Evolving Rules for Protein Degradation? Insights from the Zinc Finger

Jan 25, 2019 - Nearly 60 years later, thalidomide and its analogues (called “IMiDs” for “immunomodulatory drugs”) have seen a renewed interest...
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Evolving Rules for Protein Degradation? Insights from the Zinc Finger Degrome Alexandru D. Buhimschi† and Craig M. Crews*,†,‡,§ †

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511, United States Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, United States § Department of Pharmacology, Yale University, New Haven, Connecticut 06520-8066, United States

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‡

true, this finding would explain the sequence variability observed among neo-substrates. Since the Cys2-His2 (C2H2) ZF domains within IKZF1 and IKZF3 form the required surface turn, this raised the possibility that IMiDs degrade additional C2H2 ZF-containing transcription factors, of which there are approximately 800. Given that only a subset of ZF domains mediate degradation, Sievers et al. could characterize the structural features responsible for IMiDs’ selective degradation of neo-substrates. Such a study is of particular importance to the protein degradation community, as the structural basis for degradation selectivity has only recently become appreciated. In an approach differing from earlier proteomics studies identifying CRBN neo-substrates in single cell lines, the authors constructed a library of 6572 C2H2 ZFs fused to GFP, creating a reporter to select for peptide sequences, which afforded degradation by IMiDs. This unbiased method identified 11 C2H2 ZFs degraded under IMiD treatment (including IKZF1 and IKZF3), six of which also mediated degradation of their full-length proteins. Four of these fulllength proteins were previously unknown targets of the IMiDs. When analyzed for conservation, the proteins within the ZF degrome shared little sequence homology besides the residues stabilizing the ZF fold. To probe the plasticity of the CRBN− neo-substrate interactions, the authors tested all possible amino acids at each site within the IKZF3 ZF domain and found three nonstructural sites that contributed to IMiDinduced degradation. For example, one crucial residue is a ßhairpin glycine that packs against the IMiD phthalimide ring (Figure 2B). Crystal structures of two ZFs bound to CRBN and pomalidomide showed that, while ZF−CRBN interactions contribute to degradation specificity, the interfaces are plastic and can accommodate more diverse architectures than anticipated. Alignment of multiple CRBN−neo-substrate crystal structures showed that ZFs may be offset structurally without significant changes in binding affinity, which suggests that CRBN adjusts its interactions when presented with different amino acid combinations. The most logical follow-up to this observation is to what extent can CRBN accommodate its neo-substrates? The authors indeed found that only certain combinations of amino acids were tolerated, but the reasons still appear largely empirical. For example, a deleterious mutation for the ZF domain of IKZF3 did not seem to affect that of E4F1, another

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s perhaps the 20th century’s most infamous drug, thalidomide was initially found to induce severe birth defects when used as an antiemetic during pregnancy. Nearly 60 years later, thalidomide and its analogues (called “IMiDs” for “immunomodulatory drugs”) have seen a renewed interest for their successful treatment of proliferative hematological diseases, particularly del(5q) myelodysplastic syndrome and multiple myeloma (Figure 1). The molecular basis of this IMiD pleiotropy has been the subject of persistent inquiry, making them some of the most fascinating pharmacological agents studied to date. Early studies sought to identify the molecular target(s) of the IMiDs so that their teratogenic and anticancer activities might be separated. Along these lines, it was shown that IMiDs bind cereblon (CRBN), a substrate recognition receptor for the Cullin-4 E3 ubiquitin ligase (CRL4). The >500 E3 ubiquitin ligases in humans are used to specify substrates for ubiquitination and subsequent degradation by the proteasome. At first, it was believed that IMiDs inhibited endogenous CRBN-mediated ubiquitination. This theory persisted until several breakthrough studies demonstrated that IMiDs induced the de novo degradation of Ikaros (IKZF1) and Aiolos (IKZF3), two zinc finger (ZF) transcription factors regulating lymphoid development. This prompted the coining of “neosubstrate” to describe these IMiD-induced CRBN targets. Later, lenalidomide and its analogue, CC-885, were found to induce degradation of casein kinase 1α (CK1α) and the translation termination factor GSPT1, respectively (Figure 2A). CC-885, in particular, represented a major advance because it demonstrated that derivatizing IMiDs could be a valid approach for targeted protein degradation.1 Elegant structural studies by the group of Nicolas Thomä and the Celgene Corporation showed that IMiDs create a novel “interaction hotspot” on CRBN’s surface that supports binding of neo-substrates.1,2 Once the IMiDs hijack the CRL4CRBN complex, these neo-substrates are effectively ubiquitinated (Figure 3). However, several important questions remained. First, what is the complete substrate repertoire that can possibly be targeted using IMiDs? Second, if there is a structural basis for CRBN−neo-substrate interactions, why are the known neo-substrates seemingly unrelated and lacking significant sequence homology? In a recent issue of Science, Sievers et al. began to address these questions by identifying the IMiD “zinc finger degrome” and characterizing several neo-substrate−IMiD−CRBN structures.3 Previous work on GSPT1, IKZF1, and CK1α proposed that the critical neo-substrate degron was not a linear sequence of amino acids, but rather a surface turn of the proper shape. If © XXXX American Chemical Society

Received: December 26, 2018

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DOI: 10.1021/acs.biochem.8b01307 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Structures and clinical applications for several FDA-approved IMiDs. IMiD analogues in preclinical development are also included. In cases where clinical use is not comprehensive, the most advanced clinical trial data is provided.

Figure 2. Available crystal structures of neo-substrate−IMiD−CRBN complexes. (A) (clockwise) CK1α in complex with lenalidomide and CRBN (5FQD), GSPT1 in complex with CC-885 and CRBN (5HXB), alignment of IKZF1 ZF2 (6H0F) and ZNF692 ZF4 (6H0G) in complex with pomalidomide and cereblon. (B) Alignment of surface turn within all neo-substrates proposed to account for IMiD-induced degradation specificity. Inset illustrates the position of a critical glycine within neo-substrates relative to IMiDs’ surfaces.

ZF-containing transcription factor. This leads the authors to propose that CRBN−neo-substrate interactions are “epistatic” in that certain ZF residues alter their CRBN interactions based on others. As a result, the flexibility of the induced interface permits many possible amino acids so long as the overall ZF fold is maintained. Unfortunately, this property makes it hard to predict which mutations will be deleterious, since this may depend on the sequence context within a given neo-substrate. Using computational techniques, the authors identified several ZFs that interact with CRBN−pomalidomide in vitro, but are not degraded in cells. They suggest that small changes in

affinity lead to drastic consequences for cellular degradation and that multiple ZFs compete for the same CRBN interface with varying degrees of success. Lastly, they show that different IMiD analogues can induce degradation of specific ZFs, opening the door for tuning the selectivity of these compounds and applying them against this traditionally “undruggable” protein class. Based on the finding that a consistent surface turn motif is observed across all known neo-substrates, it is compelling to speculate that the IMiD−CRBN interface is limited to binding a confined neo-substrate repertoire bearing this ternary structure. On one hand, querying the proteome for B

DOI: 10.1021/acs.biochem.8b01307 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 3. Mechanism of IMiDs. (1) IMiD derivatives possess substituents that allow CRBN to interface with many possible neo-substrates. (2) IMiDs remodel CRBN to enable interaction with neo-substrates. An E2 enzyme catalyzes ubiquitin transfer onto the neo-substrate, forming ubiquitin chains, which are eventually recognized by the proteasome. (3) Dissociation of the neo-substrate allows recognition by the proteasome for degradation. (4) IMiDs can rebind CRBN and participate in subsequent cycles of ubiquitination and degradation (termed “catalysis”). (5) The ubiquitinated neo-substrate is threaded through the proteasome, which cleaves it into its constituent amino acids. (6) The amino acids of the degraded protein and ubiquitin are recycled by the cell.

Figure 4. PROTAC mechanism. (1) PROTACs are composed of three elements: an E3 ligand, a linker, and a ligand for the target protein. (2) An effective PROTAC enables sufficient target residence within an E3 ligase complex for ubiquitin transfer to occur. An E2 enzyme catalyzes transfer of ubiquitin onto the target protein, forming ubiquitin chains, which are recognized by the proteasome. (3) Dissociation of the target from the E3 ligase allows recognition by the proteasome for degradation. (4) PROTACs can function catalytically whereby they can induce multiple rounds of target protein ubiquitination before clearance. (5) The ubiquitin chain is recognized and the target protein is threaded through the proteasome, which cleaves the target to its constituent amino acids. (6) The amino acids of the degraded protein and ubiquitin are recycled by the cell.

possible interfaces with the same substrate when using PROTACs of different linker lengths.5,6 This “plasticity” is reminiscent of the subtly different interfaces observed within the ZF degrome and indicates that such nonevolved interfaces may be generally characterized by weak yet compatible interactions that are free to adjust when the proteins are held in proximity (Figure 5). Unlike IMiDs, PROTACs are based on high affinity ligands for both substrates, so cooperative protein−protein interactions may not always be necessary as long as sufficient residence within the E3 ligase complex is achieved for ubiquitination.7 However, inducing these ternary contacts may become more important as PROTAC ligand affinities are reduced, which may be especially relevant for undruggable targets. In addition, both technologies rely on efficient and processive ubiquitin transfer to lysine residues suitably poised within a “ubiquitination zone”. Armed with compounds that induce multiple ternary complexes with

this structural motif may yield therapeutic targets already “primed” for IMiD-induced degradation. On the other hand, the validity of such speculation may impose limitations on the generalizability of using IMiDs to degrade any target. Whatever finding holds true, the structural, biophysical, and computational approaches demonstrated by Sievers et al. will advance the understanding of IMiD-based protein degradation. However, this study also sheds light on many nuances currently observed in the design of proteolysis-targeting chimeras (PROTACs). Conceptually, PROTACs are made by joining a ligand for an E3 ubiquitin ligase to a ligand for a target protein to be degraded (Figure 4). Recently, Gadd et al. showed that their PROTACs induce a “ternary complex” between the target and E3 ligase that contributes to PROTAC potency and selectivity.4 However, work by Eric Fischer and others has challenged the necessity of a single stable complex for degradation by showing that CRBN can adopt multiple C

DOI: 10.1021/acs.biochem.8b01307 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 5. Available crystal structures of PROTAC-induced ternary complexes. (A) Ternary complex between the Von Hippel−Lindau (VHL) E3 ligase, the PROTAC MZ1, and the second bromodomain of BRD4 (5T35). (B) Alignment of CRBN−BRD4 ternary complexes illustrating poses the complex adopts with different PROTACs. PDB ID’s for the following complexes were used for the alignment: dBET6 (6BOY), dBET23 (6BN7), dBET55 (6BN8), dBET57 (6BNB), dBET70 (6BN9). cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252−257. (2) Petzold, G., Fischer, E. S., and Thomä, N. H. (2016) Structural basis of lenalidomide-induced CK1α degradation by the CRL4 CRBN ubiquitin ligase. Nature 532, 127−130. (3) Sievers, Q. L., Petzold, G., Bunker, R. D., Renneville, A., Słabicki, M., Liddicoat, B. J., Abdulrahman, W., Mikkelsen, T., Ebert, B. L., and Thomä, N. H. (2018) Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, No. eaat0572. (4) Gadd, M. S., Testa, A., Lucas, X., Chan, K. H., Chen, W., Lamont, D. J., Zengerle, M., and Ciulli, A. (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514−521. (5) Nowak, R. P., DeAngelo, S. L., Buckley, D., He, Z., Donovan, K. A., An, J., Safaee, N., Jedrychowski, M. P., Ponthier, C. M., Ishoey, M., Zhang, T., Mancias, J. D., Gray, N. S., Bradner, J. E., and Fischer, E. S. (2018) Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat. Chem. Biol. 14, 706−714. (6) Zorba, A., Nguyen, C., Xu, Y., Starr, J., Borzilleri, K., Smith, J., Zhu, H., Farley, K. A., Ding, W., Schiemer, J., Feng, X., Chang, J. S., Uccello, D. P., Young, J. A., Garcia-Irrizary, C. N., Czabaniuk, L., Schuff, B., Oliver, R., Montgomery, J., Hayward, M. M., Coe, J., Chen, J., Niosi, M., Luthra, S., Shah, J. C., El-Kattan, A., Qiu, X., West, G. M., Noe, M. C., Shanmugasundaram, V., Gilbert, A. M., Brown, M. F., and Calabrese, M. F. (2018) Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc. Natl. Acad. Sci. U. S. A. 115, E7285. (7) Bondeson, D. P., Smith, B. E., Burslem, G. M., Buhimschi, A. D., Hines, J., Jaime-Figueroa, S., Wang, J., Hamman, B. D., Ishchenko, A., and Crews, C. M. (2018) Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. Cell chemical biology 25, 78−87.

different substrates, future studies should investigate the contribution of lysine positioning to degradation. This may be an alternative explanation to the growing observation that ternary complex formation is not always predictive of PROTAC or IMiD activity. Looking forward, it is not yet clear how IMiDs, which entirely rely on de novo interactions, will be rationally designed to degrade a desired target. Computational docking approaches may hold potential to impact both the PROTAC and IMiD design spaces, which are currently confined to empirically screening large libraries to identify active compounds. Moreover, it will be interesting to follow whether the epistatic nature of IMiD- and PROTACinduced E3−neo-substrate interfaces translates to all ligases, as the repertoire of E3 ligases used continues to expand. It is clear that both IMiD and PROTAC technologies have much to learn from one another, which we expect to see as these two fields continue to intertwine. These crossdisciplinary studies will be critical as PROTACs and new IMiD derivatives make their way into the clinic in the coming years.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (203) 432-9364. Fax: (203) 432-6161. ORCID

Craig M. Crews: 0000-0002-8456-2005 Notes

The authors declare the following competing financial interest(s): C.M.C. is a consultant and shareholder for Arvinas, Inc., which supports research in his lab.

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ACKNOWLEDGMENTS We thank members of the Crews laboratory (Daniel P. Bondeson, Blake E. Smith, and George M. Burslem) for their thoughtful revisions during the preparation of this Viewpoint.

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REFERENCES

(1) 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., and Chamberlain, P. P. (2016) A novel D

DOI: 10.1021/acs.biochem.8b01307 Biochemistry XXXX, XXX, XXX−XXX