aiolos protein levels were measured by Western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured in SLE PBMC cultures treated for 7 days with iberdomide. Fiftysix healthy volunteers were randomized to a single dose of iberdomide (0.03-6mg, n=6 across seven cohorts) or placebo (n=2/cohort). CD19+ B cells, CD3+ T cells and intracellular Aiolos were measured by flow cytometry. Interleukin (IL)-2 and IL-1β prodn. was stimulated with anti-CD3 and lipopolysaccharide, resp., in an ex vivo whole blood assay. SLE patient PBMCs expressed significantly higher CRBN (1.5-fold), IKZF1 (2.1-fold) and IKZF3 (4.1- fold) mRNA levels compared with healthy volunteers. Iberdomide significantly reduced Ikaros and Aiolos protein levels in B cells, T cells and monocytes. In SLE PBMC cultures, iberdomide inhibited anti-dsDNA and anti-phospholipid autoantibody prodn. (IC50 ≈10 nM). Single doses of iberdomide (0.3-6mg) in healthy volunteers decreased intracellular Aiolos (min. mean per cent of baseline: ≈12%-28% (B cells); ≈0%-33% (T cells)), decreased abs. CD19+ B cells, increased IL-2 and decreased IL-1β prodn. ex vivo. These findings demonstrate pharmacodynamic activity of iberdomide and support its further clin. development for the treatment of SLE.
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  49. 49
    Matyskiela, 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, 535– 542,  DOI: 10.1021/acs.jmedchem.6b01921
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    49
    A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos
    Matyskiela, 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.
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    Capitosti, S. M.; Hansen, T. P.; Brown, M. L. Facile synthesis of an azido-labeled thalidomide analogue. Org. Lett. 2003, 5, 2865– 2867,  DOI: 10.1021/ol034906w
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    Facile Synthesis of an Azido-Labeled Thalidomide Analogue
    Capitosti, Scott M.; Hansen, Todd P.; Brown, Milton L.
    Organic Letters (2003), 5 (16), 2865-2867CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    A five-step synthesis of an azido-thalidomide analog I is presented. The sequence requires cheap and readily available starting materials and reagents, and only two steps require purifn. Addnl., I possesses activity comparable to that of thalidomide in inhibiting the proliferation of human microvascular endothelial cells, thus providing impetus for its use as a potential photoaffinity label of thalidomide.
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    Joossens, J.; Van der Veken, P.; Lambeir, A.-M.; Augustyns, K.; Haemers, A. Development of irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator. J. Med. Chem. 2004, 47, 2411– 2413,  DOI: 10.1021/jm0499209
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    Development of Irreversible Diphenyl Phosphonate Inhibitors for Urokinase Plasminogen Activator
    Joossens, J.; Van der Veken, P.; Lambeir, A.-M.; Augustyns, K.; Haemers, A.
    Journal of Medicinal Chemistry (2004), 47 (10), 2411-2413CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The authors report the synthesis and biochem. evaluation of selective, irreversible di-Ph phosphonate inhibitors for urokinase plasminogen activator (uPA). A di-Ph phosphonate group was introduced on the substrate-like peptide Z-D-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A kapp value in the 103 M-1 s-1 range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.
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    Kanuma, K.; Omodera, K.; Nishiguchi, M.; Funakoshi, T.; Chaki, S.; Nagase, Y.; Iida, I.; Yamaguchi, J.-i.; Semple, G.; Tran, T.-A.; Sekiguchi, Y. Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists. Bioorg. Med. Chem. 2006, 14, 3307– 3319,  DOI: 10.1016/j.bmc.2005.12.052
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    Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists
    Kanuma, Kosuke; Omodera, Katsunori; Nishiguchi, Mariko; Funakoshi, Takeo; Chaki, Shigeyuki; Nagase, Yasuko; Iida, Izumi; Yamaguchi, Jun-ichi; Semple, Graeme; Tran, Thuy-Anh; Sekiguchi, Yoshinori
    Bioorganic & Medicinal Chemistry (2006), 14 (10), 3307-3319CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    The optimization of the distance between two key pharmacophore features within our first hit compds. led to the identification of a new class of potent non-peptidic antagonists for the MCH-R1, based around 4-amino-2-cyclohexylaminoquinazolines. In particular, ATC0065, N 2-[cis-4-({2-[4-Bromo-2-(trifluoromethoxy)phenyl]ethyl}amino)cyclohexyl]-N4,N4-dimethylquinazoline-2,4-diamine dihydrochloride, bound with high affinity to the MCH-R1 (IC50 value of 16 nM) and showed good metabolic stability in liver microsomes from human and rat.
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    Nouch, R.; Cini, M.; Magre, M.; Abid, M.; Diéguez, M.; Pàmies, O.; Woodward, S.; Lewis, W. Enantioselective synthesis of 6,6-disubstituted pentafulvenes containing a chiral pendant hydroxy group. Chem.—Eur. J. 2017, 23, 17195– 17198,  DOI: 10.1002/chem.201704247
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    Enantioselective Synthesis of 6,6-Disubstituted Pentafulvenes Containing a Chiral Pendant Hydroxy Group
    Nouch, Ryan; Cini, Melchior; Magre, Marc; Abid, Mohammed; Dieguez, Montserrat; Pamies, Oscar; Woodward, Simon; Lewis, William
    Chemistry - A European Journal (2017), 23 (68), 17195-17198CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Simple enantioselective synthesis of 6,6-disubstituted pentafulvenes bearing chiral pendant hydroxy groups are attained by cascade reactivity using com. available proline-based organocatalysts. Condensation of cyclopentadiene with the acetyl function of a 1,2-formylacetophenone, followed by cyclization of a resulting fulvene-stabilized carbanion with the formyl group, generates bicyclic chiral alcs. with initial er values up to 94:6. Exceptional enantio-enrichment of the resultant alcs. results upon crystn.-even near racemic samples spontaneously de-racemize. This enables new families of substituted cyclopentadienes that are both enantiomerically and diastereomerically pure to be rapidly attained.
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    Cao, R.; Müller, P.; Lippard, S. J. Tripodal tris-tacn and tris-dpa platforms for assembling phosphate-templated trimetallic centers. J. Am. Chem. Soc. 2010, 132, 17366– 17369,  DOI: 10.1021/ja108212v
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    Tripodal Tris-tacn and Tris-dpa Platforms for Assembling Phosphate-Templated Trimetallic Centers
    Cao, Rui; Muller, Peter; Lippard, Stephen J.
    Journal of the American Chemical Society (2010), 132 (49), 17366-17369CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Multidentate tripodal ligands, N(CH2-m-C6H4-CH2tacn)3 (L1) and N(CH2-o-C6H4-CH2N(CH2py)2)3 (L2), were devised for assembling high-nuclearity metal clusters. By using the same tripodal platform with different ligand appendages, either triazacyclononanes or dipicolylamines, and functionalizing either the ortho or the meta positions on the tris(xylyl) linker arms, discrete trimetal phosphate units of relevance to phosphate-metabolizing trimetallic centers in biol. were prepd. Four such compds., [(CuIICl)3(HPO4)L1](PF6) (1), [(CuIICl)3(HAsO4)L1](PF6) (2), Na2[MnIII6MnII2(H2O)2(HPO4)6(PO4)4(L1)2] (3), and [CoII3(H2PO4)Cl2(MeCN)L2](PF6)3 (4), all contg. three metal centers bound to a central phosphate or arsenate unit bridging oxygen atoms, were synthesized and structurally characterized. These results demonstrate the propensity of this novel tripodal ligand platform, in the presence of phosphate or arsenate, to assemble {M3(EO4)} units and thus structurally mimic trimetallic active sites of proteins involved in phosphate metab. Reactivity studies reveal that the tricopper complex 1 is more efficient than monocopper analogs in catalyzing the hydrolysis of 4-nitrophenyl phosphate.
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    Steinebach, C.; Lindner, S.; Udeshi, N. D.; Mani, D. C.; Kehm, H.; Köpff, S.; Carr, S. A.; Gütschow, M.; Krönke, J. Homo-PROTACs for the chemical knockdown of cereblon. ACS Chem. Biol. 2018, 13, 2771– 2782,  DOI: 10.1021/acschembio.8b00693
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    Homo-PROTACs for the Chemical Knockdown of Cereblon
    Steinebach, Christian; Lindner, Stefanie; Udeshi, Namrata D.; Mani, Deepak C.; Kehm, Hannes; Koepff, Simon; Carr, Steven A.; Guetschow, Michael; Kroenke, Jan
    ACS Chemical Biology (2018), 13 (9), 2771-2782CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degrdn. of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degrdn. By linking two pomalidomide mols., we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degrdn. The homodimerized compd. I was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of I for CRBN degrdn. was confirmed at the proteome level by quant. mass spectrometry. Inactivation by compd. I did not affect proliferation of different cell lines, prevented pomalidomide-induced degrdn. of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the mol. mechanism of thalidomide analogs.
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Cite this: J. Med. Chem. 2019, 62, 14, 6615–6629
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Published June 28, 2019

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  • Abstract

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    Figure 1

    Figure 1. Chemical structures of thalidomide, lenalidomide, pomalidomide, avadomide, and iberdomide with their glutarimide moiety shown in red.

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    Figure 2

    Figure 2. The first panel of chemical structures, five- and six-ring based thalidomide analogues and their affinity to MsCI4 determined as Ki values in the FRET assay.

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    Scheme 1

    Scheme 1. a

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    aReagents and conditions: (a) CDI, 4-DMAP, THF, reflux, 17–48 h; (b) TFA, RT, 30 min; (c) CDI, 4-DMAP, THF, reflux, 5–20 h; and (d) 10 wt % Pd/C, EtOAc, RT, 20 h.

    Figure 3

    Figure 3. Overview of the thalidomide-binding mode and electron densities of thalidomide analogues and their hydrolysis products bound to MsCI4. Left: Cartoon representation of thalidomide-bound MsCI4 with key residues of the binding pocket shown as sticks. Right: FO–FC maps of CBG (PDB 6R0Q), 4a and hydrolyzed 4a (PDB 6R0S), hydrolyzed 4b (PDB 6R0V), 5a, and hydrolyzed 5a (PDB 6R0U) and 5b (PDB 6R11) in the MsCI4-binding pocket, contoured at 2σ.

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    Figure 4

    Figure 4. Binding mode of the initial compounds and their hydrolysis products inside the binding pocket. Thalidomide, 4a, and 5a are shown with their respective hydrolysis products. Ring opening of the phtaloyl moiety that leads to the observed hydrolysis product is indicated in red in the chemical drawings. 5b was exclusively found in the nonhydrolyzed form, whereas 4b was exclusively found as a hydrolysis product. PDB codes are 4V2Y (thalidomide), 6R0Q (CBG), 6R0S (4a), 6R0U (5a), 6R11 (5b), and 6R0V (4b).

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    Scheme 2

    Scheme 2. a

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    aReagents and conditions: (a) CDI, 4-DMAP, THF, reflux, 48 h; (b) TFA, RT, 30 min; (c) 6a–h, DIPEA, THF, 0 oC to reflux, and 2 h.

    Figure 5

    Figure 5. Compounds inspired by hydrolysis products of thalidomide analogues and their affinity data. (A) Second panel of compounds, based on the 3-amidosuccinimide scaffold. (B) Third panel of compounds, inspired by 7d. * 20a could not be purified to satisfactory levels for affinity testing.

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    Figure 6

    Figure 6. Binding modes of compounds from the second and third panel. (A) Superposition of all compounds bound to MsCI4. (B) Detailed side and top view of bound effectors based on the 3-amidosuccinimide scaffold: 7a (yellow, PDB 6R1X), 7b (sand, PDB 6R12), 7c (pink, PDB 6R1K), 7d (green, PDB 6R1D), 7f (blue, PDB 6R13), and the water soluble 11a (cyan, PDB 6R18), and 12a (light blue, PDB 6R1C), indicating interactions with N50. (C) Side and top view of compounds based on 3-amidoglutarimide, 16b (brown, PDB 6R1W) and 20b (dark green, PDB 6R1A). Although the depicted instances for this scaffold do not show the interaction with N50, this interaction was observed in one other instance for 16b.

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    Scheme 3

    Scheme 3. a

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    aReagents and conditions: (a) Phosgene solution 15 wt % in toluene, THF, 0 oC to RT, 20 h; (b) 3a or 3b, DIPEA, THF, 0 oC to reflux, 20 h; (c) TFA, DCM, 0 oC to RT, 2 h; (d) Et3N, DMF, 0 oC to RT, 20 h. Note that 13 and thereby also 14–16 are in cis conformation.

    Figure 7

    Figure 7. FO–FC maps of bound compounds shown in Figure 6. All compounds are clearly defined by their electron density maps with the exception of the prolonged extension in 12a. Crystallographic structures were refined to resolutions between 1.1 and 1.8 Å, and the maps are contoured at 2σ. PDB accession codes are 6R1X (7a), 6R12 (7b), 6R1K (7c), 6R1D (7d), 6R13 (7f), 6R18 (11a), 6R1C (12a), 6R1W (16b), 6R19 (20a), and 6R1A (20b).

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    Figure 8

    Figure 8. Compound-mediated IKFZ3 degradation in OPM-2 cells. (A) Immunoblot analysis of IKZF3 levels after treatment with compounds 7d, 5a, and 7f (100 μM) for 24 h, compared to DMSO (control) and lenalidomide (20 μM) (n = 3). Lenalidomide at 100 μM reduced IKFZ3 levels effectively by 100% (not shown). (B) Averaged IKZF3 levels from the three independent experiments, normalized against total protein loading control (Figure S2). The significance of the data was tested comparing IKZF3 levels in the presence of the compounds and DMSO (p > 0.05).

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  • References

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      Somers, G. F.
      British Journal of Pharmacology and Chemotherapy (1960), 15 (), 111-16CODEN: BJPCAL; ISSN:0366-0826.
      This new non-toxic non-barbiturate sedative was assayed on male albino rats, mice, rabbits, cats, and guinea pigs for toxicity, effect on motor activity, reflexes, narcosis, analgesis, antipyresis, autonomic system, and gastrointestinal tract. It was virtually nontoxic, and had no effect on these systems, possibly because of limited absorption. It potentiated barbiturates, alc., reserpine, chlorpromazine, and counteracted methylamphetamine and methylphenidate.
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    2. 2
      Lenz, W. A short history of thalidomide embryopathy. Teratology 1988, 38, 203– 215,  DOI: 10.1002/tera.1420380303
      2
      A short history of thalidomide embryopathy
      Lenz W
      Teratology (1988), 38 (3), 203-15 ISSN:0040-3709.
      There is no expanded citation for this reference.
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    3. 3
      Vargesson, N. Thalidomide-induced limb defects: resolving a 50-year-old puzzle. Bioessays 2009, 31, 1327– 1336,  DOI: 10.1002/bies.200900103
      3
      Thalidomide-induced limb defects: resolving a 50-year-old puzzle
      Vargesson, Neil
      BioEssays (2009), 31 (12), 1327-1336CODEN: BIOEEJ; ISSN:0265-9247. (Wiley-Blackwell)
      A review. Despite the recent discovery that thalidomide causes limb defects by targeting highly angiogenic, immature blood vessels, several challenges still remain and new ones have arisen. These include understanding the drug's species specificity, detg. mol. target(s) in the endothelial cell, shedding light on the mol. basis of phocomelia and producing a form of the drug that is clin. effective without having side effects. Now that the trigger of thalidomide-induced teratogenesis has been uncovered, a framework is proposed, incorporating and uniting previous models of thalidomide action, explaining how thalidomide causes not just limb defects, but also all the other defects it induces.
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    4. 4
      Johnston, R. E.; Abdalla, S. H. Thalidomide in low doses is effective for the treatment of resistant or relapsed multiple myeloma and for plasma cell leukaemia. Leuk. Lymphoma 2002, 43, 351– 354,  DOI: 10.1080/10428190290006143
      4
      Thalidomide in low doses is effective for the treatment of resistant or relapsed multiple myeloma and for plasma cell leukemia
      Johnston, R. E.; Abdalla, S. H.
      Leukemia & Lymphoma (2002), 43 (2), 351-354CODEN: LELYEA; ISSN:1042-8194. (Taylor & Francis Ltd.)
      Thalidomide is an effective treatment for relapsed multiple myeloma (MM), but is assocd. with a significant side effect profile at higher doses. In a recent study, only half of the enrolled patients were able to tolerate the max. dose of 800 mg/day [Singhal, S., et al. (1999) "Antitumor activity of thalidomide in refractory multiple myeloma", New Engl. J. Med. 341, 1565-1571]. Moreover, the dose-response relationship has not been defined. We report our use of low dose thalidomide in a small cohort of 12 patients-eight with relapsed or refractory MM and four with plasma cell leukemia (PCL). Five of the 12 (42%) patients had a partial response, showing a median fall in their PP/BJP of 80% (63-90%) at a median dose of 175 mg (100-300 mg) with negligible side effects. Three of four patients with PCL showed an impressive response to treatment with thalidomide as a single agent. No patient who failed to show any evidence of response at low dose (<150 mg/day) responded to higher doses. In this study, thalidomide induces a similar rate of response at a lower and better tolerated dose than previously reported and produced "best ever" responses in patients with resistant PCL.
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    5. 5
      US Thalomid Label. https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/020785s061lbl.pdf (accessed Dec 9, 2018).
      There is no corresponding record for this reference.
    6. 6
      Bertolini, F.; Mingrone, W.; Alietti, A.; Ferrucci, P. F.; Cocorocchio, E.; Peccatori, F.; Cineri, S.; Mancuso, P.; Corsini, C.; Burlini, A.; Zucca, E.; Martinelli, G. Thalidomide in multiple myeloma, myelodysplastic syndromes and histiocytosis. Analysis of clinical results and of surrogate angiogenesis markers. Ann. Oncol. 2001, 12, 987– 990,  DOI: 10.1023/a:1011141009812
      6
      Thalidomide in multiple myeloma, myelodysplastic syndromes and histiocytosis. Analysis of clinical results and of surrogate angiogenesis markers
      Bertolini F; Mingrone W; Alietti A; Ferrucci P F; Cocorocchio E; Peccatori F; Cinieri S; Mancuso P; Corsini C; Burlini A; Zucca E; Martinelli G; Cineri S
      Annals of oncology : official journal of the European Society for Medical Oncology (2001), 12 (7), 987-90 ISSN:0923-7534.
      BACKGROUND: Thalidomide, as a single agent, has been recently found to induce a clinical response in one third of refractory or relapsed myeloma patients. Although it has been reported that thalidomide significantly inhibits angiogenesis. it is still unclear whether its clinical effect is mediated, at least in part, by its anti-angiogenic properties. PATIENTS AND METHODS: We evaluated thalidomide as a single agent in myeloma, myelodysplastic syndromes (MDS) and histiocytosis, i.e. hematological diseases characterized by increased angiogenesis, and measured prospectively a number of surrogate angiogenesis markers. RESULTS: Clinical responses were observed in 7 of 17 myeloma and 2 of 5 MDS patients. The histiocytosis patient had a partial response. At the time of the best clinical response, plasma levels of angiogenic growth factors, vascular endothelial growth factor (VEGF) and basic-fibroblast growth factor (b-FGF), were significantly decreased, and flow cytometry indicated a decrease of activated endothelial cells in the bone marrow of responding MDS patients. CONCLUSIONS: These observations confirm thalidomide efficacy in myeloma, suggest a possible use in MDS and histiocytosis and may contribute to the prediction of clinical response and to understanding the mechanism of thalidomide's action.
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    7. 7
      Kale, V.; List, A. Immunomodulatory drugs (IMiDs): a new treatment option for myelodysplastic syndromes. Curr. Pharm. Biotechnol. 2006, 7, 339– 342,  DOI: 10.2174/138920106778521587
      7
      Immunomodulatory drugs (IMiDs): a new treatment option for myelodysplastic syndromes
      Kale, Vishakha; List, Alan F.
      Current Pharmaceutical Biotechnology (2006), 7 (5), 339-342CODEN: CPBUBP; ISSN:1389-2010. (Bentham Science Publishers Ltd.)
      A review. The IMiDs represent a new proprietary class of thalidomide analogs that possess greater potency and less toxicity than the parent compd. As a group, these agents share the pharmacol. property of modulating cellular response to ligand activation, the precise biol. effect of which is cell lineage and stimulant-dependent. Lenalidomide (CC-5013; Revlimid), a second generation IMiD, has shown significant erythropoietic activity in patients with lower risk MDS that have failed or are not candidates for recombinant erythropoietin treatment. Unlike cytokine therapy, lenalidomide suppresses select MDS clones and enhances erythropoietin receptor signaling to restore erythropoiesis. Activity is greatest in patients with interstitial deletions involving chromosome 5q31.1. A multicenter phase II study reported a 76% overall transfusion response rate in transfusion-dependent patients with deletion 5q, with 67% achieving transfusion independence after a median interval of 4.6 wk of treatment. Cytogenetic responses were obsd. in 73% of patients with complete cytogenetic remission in 45% patients. Both transfusion response and cytogenetic response frequency were independent of karyotype complexity, raising excitement that this new treatment strategy might favorably alter the natural history of disease in higher risk patients with deletion 5q. Lenalidomide was approved by the U.S. Food and Drug Administration on Dec. 27, 2005, for the treatment of IPSS Low and intermediate-1 risk MDS patients with del(5q) abnormality. A phase III Intergroup trial (ECOG 2905) will test the capacity to potentiate erythropoietin response by comparing response to lenalidomide monotherapy to the combination of darbepoetin and lenalidomide in non-deletion 5q MDS patients.
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    8. 8
      Wu, H.; Zhao, C.; Gu, K.; Jiao, Y.; Hao, J.; Sun, G. Thalidomide plus chemotherapy exhibit enhanced efficacy in the clinical treatment of T-cell non-Hodgkin’s lymphoma: A prospective study of 46 cases. Mol. Clin. Oncol. 2014, 2, 695– 700,  DOI: 10.3892/mco.2014.307
      8
      Thalidomide plus chemotherapy exhibit enhanced efficacy in the clinical treatment of T-cell non-Hodgkin's lymphoma: A prospective study of 46 cases
      Wu Hongyang; Zhao Chenchen; Gu Kangsheng; Jiao Yang; Hao Jiqing; Sun Guoping
      Molecular and clinical oncology (2014), 2 (5), 695-700 ISSN:2049-9450.
      The treatment of T-cell non-Hodgkin's lymphoma (T-NHL) remains challenging. There is currently no standard regimen for the treatment of T-NHL in the first- or second-line setting. Thalidomide was previously shown to exert antitumor effects through inhibiting angiogenesis, promoting apoptosis and immunomodulatory activity. However, all the previous studies on the treatment of lymphoma with thalidomide included patient samples of limited size. In the present study, 46 cases of eligible T-NHL patients were randomized into i) the control group (conventional combined chemotherapy, n=22) and ii) the thalidomide group (thalidomide plus combined chemotherapy, n=24). The median dose of thalidomide was 200 mg (range, 150-400 mg) every night, without reported severe side effects. The clinical response to treatment was as follows: Complete response (CR) in 12 cases, partial response (PR) in 7, stable disease (SD) in 1 and progressive disease (PD) in 4 cases in the thalidomide group; and CR in 8 cases, PR in 6, SD in 3 and PD in 5 cases in the control group. The CR rate was 50.0 and 36.4% in the thalidomide and the control groups, respectively (P<0.05). The median progression-free and overall survival were 12 and undefined months, respectively, in the thalidomide group and 6 and 17 months, respectively, in the control group. The toxicity profile was considered acceptable in both groups. Our results indicated that thalidomide plus combined chemotherapy may exhibit enhanced efficacy in the clinical treatment of T-NHL. In addition, this type of treatment may reduce the frequency of adverse gastrointestinal reactions and help alleviate fear of chemotherapy. Therefore, thalidomide plus combined chemotherapy may be a viable option for the clinical treatment of T-NHL.
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    9. 9
      García-Sanz, R.; González-López, T. J.; Vázquez, L.; Hermida, G.; Graciani, I. F.; San Miguel, J. F. The combination of thalidomide, cyclophosphamide and dexamethasone is potentially useful in highly resistant Hodgkin’s lymphoma. Eur. J. Haematol. 2010, 84, 266– 270,  DOI: 10.1111/j.1600-0609.2009.01375.x
      9
      The combination of thalidomide, cyclophosphamide and dexamethasone is potentially useful in highly resistant Hodgkin's lymphoma
      Garcia-Sanz, R.; Gonzalez-Lopez, T. J.; Vazquez, L.; Hermida, G.; Graciani, I. F.; San Miguel, J. F.
      European Journal of Haematology (2010), 84 (3), 266-270CODEN: EJHAEC; ISSN:0902-4441. (Wiley-Blackwell)
      Few diseases have a prognosis worse than Hodgkin's lymphoma (HL), patients relapsing after autologous or allogeneic stem cell transplantation. Here, we report two highly refractory patients with HL who successfully responded to a combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex). Despite the use of a very large no. of different drugs (>5 different schemes) including high-dose therapy and autologous and allogeneic stem cell transplantation, both patients proved to be suffering from a highly resistant disease. Fortunately, they finally responded to the ThaCyDex combination, achieving sustained complete remission that would support the running of a trial within this setting.
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    10. 10
      Kuruvilla, J.; Song, K.; Mollee, P.; Panzarella, T.; McCrae, J.; Nagy, T.; Crump, M.; Keating, A. A phase II study of thalidomide and vinblastine for palliative patients with Hodgkin’s lymphoma. Hematology 2006, 11, 25– 29,  DOI: 10.1080/10245330500276592
      10
      A phase II study of thalidomide and vinblastine for palliative patients with Hodgkin's lymphoma
      Kuruvilla, John; Song, Kevin; Mollee, Peter; Panzarella, Tony; McCrae, Jan; Nagy, Tracy; Crump, Michael; Keating, Armand
      Hematology (Abingdon, United Kingdom) (2006), 11 (1), 25-29CODEN: HMATFL; ISSN:1024-5332. (Taylor & Francis Ltd.)
      Introduction: Patients with Hodgkin's Lymphoma (HL) who relapse or progress after primary therapy and subsequent high dose chemotherapy with autologous stem cell transplantation (ASCT) cannot be cured with conventional treatment. We combined thalidomide (THAL), an agent with anti-angiogenic and immunomodulatory properties, with vinblastine, which is active after ASCT, to det. the objective response rate, improvement in B symptoms and toxicity in patients with refractory HD. Methods: Patients were eligible if they HD that progressed after chemotherapy and ASCT or had declined or were ineligible for curative therapy. Treatment consisted of THAL 200 mg orally given daily. After 2 wk, VBL 6 mg IV was given weekly × 6 doses on an eight-week cycle. Response and toxicity assessment occurred following each cycle. Results: Eleven patients were enrolled, 1 progressed within 6 days of study enrollment and was subsequently treated with alternative palliative therapy and thus 11 patients are response evaluable and 10 are evaluable for toxicity. Patient characteristics: relapsed after ASCT: 7; median no. of prior chemotherapy regimens: 3 (range 1-5); median time to progression post-ASCT: 7 mo (range 2-29). Four patients had a partial response to treatment (response rate 36%); two patients had stable disease. B symptoms were present at enrollment in four patients and resolved completely on treatment in two patients. Five had disease progression within 3 mo of starting treatment. The median duration of response was 9 mo (range 0-22 mo). Toxicity was mild and limited to grade 2 neuropathy in 6 patients and grade 2 or 3 neutropenia in 4 patients. Conclusions: In this small study in chemotherapy- refractory HL, THAL and VBL demonstrated encouraging activity with some durable responses and acceptable toxicity. These results suggest that chronic low dose chemotherapy combined with less toxic immunomodulatory or anti-angiogenic drugs warrants further study.
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    11. 11
      Seldin, D. C.; Choufani, E. B.; Dember, L. M.; Wiesman, J. F.; Berk, J. L.; Falk, R. H.; O’Hara, C.; Fennessey, S.; Finn, K. T.; Wright, D. G.; Skinner, M.; Sanchorawala, V. Tolerability and efficacy of thalidomide for the treatment of patients with light chain-associated (AL) amyloidosis. Clin. Lymphoma 2003, 3, 241– 246,  DOI: 10.3816/clm.2003.n.005
      11
      Tolerability and efficacy of thalidomide for the treatment of patients with light-chain-associated (AL) amyloidosis
      Seldin, David C.; Choufani, Elie B.; Dember, Laura M.; Wiesman, Janice F.; Berk, John L.; Falk, Rodney H.; O'Hara, Carl; Fennessey, Salli; Finn, Kathleen T.; Wright, Daniel G.; Skinner, Martha; Sanchorawala, Vaishali
      Clinical Lymphoma (2003), 3 (4), 241-246CODEN: CLLYAO; ISSN:1526-9655. (Cancer Information Group)
      A phase I/II trial was carried out with patients with AL amyloidosis, most of whom had failed prior therapy with high-dose melphalan and autologous stem cell transplantation. This trial was designed as an individualized 6-mo dose-escalation study with re-evaluation of bone marrow plasmacytosis and serum and urine monoclonal proteins after 3 and 6 mo. Sixteen patients with a median age of 62 yr (range, 37-70 yr) were enrolled. Fourteen of the patients had renal involvement, 4 had cardiac involvement, 4 had liver involvement, and 2 had predominant soft tissue or lymph node involvement. The median max. tolerated dose was 300 mg, with fatigue and other central nervous system side effects being the major dose-limiting toxicities. Side effects not frequently reported for other patient populations included exacerbation of peripheral and pulmonary edema and worsening azotemia. In all, 50% of the patients experienced grade 3/4 toxicity, and 25% had to discontinue the drug. No complete hematol. responses were seen, but 25% of the patients had a significant redn. in Bence-Jones proteinuria. Thus, while thalidomide has activity in AL amyloidosis, it also has significant toxicity in this patient population.
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    12. 12
      Baird, R.; van Zyl-Smit, R. N.; Iveson, A.; Duddy, J.; Rassam, S. M. Thalidomide is highly effective in a patient with meningeal acute myeloid leukaemia. Leuk. Lymphoma 2004, 45, 179– 181,  DOI: 10.1080/1042819031000149412
      12
      Thalidomide is highly effective in a patient with meningeal acute myeloid leukaemia
      Baird Richard; van Zyl-Smit Richard Nellis; Iveson Anne; Duddy James; Rassam Saad M B
      Leukemia & lymphoma (2004), 45 (1), 179-81 ISSN:1042-8194.
      We report a case of secondary acute myeloid leukaemia (AML) following high dose therapy for diffuse large B-cell non-Hodgkin's lymphoma (NHL) who developed meningeal leukaemia. This was refractory to systemic and intrathecal chemotherapy and cranial irradiation. Thalidomide has been reported to have anti-AML activity and appears to cross the blood brain barrier (BBB). We, therefore, attempted a trial of oral Thalidomide and achieved rapid biochemical and cytological remission with a short course. The patient, however, progressed systemically and succumbed to her illness.
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    13. 13
      Thomas, D. A.; Estey, E.; Giles, F. J.; Faderl, S.; Cortes, J.; Keating, M.; O’Brien, S.; Albitar, M.; Kantarjian, H. Single agent thalidomide in patients with relapsed or refractory acute myeloid leukaemia. Br. J. Haematol. 2003, 123, 436– 441,  DOI: 10.1046/j.1365-2141.2003.04639.x
      13
      Single agent thalidomide in patients with relapsed or refractory acute myeloid leukaemia
      Thomas, Deborah A.; Estey, Elihu; Giles, Francis J.; Faderi, Stefan; Cortes, Jorge; Keating, Michael; O'Brien, Susan; Albitar, Maher; Kantarjian, Hagop
      British Journal of Haematology (2003), 123 (3), 436-441CODEN: BJHEAL; ISSN:0007-1048. (Blackwell Publishing Ltd.)
      Thalidomide is a putative anti-angiogenesis agent that has significant anti-tumor activity in haematol. malignancies with increased bone marrow angiogenesis, including multiple myeloma (MM) and myelodysplastic syndromes (MDS). Increased levels of the mitogen for angiogenesis, vascular endothelial growth factor (VEGF), correlate with worse survival in acute myeloid leukemia (AML). A phase II trial of thalidomide was conducted in patients with relapsed- or refractory-AML previously treated with cytarabine-contg. regimens. A total of 16 patients with refractory- or relapsed-AML were treated with thalidomide 200-800 mg orally daily (median dose 400 mg daily) for a median of 27 d (range, 3-94 d). Overall, one patient (6%) achieved complete remission (CR) lasting for 36 mo, and two patients had a transient redn. in marrow blasts from 8% and 7% to less than 5% in both cases. There was no correlation between redn. in levels of angiogenesis markers and response. Toxicities related to thalidomide were significant, and precluded dose escalation beyond 400 mg orally daily in most patients. Although there appears to be some evidence of biol. activity, single agent thalidomide is not an optimal choice of therapy for salvaging patients with relapsed- or refractory-AML. Thalidomide analogs with more potent immunomodulatory activities and more favorable toxicity profiles may offer more promise as anti-AML therapy.
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    14. 14
      Hartmann, 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, 225– 232,  DOI: 10.1016/j.jsb.2014.10.010
      14
      Thalidomide mimics uridine binding to an aromatic cage in cereblon
      Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Zanini, Fabio; Lupas, Andrei N.; Hernandez Alvarez, Birte
      Journal 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.
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    15. 15
      Hartmann, M. D.; Boichenko, I.; Coles, M.; Lupas, A. N.; Hernandez Alvarez, B. Structural dynamics of the cereblon ligand binding domain. PLoS One 2015, 10, e0128342  DOI: 10.1371/journal.pone.0128342
      15
      Structural dynamics of the cereblon ligand binding domain
      Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Alvarez, Birte Hernandez
      PLoS 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.
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    16. 16
      Chamberlain, P. P.; Lopez-Girona, A.; Miller, K.; Carmel, G.; Pagarigan, B.; Chie-Leon, B.; Rychak, E.; Corral, L. G.; Ren, Y. J.; Wang, M.; Riley, M.; Delker, S. L.; Ito, T.; Ando, H.; Mori, T.; Hirano, Y.; Handa, H.; Hakoshima, T.; Daniel, T. O.; Cathers, B. E. Structure of the human cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 2014, 21, 803– 809,  DOI: 10.1038/nsmb.2874
      16
      Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs
      Chamberlain, Philip P.; Lopez-Girona, Antonia; Miller, Karen; Carmel, Gilles; Pagarigan, Barbra; Chie-Leon, Barbara; Rychak, Emily; Corral, Laura G.; Ren, Yan J.; Wang, Maria; Riley, Mariko; Delker, Silvia L.; Ito, Takumi; Ando, Hideki; Mori, Tomoyuki; Hirano, Yoshinori; Handa, Hiroshi; Hakoshima, Toshio; Daniel, Thomas O.; Cathers, Brian E.
      Nature Structural & Molecular Biology (2014), 21 (9), 803-809CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degrdn. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are crit. for antiproliferative effects.
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    17. 17
      Fischer, E. S.; Böhm, 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. J.; Harper, J. W.; Jenkins, J. L.; Thomä, N. H. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 2014, 512, 49– 53,  DOI: 10.1038/nature13527
      17
      Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide
      Fischer, 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.
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    18. 18
      Nguyen, T. V.; Lee, J. E.; Sweredoski, M. J.; Yang, S. J.; Jeon, S. J.; Harrison, J. S.; Yim, J. H.; Lee, S. G.; Handa, H.; Kuhlman, B.; Jeong, J. S.; Reitsma, J. M.; Park, C. S.; Hess, S.; Deshaies, R. J. Glutamine triggers acetylation-dependent degradation of glutamine synthetase via the thalidomide receptor cereblon. Mol. Cell 2016, 61, 809– 820,  DOI: 10.1016/j.molcel.2016.02.032
      18
      Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon
      Nguyen, T. Van; Lee, J. Eugene; Sweredoski, Michael J.; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S.; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M.; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J.
      Molecular Cell (2016), 61 (6), 809-820CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4CRBN. Upon exposing cells to high glutamine concn., GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4CRBN and degrdn. by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4CRBN that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.
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    19. 19
      Del Prete, D.; Rice, R. C.; Rajadhyaksha, A. M.; D’Adamio, L. Amyloid precursor protein (APP) may act as a substrate and a recognition unit for CRL4(CRBN) and Stub1 E3 ligases facilitating ubiquitination of proteins involved in presynaptic functions and neurodegeneration. J. Biol. Chem. 2016, 291, 17209– 17227,  DOI: 10.1074/jbc.m116.733626
      19
      Amyloid precursor protein (APP) may act as a substrate and a recognition unit for CRL4CRBN and Stub1 E3 ligases facilitating ubiquitination of proteins involved in presynaptic functions and neurodegeneration
      Del Prete, Dolores; Rice, Richard C.; Rajadhyaksha, Anjali M.; D'Adamio, Luciano
      Journal of Biological Chemistry (2016), 291 (33), 17209-17227CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the N-terminus of the ACR, and CRL4CRBN, which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the C-terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4CRBN, pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome anal. indicates that these E3 ligases are enzymically active and ubiquitinated the ACR residues Lys649/650/651/676/688. Deletion of Crbn reduces ubiquitination of Lys676 suggesting that Lys676 is physiol. ubiquitinated by CRL4CRBN. The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro. This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.
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    20. 20
      Kronke, 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, 301– 305,  DOI: 10.1126/science.1244851
      20
      Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells
      Kronke 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 L
      Science (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.
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    21. 21
      Krönke, 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.; Järås, 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, 183– 188,  DOI: 10.1038/nature14610
      21
      Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS
      Kronke, 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.
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    22. 22
      Matyskiela, M. E.; Lu, G.; Ito, T.; Pagarigan, B.; Lu, C.-C.; Miller, K.; Fang, W.; Wang, N.-Y.; Nguyen, D.; Houston, J.; Carmel, G.; Tran, T.; Riley, M.; Nosaka, L. A.; Lander, G. C.; Gaidarova, S.; Xu, S.; Ruchelman, A. L.; Handa, H.; Carmichael, J.; Daniel, T. O.; Cathers, B. E.; Lopez-Girona, A.; Chamberlain, P. P. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase. Nature 2016, 535, 252– 257,  DOI: 10.1038/nature18611
      22
      A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase
      Matyskiela, Mary E.; Lu, Gang; Ito, Takumi; Pagarigan, Barbra; Lu, Chin-Chun; Miller, Karen; Fang, Wei; Wang, Nai-Yu; Nguyen, Derek; Houston, Jack; Carmel, Gilles; Tran, Tam; Riley, Mariko; Nosaka, Lyn'Al; Lander, Gabriel C.; Gaidarova, Svetlana; Xu, Shuichan; Ruchelman, Alexander L.; Handa, Hiroshi; Carmichael, James; Daniel, Thomas O.; Cathers, Brian E.; Lopez-Girona, Antonia; Chamberlain, Philip P.
      Nature (London, United Kingdom) (2016), 535 (7611), 252-257CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4CRBN E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumor activity. The anti-tumor activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degrdn. of the translation termination factor GSPT1. Patient-derived acute myeloid leukemia tumor cells exhibit high sensitivity to CC-885, indicating the clin. potential of this mechanism. Crystallog. studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn contg. a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homol. to previously known cereblon substrates, mutational anal. and modeling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumor target rendered druggable by cereblon modulation.
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    23. 23
      Matyskiela, M. E.; Couto, S.; Zheng, X.; Lu, G.; Hui, J.; Stamp, K.; Drew, C.; Ren, Y.; Wang, M.; Carpenter, A.; Lee, C.-W.; Clayton, T.; Fang, W.; Lu, C.-C.; Riley, M.; Abdubek, P.; Blease, K.; Hartke, J.; Kumar, G.; Vessey, R.; Rolfe, M.; Hamann, L. G.; Chamberlain, P. P. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 2018, 14, 981– 987,  DOI: 10.1038/s41589-018-0129-x
      23
      SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate
      Matyskiela, Mary E.; Couto, Suzana; Zheng, Xinde; Lu, Gang; Hui, Julia; Stamp, Katie; Drew, Clifton; Ren, Yan; Wang, Maria; Carpenter, Aaron; Lee, Chung-Wein; Clayton, Thomas; Fang, Wei; Lu, Chin-Chun; Riley, Mariko; Abdubek, Polat; Blease, Kate; Hartke, James; Kumar, Gondi; Vessey, Rupert; Rolfe, Mark; Hamann, Lawrence G.; Chamberlain, Philip P.
      Nature Chemical Biology (2018), 14 (10), 981-987CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Targeted protein degrdn. via small-mol. modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clin. presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the mol. basis of thalidomide-induced teratogenicity.
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    24. 24
      Donovan, K. A.; An, J.; Nowak, R. P.; Yuan, J. C.; Fink, E. C.; Berry, B. C.; Ebert, B. L.; Fischer, E. S., Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray Syndrome. Elife 2018, 7. DOI: 10.7554/elife.38430
      There is no corresponding record for this reference.
    25. 25
      Petzold, G.; Fischer, E. S.; Thomä, N. H. Structural basis of lenalidomide-induced CK1alpha degradation by the CRL4(CRBN) ubiquitin ligase. Nature 2016, 532, 127– 130,  DOI: 10.1038/nature16979
      25
      Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase
      Petzold, 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.
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    26. 26
      Sievers, Q. L.; Petzold, G.; Bunker, R. D.; Renneville, A.; Słabicki, M.; Liddicoat, B. J.; Abdulrahman, W.; Mikkelsen, T.; Ebert, B. L.; Thomä, N. H. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 2018, 362, eaat0572  DOI: 10.1126/science.aat0572
      There is no corresponding record for this reference.
    27. 27
      Lai, A. C.; Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discovery 2017, 16, 101– 114,  DOI: 10.1038/nrd.2016.211
      27
      Induced protein degradation: an emerging drug discovery paradigm
      Lai, 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.
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    28. 28
      Sakamoto, 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, 8554– 8559,  DOI: 10.1073/pnas.141230798
      28
      Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation
      Sakamoto, 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.
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    29. 29
      Hu, J.; Hu, B.; Wang, M.; Xu, F.; Miao, B.; Yang, C.-Y.; Wang, M.; Liu, Z.; Hayes, D. F.; Chinnaswamy, K.; Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Estrogen Receptor (ER). J. Med. Chem. 2019, 62, 1420– 1442,  DOI: 10.1021/acs.jmedchem.8b01572
      29
      Discovery of ERD-308 as a highly potent proteolysis targeting chimera (PROTAC) degrader of estrogen receptor (ER)
      Hu, Jiantao; Hu, Biao; Wang, Mingliang; Xu, Fuming; Miao, Bukeyan; Yang, Chao-Yie; Wang, Mi; Liu, Zhaomin; Hayes, Daniel F.; Chinnaswamy, Krishnapriya; Delproposto, James; Stuckey, Jeanne; Wang, Shaomeng
      Journal of Medicinal Chemistry (2019), 62 (3), 1420-1442CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The estrogen receptor (ER) is a validated target for the treatment of estrogen receptor-pos. (ER+) breast cancer. Here, we describe the design, synthesis, and extensive structure-activity relationship (SAR) studies of small-mol. ERα degraders based on the proteolysis targeting chimeras (PROTAC) concept. Our efforts have resulted in the discovery of highly potent and effective PROTAC ER degraders, as exemplified by ERD-308 I. I achieves DC50 (concn. causing 50% of protein degrdn.) values of 0.17 and 0.43 nM in MCF-7 and T47D ER+ breast cancer cell lines, resp., and induces >95% of ER degrdn. at concns. as low as 5 nM in both cell lines. Significantly, I induces more complete ER degrdn. than fulvestrant, the only approved selective ER degrader (SERD), and is more effective in inhibition of cell proliferation than fulvestrant in MCF-7 cells. Further optimization of I may lead to a new therapy for advanced ER+ breast cancer.
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    30. 30
      Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.; Yang, C.-Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.; Delproposto, J.; Stuckey, J.; Wang, S. Discovery of ARD-69 as a highly potent proteolysis targeting chimera (PROTAC) degrader of androgen receptor (AR) for the treatment of prostate cancer. J. Med. Chem. 2019, 62, 941– 964,  DOI: 10.1021/acs.jmedchem.8b01631
      30
      Discovery of ARD-69 as a highly potent proteolysis targeting chimera (PROTAC) degrader of androgen receptor (AR) for the treatment of prostate cancer
      Han, Xin; Wang, Chao; Qin, Chong; Xiang, Weiguo; Fernandez-Salas, Ester; Yang, Chao-Yie; Wang, Mi; Zhao, Lijie; Xu, Tianfeng; Chinnaswamy, Krishnapriya; Delproposto, James; Stuckey, Jeanne; Wang, Shaomeng
      Journal of Medicinal Chemistry (2019), 62 (2), 941-964CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We report herein the discovery of highly potent PROTAC degraders of androgen receptor (AR), as exemplified by IARD-69. I induces degrdn. of AR protein in AR-pos. prostate cancer cell lines in a dose- and time-dependent manner. I achieves DC50 values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1 AR+ prostate cancer cell lines, resp. I is capable of reducing the AR protein level by >95% in these prostate cancer cell lines and effectively suppressing AR-regulated gene expression. I potently inhibits cell growth in these AR-pos. prostate cancer cell lines and is >100 times more potent than AR antagonists. A single dose of I effectively reduces the level of AR protein in xenograft tumor tissue in mice. Further optimization of I may ultimately lead to a new therapy for AR+, castration-resistant prostate cancer.
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    31. 31
      Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza, A.; Dhe-Paganon, S.; Bradner, J. E. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 2015, 348, 1376– 1381,  DOI: 10.1126/science.aab1433
      31
      Phthalimide conjugation as a strategy for in vivo target protein degradation
      Winter, Georg E.; Buckley, Dennis L.; Paulk, Joshiawa; Roberts, Justin M.; Souza, Amanda; Dhe-Paganon, Sirano; Bradner, James E.
      Science (Washington, DC, United States) (2015), 348 (6241), 1376-1381CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The development of effective pharmacol. inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-mol. antagonists disrupt the activity of only one domain in the target protein. The authors devised a chem. strategy that promotes ligand-dependent target protein degrdn. using as an example the transcriptional coactivator BRD4, a protein crit. for cancer cell growth and survival. The authors appended a competitive antagonist of BET bromodomains to a phthalimide moiety to hijack the cereblon E3 ubiquitin ligase complex. The resultant compd., dBET1, induced highly selective cereblon-dependent BET protein degrdn. in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degrdn. of the cytosolic protein FKBP12. This chem. strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins.
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    32. 32
      Steinebach, C.; Kehm, H.; Lindner, S.; Vu, L. P.; Köpff, S.; López Mármol, Á.; Weiler, C.; Wagner, K. G.; Reichenzeller, M.; Krönke, J.; Gütschow, M. PROTAC-mediated crosstalk between E3 ligases. Chem. Commun. 2019, 55, 1821– 1824,  DOI: 10.1039/c8cc09541h
      32
      PROTAC-mediated crosstalk between E3 ligases
      Steinebach, Christian; Kehm, Hannes; Lindner, Stefanie; Vu, Lan Phuong; Koepff, Simon; Lopez Marmol, Alvaro; Weiler, Corinna; Wagner, Karl G.; Reichenzeller, Michaela; Kroenke, Jan; Guetschow, Michael
      Chemical 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.
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    33. 33
      Lu, M.; Liu, T.; Jiao, Q.; Ji, J.; Tao, M.; Liu, Y.; You, Q.; Jiang, Z. Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway. Eur. J. Med. Chem. 2018, 146, 251– 259,  DOI: 10.1016/j.ejmech.2018.01.063
      33
      Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway
      Lu, Mengchen; Liu, Tian; Jiao, Qiong; Ji, Jianai; Tao, Mengmin; Liu, Yijun; You, Qidong; Jiang, Zhengyu
      European Journal of Medicinal Chemistry (2018), 146 (), 251-259CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      Induced protein degrdn. by PROTACs has emerged as a promising strategy to target nonenzymic proteins inside the cell. The aim of this study was to identify Keap1, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degrdn. of the nonenzymic protein Tau. A peptide PROTAC by recruiting Keap1-Cul3 ubiquitin E3 ligase was developed and applied in the degrdn. of intracellular Tau. Peptide 1 showed strong in vitro binding with Keap1 and Tau. With proper cell permeability, peptide 1 was found to colocalize with cellular Keap1 and resulted in the coimmunopptn. of Tau and Keap1. The results of flow cytometry and western blotting assays showed that peptide 1 can downregulate the intracellular Tau level in both time- and concn.-dependent manner. The application of Keap1 siRNA silencing and the proteasome inhibitor MG132 confirmed that peptide 1 could promote the Keap1-dependent poly-ubiquitination and proteasome-dependent degrdn. of Tau. The results suggested that using PROTACs to recruit Keap1 to induce the degrdn. of Tau may show promising character in the treatment of neurodegenerative disease. In addn., our research demonstrated that Keap1 should be a promising E3 ligase adaptor to be used in the design of novel PROTACs.
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    34. 34
      Kargbo, R. B. Treatment of Alzheimer’s by PROTAC-Tau protein degradation. ACS Med. Chem. Lett. 2019, 10, 699– 700,  DOI: 10.1021/acsmedchemlett.9b00083
      34
      Treatment of Alzheimer's by PROTAC-Tau Protein Degradation
      Kargbo, Robert B.
      ACS Medicinal Chemistry Letters (2019), 10 (5), 699-700CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      There is no expanded citation for this reference.
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    35. 35
      Chu, T.-T.; Gao, N.; Li, Q.-Q.; Chen, P.-G.; Yang, X.-F.; Chen, Y.-X.; Zhao, Y.-F.; Li, Y.-M. Specific knockdown of endogenous Tau protein by peptide-directed ubiquitin-proteasome degradation. Cell Chem. Biol. 2016, 23, 453– 461,  DOI: 10.1016/j.chembiol.2016.02.016
      35
      Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation
      Chu, Ting-Ting; Gao, Na; Li, Qian-Qian; Chen, Pu-Guang; Yang, Xi-Fei; Chen, Yong-Xiang; Zhao, Yu-Fen; Li, Yan-Mei
      Cell Chemical Biology (2016), 23 (4), 453-461CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
      Tau, an important pathol. protein of Alzheimer's disease (AD), can mediate the toxicity of amyloid β (Aβ). Thus, redn. of Tau with chem. mols. may offer a novel strategy for treating AD. Here, we designed and synthesized a series of multifunctional mols. that contained Tau-recognition moieties and E3 ligase-binding moieties to enhance Tau degrdn. Among these mols., TH006 had the highest activity of inducing Tau degrdn. by increasing its poly-ubiquitination. The decrement in Tau induced by TH006 could decrease the cytotoxicity caused by Aβ. Furthermore, TH006 could regulate the Tau level in the brain of an AD mouse model. Therefore, partial redn. of Tau with such multifunctional peptides may open up a novel therapeutic strategy for AD treatment.
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    36. 36
      Boichenko, I.; Bär, 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, 11163– 11171,  DOI: 10.1021/acsomega.8b00959
      36
      Chemical Ligand Space of Cereblon
      Boichenko, 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.
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    37. 37
      Burslem, G. M.; Ottis, P.; Jaime-Figueroa, S.; Morgan, A.; Cromm, P. M.; Toure, M.; Crews, C. M. Efficient synthesis of immunomodulatory drug analogues enables exploration of structure-degradation relationships. ChemMedChem 2018, 13, 1508– 1512,  DOI: 10.1002/cmdc.201800271
      37
      Efficient Synthesis of Immunomodulatory Drug Analogues Enables Exploration of Structure-Degradation Relationships
      Burslem, George M.; Ottis, Philipp; Jaime-Figueroa, Saul; Morgan, Alicia; Cromm, Philipp M.; Toure, Momar; Crews, Craig M.
      ChemMedChem (2018), 13 (15), 1508-1512CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The immunomodulatory drugs (IMiDs) thalidomide, pomalidomide, and lenalidomide have been approved for the treatment of multiple myeloma for many years. Recently, their use as E3 ligase recruiting elements for small-mol.-induced protein degrdn. has led to a resurgence in interest in IMiD synthesis and functionalization. Traditional IMiD synthesis follows a stepwise route with multiple purifn. steps. Herein we describe a novel one-pot synthesis without purifn. that provides rapid access to a multitude of IMiD analogs. Binding studies with the IMiD target protein cereblon (CRBN) reveals a narrow structure-activity relationship with only a few compds. showing sub-micromolar binding affinity in the range of pomalidomide and lenalidomide. However, anti-proliferative activity as well as Aiolos degrdn. could be identified for two IMiD analogs. This study provides useful insight into the structure-degrdn. relationships for mols. of this type as well as a rapid and robust method for IMiD synthesis.
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    38. 38
      Bartlett, J. B.; Dredge, K.; Dalgleish, A. G. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat. Rev. Cancer 2004, 4, 314– 322,  DOI: 10.1038/nrc1323
      38
      Timeline: The evolution of thalidomide and its IMiD derivatives as anticancer agents
      Bartlett, J. Blake; Dredge, Keith; Dalgleish, Angus G.
      Nature Reviews Cancer (2004), 4 (4), 314-322CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. Thalidomide was originally used to treat morning sickness, but was banned in the 1960s for causing serious congenital birth defects. Remarkably, thalidomide was subsequently discovered to have anti-inflammatory and anti-angiogenic properties, and was identified as an effective treatment for multiple myeloma. A series of immunomodulatory drugs - created by chem. modification of thalidomide - have been developed to overcome the original devastating side effects. Their powerful anticancer properties mean that these drugs are now emerging from thalidomide's shadow as useful anticancer agents.
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    39. 39
      Reist, M.; Carrupt, P.-A.; Francotte, E.; Testa, B. Chiral inversion and hydrolysis of thalidomide: mechanisms and catalysis by bases and serum albumin, and chiral stability of teratogenic metabolites. Chem. Res. Toxicol. 1998, 11, 1521– 1528,  DOI: 10.1021/tx9801817
      39
      Chiral Inversion and Hydrolysis of Thalidomide: Mechanisms and Catalysis by Bases and Serum Albumin, and Chiral Stability of Teratogenic Metabolites
      Reist, Marianne; Carrupt, Pierre-Alain; Francotte, Eric; Testa, Bernard
      Chemical Research in Toxicology (1998), 11 (12), 1521-1528CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      The chiral inversion and hydrolysis of thalidomide and the catalysis by bases and human serum albumin were investigated by using a stereoselective HPLC assay. Chiral inversion was catalyzed by albumin, hydroxyl ions, phosphate, and amino acids. Basic amino acids (Arg and Lys) had a superior potency in catalyzing chiral inversion compared to acid and neutral ones. The chiral inversion of thalidomide is thus subject to specific and general base catalysis, and it is suggested that the ability of HSA to catalyze the reaction is due to the basic groups of the amino acids Arg and Lys and not to a single catalytic site on the macromol. The hydrolysis of thalidomide was also base-catalyzed. However, albumin had no effect on hydrolysis, and there was no difference between the catalytic potencies of acidic, neutral, and basic amino acids. This may be explained by different reaction mechanisms of the chiral inversion and hydrolysis of thalidomide. Chiral inversion is deduced to occur by electrophilic substitution involving specific and general base catalysis, whereas hydrolysis is thought to occur by nucleophilic substitution involving specific and general base as well as nucleophilic catalysis. As nucleophilic attack is sensitive to steric properties of the catalyst, steric hindrance might be the reason albumin is not able to catalyze hydrolysis. 1H NMR expts. revealed that the three teratogenic metabolites of thalidomide, in sharp contrast to the drug itself, had complete chiral stability. This leads to the speculation that, were some enantioselectivity to exist in the teratogenicity of thalidomide, it could result from fast hydrolysis to chirally stable teratogenic metabolites.
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    40. 40
      Chen, T. L.; Vogelsang, G. B.; Petty, B. G.; Brundrett, R. B.; Noe, D. A.; Santos, G. W.; Colvin, O. M. Plasma pharmacokinetics and urinary excretion of thalidomide after oral dosing in healthy male volunteers. Drug Metab Dispos 1989, 17, 402– 405
      40
      Plasma pharmacokinetics and urinary excretion of thalidomide after oral dosing in healthy male volunteers
      Chen, Tian Ling; Vogelsang, Georgia B.; Petty, Brent G.; Brundrett, Robert B.; Noe, Dennis A.; Santos, George W.; Colvin, O. Michael
      Drug Metabolism and Disposition (1989), 17 (4), 402-5CODEN: DMDSAI; ISSN:0090-9556.
      The plasma pharmacokinetics and urinary excretion of thalidomide were evaluated in healthy male volunteers receiving a single oral dose of 200 mg. Concns. of thalidomide were detd. by a new HPLC assay. Plasma concn. vs. time data were well fit by a 1-compartment model. The mean peak concn., 1.15 μg/mL, was achieved at 4.39 h. Absorption and elimination half-lives were 1.70 and 8.70 h, resp., with a lag time of 0.41 h obsd. in 6 of 8 subjects. The apparent vol. of distribution and total body clearance rate, based on assumed complete bioavailability, were 120.69 L and 10.41 L/h. The urinary excretion of thalidomide accounted for only 0.6% of the total dose administered over 24 h, and the renal clearance rate was 0.08 L/h. Thus, the major route of elimination of thalidomide is nonrenal.
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    41. 41
      Schumacher, H.; Smith, R. L.; Williams, R. T. The metabolism of thalidomide: the fate of thalidomide and some of its hydrolysis products in various species. Br. J. Pharmacol. Chemother. 1965, 25, 338– 351,  DOI: 10.1111/j.1476-5381.1965.tb02054.x
      41
      The metabolism of thalidomide. The fate of thalidomide and some of its hydrolysis products in various species
      Schumacher, H.; Smith, R. L.; Williams, R. T.
      British Journal of Pharmacology and Chemotherapy (1965), 25 (2), 338-51CODEN: BJPCAL; ISSN:0366-0826.
      cf. preceding abstr. The metabolism of thalidomide in the rabbit, rat, mouse, and guinea pig was investigated. The metabolites of thalidomide present in the urine, blood, and tissues of various species dosed with the drug were characterized by comparing their chromatographic mobility and color reactions with those given by authentic samples of the compds. The rabbit urinary metabolites were isolated in cryst. form by solvent extn. and adsorption chromatography, and their identity was established by analysis, m.p. behavior, and comparison of their ir spectra with those of the authentic compds. When thalidomide is fed to rabbits, rats, mice, and guinea pigs a no. of hydrolysis products appear in the urine. These hydrolysis products are formed by the spontaneous hydrolysis of thalidomide. In addn. the urine of rabbits dosed with thalidomide contains derivs. of 3- and 4-hydroxyphthalic acid; these minor metabolites were not identified. The hydrolysis products appear to be derived by spontaneous breakdown of thalidomide in the body, although it is possible that any of the hydrolytic reactions of thalidomide may be assisted by hydrolases in the body. In rats, some breakdown of thalidomide occurs in the gut before absorption; hydrolysis products are present in the gastrointestinal tract following the oral administration of the drug. Thalidomide and some of its hydrolysis products can be detected in the plasma and brain of rats dosed orally with thalidomide.
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    42. 42
      Teo, S. K.; Colburn, W. A.; Tracewell, W. G.; Kook, K. A.; Stirling, D. I.; Jaworsky, M. S.; Scheffler, M. A.; Thomas, S. D.; Laskin, O. L. Clinical pharmacokinetics of thalidomide. Clin. Pharmacokinet. 2004, 43, 311– 327,  DOI: 10.2165/00003088-200443050-00004
      42
      Clinical pharmacokinetics of thalidomide
      Teo, Steve K.; Colburn, Wayne A.; Tracewell, William G.; Kook, Karin A.; Stirling, David I.; Jaworsky, Markian S.; Scheffler, Michael A.; Thomas, Steve D.; Laskin, Oscar L.
      Clinical Pharmacokinetics (2004), 43 (5), 311-327CODEN: CPKNDH; ISSN:0312-5963. (Adis International Ltd.)
      A review. Thalidomide is a racemic glutamic acid deriv. approved in the US for erythema nodosum leprosum, a complication of leprosy. In addn., its use in various inflammatory and oncol. conditions is being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, resp. More than 90% of the absorbed drug is excreted in the urine and feces within 48 h. Thalidomide is minimally metabolized by the liver, but is spontaneously hydrolyzed into numerous renally excreted products. After a single oral dose of thalidomide 200mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concn. (Cmax) of 1-2 mg/L at 3-4 h after administration, absorption lag time of 30 min, total exposure (AUC∞) of 18 mg • h/L, apparent elimination half-life of 6 h and apparent systemic clearance of 10 L/h. Thalidomide pharmacokinetics are best described by a one-compartment model with first-order absorption and elimination. Because of the low soly. of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacokinetics (the "flip-flop" phenomenon), with its elimination rate being faster than its absorption rate. The apparent elimination half-life of 6 h therefore represents absorption, not elimination. The "true" apparent vol. of distribution was estd. to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state Cmax (Cssmax) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accumulation, with Cssmax of 3.5 and 6.0 mg/L, resp. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400mg. Because of the low soly. of thalidomide, Cmax is less than proportional to dose, and tmax is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolyzed and passively excreted, its pharmacokinetics are not expected to change in patients with impaired liver or kidney function.
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    43. 43
      Beckmann, R. Ueber das Verhalten von Thalidomid im Organismus. Arzneimittelforschung 1963, 13, 185– 191
      43
      C-14])
      BECKMANN R
      Arzneimittel-Forschung (1963), 13 (), 185-91 ISSN:0004-4172.
      There is no expanded citation for this reference.
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    44. 44
      Chung, F.; Lu, J.; Palmer, B. D.; Kestell, P.; Browett, P.; Baguley, B. C.; Tingle, M.; Ching, L. M. Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients. Clin. Cancer Res. 2004, 10, 5949– 5956,  DOI: 10.1158/1078-0432.ccr-04-0421
      44
      Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients
      Chung, Francisco; Lu, Jun; Palmer, Brian D.; Kestell, Philip; Browett, Peter; Baguley, Bruce C.; Tingle, Malcolm; Ching, Lai-Ming
      Clinical Cancer Research (2004), 10 (17), 5949-5956CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      Thalidomide has a variety of biol. effects that vary considerably according to the species tested. The authors sought to establish whether differences in pharmacokinetics could form a basis for the species-specific effects of thalidomide. Mice and rabbits were administered thalidomide (2 mg/kg) p.o. or i.v., and plasma concns. of thalidomide were measured after drug administration using high performance liq. chromatog. Plasma samples from five multiple myeloma patients over 24 h after their first dose of thalidomide (200 mg) were similarly analyzed and all data were fitted to a one-compartment model. Metabolites of thalidomide in plasma were identified simultaneously using liq. chromatog.-mass spectrometry. Plasma concn.-time profiles for the individual patients were very similar to each other, but widely different pharmacokinetic properties were found between patients compared with those in mice or rabbits. Area under the concn. curve values for mice, rabbits, and multiple myeloma patients were 4, 8, and 81 μmol/L · hour, resp., and corresponding elimination half-lives were 0.5, 2.2, and 7.3 h, resp. Large differences were also obsd. between the metabolite profiles from the three species. Hydrolysis products were detected for all species, and the proportion of hydroxylated metabolites was higher in mice than in rabbits and undetectable in patients. Our results show major interspecies differences in the pharmacokinetics of thalidomide that are related to the altered degree of metab. The authors suggest that the interspecies differences in biol. effects of thalidomide may be attributable, at least in part, to the differences in its metab. and hence pharmacokinetics.
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    45. 45
      Nakamura, T.; Noguchi, T.; Miyachi, H.; Hashimoto, Y. Hydrolyzed metabolites of thalidomide: synthesis and TNF-alpha production-inhibitory activity. Chem. Pharm. Bull. 2007, 55, 651– 654,  DOI: 10.1248/cpb.55.651
      45
      Hydrolyzed metabolites of thalidomide: synthesis and TNF-α production-inhibitory activity
      Nakamura, Takanori; Noguchi, Tomomi; Miyachi, Hiroyuki; Hashimoto, Yuichi
      Chemical & Pharmaceutical Bulletin (2007), 55 (4), 651-654CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)
      Putative hydrolyzed metabolites of thalidomide (I) were prepd. and characterized, and their inhibitory activity on tumor necrosis factor (TNF)-α prodn. in the human monocytic leukemia cell line THP-1 was evaluated. α-(2-Carboxybenzamido)glutarimide (II) was a more potent TNF-α prodn. inhibitor than I.
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    46. 46
      Otogawa, K.; Ogino, Y.; Ishikawa, K.; Tanaka, M.; Shiro, M.; Osaka, T.; Asahi, T. Structural and thermal analyses of a hydrolysis compound of thalidomide. Acta Crystallogr., Sect. A: Found. Adv. 2014, 70, C113,  DOI: 10.1107/s2053273314098866
      There is no corresponding record for this reference.
    47. 47
      Krönke, J.; Hurst, S. N.; Ebert, B. L. Lenalidomide induces degradation of IKZF1 and IKZF3. OncoImmunology 2014, 3, e941742  DOI: 10.4161/21624011.2014.941742
      There is no corresponding record for this reference.
    48. 48
      Schafer, P. H.; Ye, Y.; Wu, L.; Kosek, J.; Ringheim, G.; Yang, Z.; Liu, L.; Thomas, M.; Palmisano, M.; Chopra, R. Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus. Ann. Rheum. Dis. 2018, 77, 1516– 1523,  DOI: 10.1136/annrheumdis-2017-212916
      48
      Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus
      Schafer, Peter H.; Ye, Ying; Wu, Lei; Kosek, Jolanta; Ringheim, Garth; Yang, Zhihong; Liu, Liangang; Thomas, Michael; Palmisano, Maria; Chopra, Rajesh
      Annals of the Rheumatic Diseases (2018), 77 (10), 1516-1523CODEN: ARDIAO; ISSN:0003-4967. (BMJ Publishing Group)
      IKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacol. of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos, was studied in SLE patient cells and in a phase 1 healthy volunteer study. CRBN, IKZF1 and IKZF3 gene expression was measured in peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy volunteers. Ikaros and Aiolos protein levels were measured by Western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured in SLE PBMC cultures treated for 7 days with iberdomide. Fiftysix healthy volunteers were randomized to a single dose of iberdomide (0.03-6mg, n=6 across seven cohorts) or placebo (n=2/cohort). CD19+ B cells, CD3+ T cells and intracellular Aiolos were measured by flow cytometry. Interleukin (IL)-2 and IL-1β prodn. was stimulated with anti-CD3 and lipopolysaccharide, resp., in an ex vivo whole blood assay. SLE patient PBMCs expressed significantly higher CRBN (1.5-fold), IKZF1 (2.1-fold) and IKZF3 (4.1- fold) mRNA levels compared with healthy volunteers. Iberdomide significantly reduced Ikaros and Aiolos protein levels in B cells, T cells and monocytes. In SLE PBMC cultures, iberdomide inhibited anti-dsDNA and anti-phospholipid autoantibody prodn. (IC50 ≈10 nM). Single doses of iberdomide (0.3-6mg) in healthy volunteers decreased intracellular Aiolos (min. mean per cent of baseline: ≈12%-28% (B cells); ≈0%-33% (T cells)), decreased abs. CD19+ B cells, increased IL-2 and decreased IL-1β prodn. ex vivo. These findings demonstrate pharmacodynamic activity of iberdomide and support its further clin. development for the treatment of SLE.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjtVektbw%253D&md5=200098adf4787ee935df528d18a5508d
    49. 49
      Matyskiela, 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, 535– 542,  DOI: 10.1021/acs.jmedchem.6b01921
      49
      A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos
      Matyskiela, 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.
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    50. 50
      Capitosti, S. M.; Hansen, T. P.; Brown, M. L. Facile synthesis of an azido-labeled thalidomide analogue. Org. Lett. 2003, 5, 2865– 2867,  DOI: 10.1021/ol034906w
      50
      Facile Synthesis of an Azido-Labeled Thalidomide Analogue
      Capitosti, Scott M.; Hansen, Todd P.; Brown, Milton L.
      Organic Letters (2003), 5 (16), 2865-2867CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A five-step synthesis of an azido-thalidomide analog I is presented. The sequence requires cheap and readily available starting materials and reagents, and only two steps require purifn. Addnl., I possesses activity comparable to that of thalidomide in inhibiting the proliferation of human microvascular endothelial cells, thus providing impetus for its use as a potential photoaffinity label of thalidomide.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlt1Cgs70%253D&md5=fdefbdcf05d15f638e322bc9e5687ebb
    51. 51
      Joossens, J.; Van der Veken, P.; Lambeir, A.-M.; Augustyns, K.; Haemers, A. Development of irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator. J. Med. Chem. 2004, 47, 2411– 2413,  DOI: 10.1021/jm0499209
      51
      Development of Irreversible Diphenyl Phosphonate Inhibitors for Urokinase Plasminogen Activator
      Joossens, J.; Van der Veken, P.; Lambeir, A.-M.; Augustyns, K.; Haemers, A.
      Journal of Medicinal Chemistry (2004), 47 (10), 2411-2413CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The authors report the synthesis and biochem. evaluation of selective, irreversible di-Ph phosphonate inhibitors for urokinase plasminogen activator (uPA). A di-Ph phosphonate group was introduced on the substrate-like peptide Z-D-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A kapp value in the 103 M-1 s-1 range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXisl2it7k%253D&md5=a5e0eb9791dc09a1ac71b6cf8415af29
    52. 52
      Kanuma, K.; Omodera, K.; Nishiguchi, M.; Funakoshi, T.; Chaki, S.; Nagase, Y.; Iida, I.; Yamaguchi, J.-i.; Semple, G.; Tran, T.-A.; Sekiguchi, Y. Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists. Bioorg. Med. Chem. 2006, 14, 3307– 3319,  DOI: 10.1016/j.bmc.2005.12.052
      52
      Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists
      Kanuma, Kosuke; Omodera, Katsunori; Nishiguchi, Mariko; Funakoshi, Takeo; Chaki, Shigeyuki; Nagase, Yasuko; Iida, Izumi; Yamaguchi, Jun-ichi; Semple, Graeme; Tran, Thuy-Anh; Sekiguchi, Yoshinori
      Bioorganic & Medicinal Chemistry (2006), 14 (10), 3307-3319CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      The optimization of the distance between two key pharmacophore features within our first hit compds. led to the identification of a new class of potent non-peptidic antagonists for the MCH-R1, based around 4-amino-2-cyclohexylaminoquinazolines. In particular, ATC0065, N 2-[cis-4-({2-[4-Bromo-2-(trifluoromethoxy)phenyl]ethyl}amino)cyclohexyl]-N4,N4-dimethylquinazoline-2,4-diamine dihydrochloride, bound with high affinity to the MCH-R1 (IC50 value of 16 nM) and showed good metabolic stability in liver microsomes from human and rat.
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    53. 53
      Nouch, R.; Cini, M.; Magre, M.; Abid, M.; Diéguez, M.; Pàmies, O.; Woodward, S.; Lewis, W. Enantioselective synthesis of 6,6-disubstituted pentafulvenes containing a chiral pendant hydroxy group. Chem.—Eur. J. 2017, 23, 17195– 17198,  DOI: 10.1002/chem.201704247
      53
      Enantioselective Synthesis of 6,6-Disubstituted Pentafulvenes Containing a Chiral Pendant Hydroxy Group
      Nouch, Ryan; Cini, Melchior; Magre, Marc; Abid, Mohammed; Dieguez, Montserrat; Pamies, Oscar; Woodward, Simon; Lewis, William
      Chemistry - A European Journal (2017), 23 (68), 17195-17198CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Simple enantioselective synthesis of 6,6-disubstituted pentafulvenes bearing chiral pendant hydroxy groups are attained by cascade reactivity using com. available proline-based organocatalysts. Condensation of cyclopentadiene with the acetyl function of a 1,2-formylacetophenone, followed by cyclization of a resulting fulvene-stabilized carbanion with the formyl group, generates bicyclic chiral alcs. with initial er values up to 94:6. Exceptional enantio-enrichment of the resultant alcs. results upon crystn.-even near racemic samples spontaneously de-racemize. This enables new families of substituted cyclopentadienes that are both enantiomerically and diastereomerically pure to be rapidly attained.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVKmurjI&md5=ed815c390637d2d2ca7c6322180f583b
    54. 54
      Cao, R.; Müller, P.; Lippard, S. J. Tripodal tris-tacn and tris-dpa platforms for assembling phosphate-templated trimetallic centers. J. Am. Chem. Soc. 2010, 132, 17366– 17369,  DOI: 10.1021/ja108212v
      54
      Tripodal Tris-tacn and Tris-dpa Platforms for Assembling Phosphate-Templated Trimetallic Centers
      Cao, Rui; Muller, Peter; Lippard, Stephen J.
      Journal of the American Chemical Society (2010), 132 (49), 17366-17369CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Multidentate tripodal ligands, N(CH2-m-C6H4-CH2tacn)3 (L1) and N(CH2-o-C6H4-CH2N(CH2py)2)3 (L2), were devised for assembling high-nuclearity metal clusters. By using the same tripodal platform with different ligand appendages, either triazacyclononanes or dipicolylamines, and functionalizing either the ortho or the meta positions on the tris(xylyl) linker arms, discrete trimetal phosphate units of relevance to phosphate-metabolizing trimetallic centers in biol. were prepd. Four such compds., [(CuIICl)3(HPO4)L1](PF6) (1), [(CuIICl)3(HAsO4)L1](PF6) (2), Na2[MnIII6MnII2(H2O)2(HPO4)6(PO4)4(L1)2] (3), and [CoII3(H2PO4)Cl2(MeCN)L2](PF6)3 (4), all contg. three metal centers bound to a central phosphate or arsenate unit bridging oxygen atoms, were synthesized and structurally characterized. These results demonstrate the propensity of this novel tripodal ligand platform, in the presence of phosphate or arsenate, to assemble {M3(EO4)} units and thus structurally mimic trimetallic active sites of proteins involved in phosphate metab. Reactivity studies reveal that the tricopper complex 1 is more efficient than monocopper analogs in catalyzing the hydrolysis of 4-nitrophenyl phosphate.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVGksbzJ&md5=26ced8d2ad3686926092f1940b96a9cc
    55. 55
      Steinebach, C.; Lindner, S.; Udeshi, N. D.; Mani, D. C.; Kehm, H.; Köpff, S.; Carr, S. A.; Gütschow, M.; Krönke, J. Homo-PROTACs for the chemical knockdown of cereblon. ACS Chem. Biol. 2018, 13, 2771– 2782,  DOI: 10.1021/acschembio.8b00693
      55
      Homo-PROTACs for the Chemical Knockdown of Cereblon
      Steinebach, Christian; Lindner, Stefanie; Udeshi, Namrata D.; Mani, Deepak C.; Kehm, Hannes; Koepff, Simon; Carr, Steven A.; Guetschow, Michael; Kroenke, Jan
      ACS Chemical Biology (2018), 13 (9), 2771-2782CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degrdn. of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degrdn. By linking two pomalidomide mols., we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degrdn. The homodimerized compd. I was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of I for CRBN degrdn. was confirmed at the proteome level by quant. mass spectrometry. Inactivation by compd. I did not affect proliferation of different cell lines, prevented pomalidomide-induced degrdn. of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the mol. mechanism of thalidomide analogs.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFejsLrO&md5=71bf2d53ad23c2b873f754de5144883c
    56. 56
      Boichenko, I.; Deiss, S.; Bär, K.; Hartmann, M. D.; Hernandez Alvarez, B. A FRET-based assay for the identification and characterization of cereblon ligands. J. Med. Chem. 2016, 59, 770– 774,  DOI: 10.1021/acs.jmedchem.5b01735
      56
      A FRET-Based Assay for the Identification and Characterization of Cereblon Ligands
      Boichenko, Iuliia; Deiss, Silvia; Baer, Kerstin; Hartmann, Marcus D.; Hernandez Alvarez, Birte
      Journal 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.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1yktg%253D%253D&md5=f0400506428f439e81f579204e0bd39d
    57. 57
      Kabsch, W. Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132,  DOI: 10.1107/s0907444909047337
      57
      Software XDS for image rotation, recognition and crystal symmetry assignment
      Kabsch, Wolfgang
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1SisLc%253D&md5=1aa9a38aeb3ce95af4ffb7d8b8a142bd
    58. 58
      Vagin, A.; Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 22– 25,  DOI: 10.1107/s0907444909042589
      58
      Molecular replacement with MOLREP
      Vagin, Alexei; Teplyakov, Alexei
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 22-25CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      MOLREP is an automated program for mol. replacement that utilizes a no. of original approaches to rotational and translational search and data prepn. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron d., structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calcd. from the input data.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktw%253D%253D&md5=820d114719aca209994ffb0403e3b20d
    59. 59
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501,  DOI: 10.1107/s0907444910007493
      59
      Features and development of Coot
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910
    60. 60
      Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 2.2.1.
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00454.

    • HPLC traces of tested compounds, western blot quantification and normalization IKZF3, concentration response curve for IKZF3 mAb, and Ki values of tested compounds (PDF)

    • Molecular formula strings (CSV)

    Accession Codes

    Crystal structures have been deposited in the protein data bank (PDB) under the accession codes 6R0S (4a), 6R0V (4b), 6R0U (5a), 6R11 (5b), 6R1X (7a), 6R12 (7b), 6R1K (7c), 6R1D (7d), 6R13 (7f), 6R18 (11a), 6R1C (12a), 6R1W (16b), 6R19 (20a), and 6R1A (20b), 6R0Q (CBG). The authors will release the atomic coordinates and experimental data upon article publication.


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