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• W3024223733 abstract "•Cell-based assay developed for evaluating the binding affinity of CRBN E3 ligands•A small library of CRBN E3 ligands was synthesized and examined by this assay•Selected E3 ligands were used to construct cellular active HDAC6 degraders Proteolysis-targeting chimeras (PROTACs) is a paradigm shift for small-molecule drug discovery. However, limited E3 ubiquitin ligase ligands with cellular activity are available. In vitro binding assays involve the expression and purification of a large amount of proteins and they often yield ligands that are inactive in cell-based assays due to poor cell permeability, stability, and other reasons. Herein, we report the development of a practical and efficient cell-based target engagement assay to evaluate the binding affinity of a small library of cereblon ligands to its E3 ligase in cells. Selected cell-permeable E3 ligase ligands derived from this assay are then used to construct HDAC6 degraders with cellular protein degradation activity. Because the assay does not involve any genetic engineering, it is relatively easy to transfer from one cell type to a different one. Proteolysis-targeting chimeras (PROTACs) is a paradigm shift for small-molecule drug discovery. However, limited E3 ubiquitin ligase ligands with cellular activity are available. In vitro binding assays involve the expression and purification of a large amount of proteins and they often yield ligands that are inactive in cell-based assays due to poor cell permeability, stability, and other reasons. Herein, we report the development of a practical and efficient cell-based target engagement assay to evaluate the binding affinity of a small library of cereblon ligands to its E3 ligase in cells. Selected cell-permeable E3 ligase ligands derived from this assay are then used to construct HDAC6 degraders with cellular protein degradation activity. Because the assay does not involve any genetic engineering, it is relatively easy to transfer from one cell type to a different one. Thalidomide and its analogs, such as pomalidomide and lenalidomide, belong to the class of immunomodulatory drugs (IMiDs) for the treatment of multiple myeloma (Holstein et al., 2018Holstein S.A. Hillengass J. McCarthy P.L. Next-generation drugs targeting the cereblon ubiquitin ligase.J. Clin. Oncol. 2018; 36: 2101-2104Crossref PubMed Scopus (4) Google Scholar) (Figure 1A). Thalidomide was once notorious for its teratogenic effect in infants when the drug was prescribed to pregnant women for relieving pregnancy nausea (Matyskiela et al., 2018aMatyskiela M.E. Couto S. Zheng X. Lu G. Hui J. Stamp K. Drew C. Ren Y. Wang M. Carpenter A. et al.SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.Nat. Chem. Biol. 2018; 14: 981-987Crossref PubMed Scopus (107) Google Scholar). After being withdrawn from the market, thalidomide and its analogs were found to possess multiple other functions, such as anti-inflammation, anti-angiogenesis, and immune modulation, including T cell co-stimulation and natural killer cell activation (Quach et al., 2010Quach H. Ritchie D. Stewart A.K. Neeson P. Harrison S. Smyth M.J. Prince H.M. Mechanism of action of immunomodulatory drugs (IMiDS) in multiple myeloma.Leukemia. 2010; 24: 22-32Crossref PubMed Scopus (374) Google Scholar). After years of carefully designed clinical trials, thalidomide and several of its analogs were approved as IMiDs for the treatment of multiple myeloma (Palumbo et al., 2008Palumbo A. Facon T. Sonneveld P. Bladè J. Offidani M. Gay F. Moreau P. Waage A. Spencer A. Ludwig H. et al.Thalidomide for treatment of multiple myeloma: 10 years later.Blood. 2008; 111: 3968-3977Crossref PubMed Scopus (243) Google Scholar). However, their molecular target and the mechanism of action of their anticancer effect were uncovered years later. Cereblon (CRBN), a component of E3 complex with other proteins, such as DDB1, was identified as the cellular target of IMiDs (Ito et al., 2010Ito T. Ando H. Suzuki T. Ogura T. Hotta K. Imamura Y. Yamaguchi Y. Handa H. Identification of a primary target of thalidomide teratogenicity.Science. 2010; 327: 1345-1350Crossref PubMed Scopus (1069) Google Scholar, Lopez-Girona et al., 2012Lopez-Girona A. Mendy D. Ito T. Miller K. Gandhi A.K. Kang J. Karasawa S. Carmel G. Jackson P. Abbasian M. et al.Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide.Leukemia. 2012; 26: 2326-2335Crossref PubMed Scopus (476) Google Scholar). Upon binding to CRBN, the IMiDs can induce interactions between the ligand-bound CRBN E3 ligase and some of the physiologically irrelevant protein substrates, including IKZF1, IKZF3, SALL4, and GSPT1 (Lu et al., 2014Lu G. Middleton R.E. Sun H. Naniong M. Ott C.J. Mitsiades C.S. Wong K.-K. Bradner J.E. Kaelin W.G. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins.Science. 2014; 343: 305-309Crossref PubMed Scopus (775) Google Scholar). IMiDs were therefore described as “molecular glues” for this type of mechanism of action and the above protein substrates are often termed as “neo-substrates” of CRBN. Specifically, the degradation of IKZF1/3 will downregulate cMYC, which is an essential transcription factor for the growth of multiple myeloma. The downregulation of IRF4/cMYC was proposed as the mechanism of action for the anti-myeloma activity of IMiDs (Shaffer et al., 2008Shaffer A.L. Emre N.C.T. Lamy L. Ngo V.N. Wright G. Xiao W. Powell J. Dave S. Yu X. Zhao H. et al.IRF4 addiction in multiple myeloma.Nature. 2008; 454: 226-231Crossref PubMed Scopus (456) Google Scholar, Zhu et al., 2011Zhu Y.X. Braggio E. Shi C.-X. Bruins L.A. Schmidt J.E. Van Wier S. Chang X.-B. Bjorklund C.C. Fonseca R. Bergsagel P.L. et al.Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide.Blood. 2011; 118: 4771-4779Crossref PubMed Scopus (422) Google Scholar). The degradation of SALL4 was proposed to be responsible for the teratogenic effect (Donovan et al., 2018Donovan 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; 7https://doi.org/10.7554/eLife.38430Crossref PubMed Scopus (139) Google Scholar, Matyskiela et al., 2018aMatyskiela M.E. Couto S. Zheng X. Lu G. Hui J. Stamp K. Drew C. Ren Y. Wang M. Carpenter A. et al.SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.Nat. Chem. Biol. 2018; 14: 981-987Crossref PubMed Scopus (107) Google Scholar). In addition to being used as molecular glues, thalidomide and its derivatives are widely used as the ligand of CRBN E3 ligase for the design of bifunctional proteolysis-targeting chimera (PROTAC), which can efficiently recruit CRBN E3 ligase to a protein of interest (POI) and induce the polyubiquitination, followed by degradation of the POI (An et al., 2019An Z. Lv W. Su S. Wu W. Rao Y. Developing potent PROTACs tools for selective degradation of HDAC6 protein.Protein Cell. 2019; 10: 606-609Crossref PubMed Scopus (38) Google Scholar, Bondeson et al., 2018Bondeson D.P. Smith B.E. Burslem G.M. Buhimschi A.D. Hines J. Jaime-Figueroa S. Wang J. Hamman B.D. Ishchenko A. Crews C.M. Lessons in PROTAC design from selective degradation with a promiscuous warhead.Cell Chem. Biol. 2018; 25: 78-87Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, Nabet et al., 2018Nabet B. Roberts J.M. Buckley D.L. Paulk J. Dastjerdi S. Yang A. Leggett A.L. Erb M.A. Lawlor M.A. Souza A. et al.The dTAG system for immediate and target-specific protein degradation.Nat. Chem. Biol. 2018; 14: 431-441Crossref PubMed Scopus (210) Google Scholar, Schiedel et al., 2018Schiedel M. Herp D. Hammelmann S. Swyter S. Lehotzky A. Robaa D. Oláh J. Ovádi J. Sippl W. Jung M. Chemically induced degradation of sirtuin 2 (Sirt2) by a proteolysis targeting chimera (PROTAC) based on sirtuin rearranging ligands (SirReals).J. Med. Chem. 2018; 61: 482-491Crossref PubMed Scopus (132) Google Scholar, Zhou et al., 2018Zhou B. Hu J. Xu F. Chen Z. Bai L. Fernandez-Salas E. Lin M. Liu L. Yang C.Y. Zhao Y. et al.Discovery of a small-molecule degrader of bromodomain and extra-terminal (BET) proteins with picomolar cellular potencies and capable of achieving tumor regression.J. Med. Chem. 2018; 61: 462-481Crossref PubMed Scopus (179) Google Scholar) (Figure 1B). PROTAC is an emerging novel technology for the development of small-molecule therapeutics that can induce the degradation of abnormally expressed or mutated disease-associated proteins (Pettersson and Crews, 2019Pettersson M. Crews C.M. PROteolysis TArgeting Chimeras (PROTACs)—past, present and future.Drug Discov. Today Technol. 2019; 31: 15-27Crossref PubMed Scopus (185) Google Scholar). To expand the toolbox of targeted protein degradation (TPD), it is important to develop novel ligands for well-known and also novel E3 ligases. Due to the ease of synthesis of thalidomide and its derivatives, most reported PROTACs use them as the ligand to recruit CRBN E3 ligase. However, PROTACs based on thalidomide and its derivatives, such as pomalidomide and lenalidomide can still degrade some of the neo-substrates, including IKZFs (Matyskiela et al., 2016Matyskiela M.E. Lu G. Ito T. Pagarigan B. Lu C.-C. Miller K. Fang W. Wang N.-Y. Nguyen D. Houston J. et al.A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase.Nature. 2016; 535: 252-257Crossref PubMed Scopus (200) Google Scholar, Nabet et al., 2018Nabet B. Roberts J.M. Buckley D.L. Paulk J. Dastjerdi S. Yang A. Leggett A.L. Erb M.A. Lawlor M.A. Souza A. et al.The dTAG system for immediate and target-specific protein degradation.Nat. Chem. Biol. 2018; 14: 431-441Crossref PubMed Scopus (210) Google Scholar, Nowak et al., 2018Nowak R.P. DeAngelo S.L. Buckley D. He Z. Donovan K.A. An J. Safaee N. Jedrychowski M.P. Ponthier C.M. Ishoey M. et al.Plasticity in binding confers selectivity in ligand-induced protein degradation.Nat. Chem. Biol. 2018; 14: 706-714Crossref PubMed Scopus (164) Google Scholar, Zorba et al., 2018Zorba A. Nguyen C. Xu Y. Starr J. Borzilleri K. Smith J. Zhu H. Farley K.A. Ding W. Schiemer J. et al.Delineating the role of cooperativity in the design of potent PROTACs for BTK.Proc. Natl. Acad. Sci. U S A. 2018; 115: 7285-7292Crossref PubMed Scopus (136) Google Scholar) or GSPT1(Yang et al., 2019Yang J. Li Y. Aguilar A. Liu Z. Yang C.-Y. Wang S. Simple structural modifications converting a bona fide MDM2 PROTAC degrader into a molecular glue molecule: a cautionary tale in the design of PROTAC degraders.J. Med. Chem. 2019; 62: 9471-9487Crossref PubMed Scopus (41) Google Scholar). The off-target effect may lead to downstream effects that are irrelevant to the intended target of PROTACs and complicate many studies. In vitro binding assays involve the expression and purification of a large amount of proteins and they often yield ligands that are inactive in cell-based assays due to poor cell permeability, stability, and other reasons. For example, CRBN co-exists with many other protein partners in cells to form the functional E3 ubiquitin ligase complex and the binding mode of small-molecule ligands to recombinant CRBN may not be the same as in the protein complex. It is important to ensure the engagement of the native stage of the protein target in the relevant cellular context for the development of chemical probes and therapeutics (Simon et al., 2013Simon G.M. Niphakis M.J. Cravatt B.F. Determining target engagement in living systems.Nat. Chem. Biol. 2013; 9: 200-205Crossref PubMed Scopus (161) Google Scholar). Numerous assays are available for the study of target engagement, such as fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer, affinity-based chemoproteomics, ligand-directed protein labeling, enzyme fragment complementation assay, and cellular thermal shift assay (CETSA) (Schürmann et al., 2016Schürmann M. Janning P. Ziegler S. Waldmann H. Small-molecule target engagement in cells.Cell Chem. Biol. 2016; 23: 435-441Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). As for PROTAC, methods to assess the degradation, ubiquitination, and interactions among all participating partners have also been used to understand the detailed mechanism of action. For example, Promega has applied its NanoLuc technology to study the target engagement for both the targeted protein and the E3 ligase (Riching et al., 2018Riching K.M. Mahan S. Corona C.R. McDougall M. Vasta J.D. Robers M.B. Urh M. Daniels D.L. Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action.ACS Chem. Biol. 2018; 13: 2758-2770Crossref PubMed Scopus (94) Google Scholar). However, this method involves transfection, validation, and potential interference with endogenous targets. The transfection requires extensive optimization for each protein target and each cell type. To confirm the binding of a chemical probe (CCT251236) to its putative transcription factor regulator Pirin in living cells, a library of PROTAC small molecules based on this chemical probe was developed to support the intracellular target engagement (Chessum et al., 2018Chessum N.E.A.A. Sharp S.Y. Caldwell J.J. Pasqua A.E. Wilding B. Colombano G. Collins I. Ozer B. Richards M. Rowlands M. et al.Demonstrating in-cell target engagement using a Pirin protein degradation probe (CCT367766).J. Med. Chem. 2018; 61: 918-933Crossref PubMed Scopus (52) Google Scholar). We (Yang et al., 2018Yang K. Song Y. Xie H. Wu H. Wu Y.-T. Leisten E.D. Tang W. Development of the first small molecule histone deacetylase 6 (HDAC6) degraders.Bioorg. Med. Chem. Lett. 2018; 28: 2493-2497Crossref PubMed Scopus (72) Google Scholar) recently prepared a small library of PROTACs by tethering a pan histone deacetylase (HDAC) inhibitor as the ligand for all 11 isoforms of HDACs with pomalidomide, a well-known member of the IMiDs as the ligand for CRBN E3 ligase. Then, we discovered that HDAC6 was a viable target for TPD by CRBN E3 ubiquitin ligase. Later, we developed multifunctional HDAC6-selective degraders by tethering Nexturastat A (Next-A), a selective HDAC6 inhibitor, with pomalidomide (Figure 2A). These PROTACs efficiently depleted HDAC6 and inhibited cell growth of multiple myeloma. A high-throughput in-cell ELISA was developed to assess the degradation efficiency of the above HDAC6 degraders. Herein, we describe our efforts on the development of a cellular target engagement assay based on this in-cell ELISA for the evaluation of binding affinity of various analogs of thalidomide, including those with partial linkers to the native stage of its CRBN E3 ligase in cells. It has been reported that the type and length of linkers are critical factors for the development of PROTACs (Chan et al., 2017Chan K.-H. Zengerle M. Testa A. Ciulli A. Impact of target warhead and linkage vector on inducing protein degradation: comparison of bromodomain and extra-terminal (BET) degraders derived from triazolodiazepine (JQ1) and tetrahydroquinoline (I-BET726) BET inhibitor scaffolds.J. Med. Chem. 2017; 61: 504-513Crossref PubMed Scopus (85) Google Scholar, Cyrus et al., 2011Cyrus K. Wehenkel M. Choi E.-Y. Han H.-J. Lee H. Swanson H. Kim K.-B. Impact of linker length on the activity of PROTACs.Mol. Biosyst. 2011; 7: 359-364Crossref PubMed Scopus (62) Google Scholar, Smith et al., 2019Smith B.E. Wang S.L. Jaime-Figueroa S. Harbin A. Wang J. Hamman B.D. Crews C.M. Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase.Nat. Commun. 2019; 10: 131Crossref PubMed Scopus (140) Google Scholar). When the same pair of the ligand of the POI and the ligand of the E3 ligase was tethered by different linkers, the resulting biological outcomes can be very different. Although the types of linkers can be evaluated by the degradation efficiency of the complete PROTACs in cells, it is difficult to generalize the linker-activity relationship for the development of PROTACs against different targets. Using the cellular target engagement assay described here, it is possible to systematically evaluate the binding of thalidomide analogs with or without partial linkers to CRBN, which can yield useful information for the design of PROTACs against HDAC6 and possibly many other different targets. We showed previously that pomalidomide or HDAC inhibitor could abolish the degradation of HDAC6 induced by the degraders (Wu et al., 2019Wu H. Yang K. Zhang Z. Leisten E.D. Li Z. Xie H. Liu J. Smith K.A. Novakova Z. Barinka C. et al.Development of multifunctional histone deacetylase 6 degraders with potent antimyeloma activity.J. Med. Chem. 2019; 62: 7042-7057Crossref PubMed Scopus (51) Google Scholar). These ligands could compete with the degraders for the binding sites of either CRBN E3 or HDAC6. This competition led to the formation of fewer ternary complexes when the degraders are introduced to the cellular system. These co-treatment experiments used by us and many others supported the involvement of both POI and E3 ligase in PROTAC-induced degradation. We envisioned that, upon further optimization, we might be able to use this type of co-treatment experiments to develop a cell-based assay to systematically evaluate the binding of various potential E3 ligase ligands to the native stage of its cellular target. In theory, ligands with higher affinity to their cellular targets should compete for the binding sites of CRBN more effectively and abolish the effect of PROTAC degrader more efficiently (Figure 1B). Therefore, we hypothesize that we may be able to evaluate the binding affinity of a synthetic library of thalidomide analogs to cellular CRBN E3 ligase using degrader 1 and the in-cell ELISA upon further optimization. For cell-based assays, many other factors, such as permeability and stability of the small molecules may also contribute to the observed results, in addition to binding affinity. More active ligands or motifs identified from cell-based assays may be the combination of several factors and should be analyzed carefully in each case. To optimize the assay condition, we pre-treated MM1S cells with different concentrations of pomalidomide, lenalidomide, and Next-A (HDAC6 inhibitor) for 1 h and then added 100 nM degrader 1 for 5 h of treatment. The resulting cells were analyzed by in-cell ELISA (Figure 2B). We observed the expected dose responses for all three compounds. Compared with the HDAC6 inhibitor, pomalidomide and lenalidomide are less effective to abolish the effect of degrader-induced degradation of HDAC6. With pre-treatment of 10 μM of the CRBN ligand (100 times of the degrader concentration), there was still 10%–20% of less HDAC6 than vehicle control. At 3 μM of pomalidomide or lenalidomide, about 50% of HDAC6 remained compared with the vehicle control. We decided to use this condition for the screening of analogs of thalidomide because this concentration provides an ideal window to compare the analogs with the parent compounds. At this concentration, an analog that is more potent than pomalidomide/lenalidomide for binding to cellular CRBN will abolish more HDAC6 degradation and have over 50% of HDAC6 level. A weaker ligand would have lower than 50% of the HDAC6 level, assuming other properties of the analogs are similar to pomalidomide/lenalidomide. The proof-of-concept study for cellular target engagement assay is shown in Figure 2C. Ligands that can bind to either CRBN (e.g., deactivated degrader 2, thalidomide, lenalidomide, and pomalidomide) or HDAC6 (e.g., deactivated degrader 3) can abolish the degradation effect of HDAC6 induced by degrader 1. The HDAC6 level for cells co-treated with any one of these ligands and degrader 1 was, therefore, higher than the HDAC6 level in cells that were treated only with degrader 1. The highest level of HDAC6 was observed for compound 2, while the lowest level of HDAC6 was observed for N-methyl pomalidomide. The binding affinities of thalidomide, pomalidomide, and lenalidomide to CRBN have been reported in the literature (Chamberlain et al., 2014Chamberlain P.P. Lopez-Girona A. Miller K. Carmel G. Pagarigan B. Chie-Leon B. Rychak E. Corral L.G. Ren Y.J. Wang M. et al.Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs.Nat. Struct. Mol. Biol. 2014; 21: 803-809Crossref PubMed Scopus (255) Google Scholar, Fischer et al., 2014Fischer E.S. Böhm K. Lydeard J.R. Yang H. Stadler M.B. Cavadini S. Nagel J. Serluca F. Acker V. Lingaraju G.M. et al.Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.Nature. 2014; 512: 49-53Crossref PubMed Scopus (436) Google Scholar, Lopez-Girona et al., 2012Lopez-Girona A. Mendy D. Ito T. Miller K. Gandhi A.K. Kang J. Karasawa S. Carmel G. Jackson P. Abbasian M. et al.Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide.Leukemia. 2012; 26: 2326-2335Crossref PubMed Scopus (476) Google Scholar, Sievers et al., 2018Sievers 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: eaat0572Crossref PubMed Scopus (127) Google Scholar). Pomalidomide is slightly more potent than lenalidomide, while thalidomide is the weakest binder among the three. This potency order was confirmed by our cellular assay in Figure 2C. Thalidomide had the least HDAC6 recovery compared with the other two at both 3 and 1 μM concentrations. Our results also suggested that pomalidomide and lenalidomide were almost equally potent in cell-based assays. Deactivated degrader 2 can still bind to CRBN but not to HDAC6. The strongest effect for abolishing HDAC6 degradation by 2 suggests that the linker part may contribute to the binding of 2 to CRBN. The known negative control, N-methylated pomalidomide, could not bind to CRBN E3 (Lu et al., 2015Lu J. Qian Y. Altieri M. Dong H. Wang J. Raina K. Hines J. Winkler J.D. Crew A.P. Coleman K. et al.Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4.Chem. Biol. 2015; 22: 755-763Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). As expected, this compound has almost no effect on the degrader 1-induced degradation of HDAC6. Deactivated degrader 3 cannot bind to CRBN, but it can still bind to HDAC6 and abolish part of degrader 1-induced HDAC6 degradation. Then, we evaluated several other known thalidomide analogs, including CC-122 (Avadomide) (Hagner et al., 2015Hagner P.R. Man H.-W. Fontanillo C. Wang M. Couto S. Breider M. Bjorklund C. Havens C.G. Lu G. Rychak E. et al.CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL.Blood. 2015; 126: 779-789Crossref PubMed Scopus (91) Google Scholar), CC-220 (Iberdomide) (Matyskiela et al., 2018bMatyskiela M.E. Zhang W. Man H.-W. Muller G. Khambatta G. Baculi F. Hickman M. LeBrun L. Pagarigan B. Carmel G. et al.A cereblon modulator (CC-220) with improved degradation of Ikaros and Aiolos.J. Med. Chem. 2018; 61: 535-542Crossref PubMed Scopus (89) Google Scholar), and CC-885 (Matyskiela et al., 2016Matyskiela M.E. Lu G. Ito T. Pagarigan B. Lu C.-C. Miller K. Fang W. Wang N.-Y. Nguyen D. Houston J. et al.A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase.Nature. 2016; 535: 252-257Crossref PubMed Scopus (200) Google Scholar) (Figures 3A and 3B ). A half maximal inhibitory concentration [IC50] of 60 nM was reported for CC-220 in the TR-FRET binding assay to CRBN, while the IC50 values for pomalidomide and lenalidomide were 1.2 and 1.5 μM, respectively, from the same assay (Matyskiela et al., 2018bMatyskiela M.E. Zhang W. Man H.-W. Muller G. Khambatta G. Baculi F. Hickman M. LeBrun L. Pagarigan B. Carmel G. et al.A cereblon modulator (CC-220) with improved degradation of Ikaros and Aiolos.J. Med. Chem. 2018; 61: 535-542Crossref PubMed Scopus (89) Google Scholar). We evaluated these three compounds in our cell-based assay. CC-122 was found to be less potent than pomalidomide. Both CC-220 and CC-885 remarkably reduced HDAC6 degradation, which is consistent with their high affinity to CRBN, as reported in the literature. However, CC-885 can recruit both IKZFs and GSPT1 as neo-substrates to CRBN and promote their degradation (Ishoey et al., 2018Ishoey M. Chorn S. Singh N. Jaeger M.G. Brand M. Paulk J. Bauer S. Erb M.A. Parapatics K. Müller A.C. et al.Translation termination factor GSPT1 is a phenotypically relevant off-target of heterobifunctional phthalimide degraders.ACS Chem. Biol. 2018; 13: 553-560Crossref PubMed Scopus (57) Google Scholar, Matyskiela et al., 2016Matyskiela M.E. Lu G. Ito T. Pagarigan B. Lu C.-C. Miller K. Fang W. Wang N.-Y. Nguyen D. Houston J. et al.A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase.Nature. 2016; 535: 252-257Crossref PubMed Scopus (200) Google Scholar), suggesting that using CC-885 as the ligand for PROTACs will have off-target effects. Because the above cell-based assay does not involve genetic manipulations, it should be relatively easy to transform from one cell to another. To further demonstrate the utility and generality of this assay, we applied it to a commonly used T cell model, Jurkat cell line, which is difficult to undergo efficient transfection due to low content of proteoglycans (Riedl et al., 2018Riedl S. Kaiser P. Raup A. Synatschke C. Jérôme V. Freitag R. Non-viral transfection of human T lymphocytes.Processes. 2018; 6: 188Crossref Scopus (10) Google Scholar). We treated the Jurkat cells with different concentrations of reported IMiDs and then 100 nM of degrader 1 (Figure S1). As expected, we observed a similar trend of potency for these compounds as in MM1S cells. One exception was that the CC885 recovered less HDAC6 degradation than it did in MM1S. Considering CC220 and CC885 were reported with similar in vitro binding affinity to CRBN and their similar activities in MM1S cells (Figure 3B), the discrepancy may be due to the compound's other properties, such as cell permeability, metabolic stability, or in-cell retention time, in this particular cell line. This observation further highlights the utility of the cell-based target engagement assay for the development of E3 ligases ligands in its relevant cellular context. Overall, the above results suggested that the optimized in-cell ELISA was able to differentiate the binding affinity of thalidomide derivatives through cellular target engagement, assuming other properties of these thalidomide derivatives are close to the parent pomalidomide/lenalidomide in a given cellular context. The higher potency of any analog than its parent compounds is the reflection of its overall properties, including the affinity to its cellular target at the native conformation, permeability, stability, and other factors. With a validated cell-based assay in hand, we then screened a library of synthetic thalidomide analogs, including those with partial linkers (Figures 3C, 3D, and S2). Among them, we discovered several classes of promising ligands for CRBN E3 ligase. Compounds 4a-c were pomalidomide derivatives linked with a terminal alkyne via methylene linkers of different lengths (Figure 3C). All of them showed more HDAC6 recovery than pomalidomide. Compound 4b with a linker of two methylene units had the highest potency. These results suggest that the linker part of the ligand may contribute to its binding to the CRBN E3 ligase (Figure 3D), assuming their other properties are similar. Compounds 5a and 5b are lenalidomide derivatives with an alkyne motif directly attached to the benzene ring and an alcohol at the end of the partial linker. Both of them showed promising activity. Interestingly, compound 5b, which is only one methylene different from 5a, showed much higher activity than the parent thalidomide. Compound 5b, which lacks the aniline nitrogen compared with pomalidomide, is even more active than pomalidomide. These results suggest that the distance between the terminal hydroxyl group and the phthalimidine ring is critical for their cellular activity, which is likely due to different binding affinity to CRBN, considering their overall structural similarity to pomalidomide and lenalidomide. Overall, the in-cell ELISA allowed us to systematically evaluate the effect of modifications on thalidomide, including the linker part for the degradation of HDAC6 in relevant cellular context. Based on our preliminary results with 4a-c and 5a-b, we synthesized more lenalidomide derivatives with an alkyne or a phenyl group directly attached to the benzene ring of lenalidomide (compounds 6a-i, 7a-b, 8, 9a-f, and 10) and other types of analogs as shown in Figure 4A. Compared with pomalidomide, several of them have a higher activity (Figure 4B). Compounds 6a-i all have an alkyne attachment and they differ in the linker length and the bridging atom (nitrogen or oxygen) between the alkyne and the terminal phenyl ring. Compounds 6d-f (X = O) appear to have decreased activity as the linker lengths increase. Aldehyde substitution enhanced the activity as suggested by the higher activity of 6g and 6i over 6f and 6h against HDAC6, respectively. Compounds 6g and 6i showed the highest recovery of HDAC6 protein level and both of them are more potent than pomalidomide. The HDAC6 recovery percentage in response to various concentrations of compounds 6g and 6i was also examined (Figure 4C). The EC50 values (concentrations at which half of degraded HDAC6 was recovered) were 6.4, 1.3, and 1.5 μM for pomalidomide, 6g, and 6i, respectively. It confirmed the improved activities of these ligands to CRBN in cells. Compounds 7a-b hav" @default.
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• W3024223733 date "2020-07-01" @default.
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• W3024223733 title "A Cell-Based Target Engagement Assay for the Identification of Cereblon E3 Ubiquitin Ligase Ligands and Their Application in HDAC6 Degraders" @default.
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• W3024223733 doi "https://doi.org/10.1016/j.chembiol.2020.04.008" @default.
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