Vol. 31, 2019 Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Boston, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY TODAY TECHNOLOGIES Protein degradation for drug discovery Cereblon modulators: Low molecular weight inducers of protein degradation Philip P. Chamberlain*, Brian E. Cathers Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, CA, United States Targeted protein degradation has become an exciting weight degraders are not unique to cereblon. The new paradigm in drug discovery with the potential to groundbreaking work on cereblon represents a case target new protein families for therapeutic interven- study for the discovery and characterization of low tion. In 2010, Hiroshi Handa and colleagues discovered molecular weight protein degraders for other ligases. that the drug thalidomide binds to the protein cere- CRBN blon, a component of the CRL4 E3 ubiquitin ligase. Section editors: Alessio Ciulli, FRSC – Professor of Chemical & Structural In contrast to the heterobifunctional small molecule Biology, School of Life Sciences, University of Dundee, degraders reported in the literature, thalidomide is of Division of Biological Chemistry and Drug Discovery, very low molecular weight ( 258Da) with molecular James Black Centre, Dow Street, Dundee DD1 5EH, United Kingdom. properties (solubility, metabolic stability, permeability William Farnaby – Professor of Chemical & Structural etc) that readily support pharmaceutical dosing. It was Biology, School of Life Sciences, University of Dundee, subsequently shown that thalidomide and the analo- Division of Biological Chemistry and Drug Discovery, University of Dundee, James Black Centre, Dow Street, gues lenalidomide and pomalidomide are able to de- Dundee DD1 5EH, United Kingdom. grade the transcription factors Ikaros and Aiolos. CK1a and GSPT1 were subsequently identified as substrates for specific ligands, indicating that this molecular class Introduction The ubiquitin proteasome system (UPS) has proven to be a could be tuned for selective protein degradation. Struc- challenging but extremely promising area of biology for drug tural studies showed that the thalidomide analogues discovery and development. There are several drugs targeting bind to a shallow hydrophobic pocket on the surface of the proteasome itself, but considering that there are >700 cereblon, and scaffold a protein-protein interaction potential drug targets across the combined ubiquitin ligase and deubiquitinating enzyme space, there remains relatively with target proteins. Target proteins do not need few agents in clinical development and even fewer approved any affinity for the cereblon modulators, and as such drugs that target these enzymes (Fig. 1). The ÀE3 ligases are the undruggable, or even unligandable, proteins can be most numerous membersoftheUPS andhave proventobevery targeted for degradation. A similar mechanism of challenging as drug targets. Developing complex enzyme screening cascades is difficult and discovering bona fide inhi- action was subsequently identified for the clinical bitors of E3 ligases even more so. Only inhibitors of the p53- molecule indisulam, indicating that low molecular mdm2 interaction [1] or SMAC mimetics targeting the IAPs [2] have reached the clinical stage. As the field endeavors to find *Corresponding author: P.P. Chamberlain ([email protected]) 1740-6749/$ © 2019 Published by Elsevier Ltd. https://doi.org/10.1016/j.ddtec.2019.02.004 29 Drug Discovery Today: Technologies | Protein degradation for drug discovery Vol. 31, 2019 CB-5083 Proteasome Inhibitors Bortezomib (Velcade) p97 VLX1570 Carfilzomib (Kyprolis) Ixazomib (Ninlaro) Marizomib Oprozomib Delanzomib E1 Inhibitors MLN7243 MLN4924 Cereblon Thalidomide ® ATP IMiD compounds ATP + Pi Lenalidomide DCAF15 Pomalidomide Indisulam Mdm2 CELMoDs Nutlins CC-122, CC-220 CC-90009, CC-92480 IAPs SMAC mimetics TL32711, LCL161 GDC-0917, HGS1029, SM-406 Drug Discovery Today: Technologies Fig. 1. Clinical stage drugs targeting the ubiquitin proteasome system (UPS). inhibitors of E3 ligases, an alternate approach has sparked and selectivity of the thalidomide analogs was demonstrated enormous interest in recent years. In 2001 it was shown that when it was shown that lenalidomide can induce the degra- an E3 ubiquitin ligase could be redirected from endogenous dation of CK1a while the structurally similar pomalidomide substratesto selectively ubiquitinate aproteinofinterestresult- cannot [10]. More recently, related molecules referred to as ing in proteasomal degradation [3]. Crews and Deshaies had Cereblon E3 Ligase Modulating Drugs (CELMoDs) have ex- pioneered a technique whereby an E3 ligase is co-opted to panded the range of potential target substrates even further ubiquitinate a non-natural protein substrate and coined the when the translation termination factor GSPT1 was shown to term PROTACs (Proteolysis Targeting Chimeras). In this ap- be degraded by CC-885 [11]. While these types of molecules proach, discrete substrate binding moieties are connected to an are much smaller than the chimeric PROTACs, the breadth or E3 ligase recognition element via a linker. Early examples of scope of CELMoDs may be limited to a smaller set of target proteins to be targeted using this approach include the andro- proteins based on the requirement for highly compatible gen receptor, the estrogen receptor-alpha, and further work has protein surfaces and a short glycine-containing hairpin loop successfully targeted BRD4, and identified a broad spectrum of [11,12]. However, the exciting possibility that other ligases CRBN kinases vulnerable to this strategy [4–8]. The requirement for beyond CRL4 are capable of a similar ligand-induced or two independent binding moieties has thus far generated molecular glue substrate recognition is supported by the rather large molecules from a drug discovery perspective, but discovery that indisulam, an investigational anti-cancer drug, the approach holds considerable promise. also operates via redirection of an E3 ubiquitin ligase [13,14]. Recently, the discovery that the molecular mechanism of CRBN action of thalidomide analogs, namely the IMiD drugs lena- Discovery of the E3 ligase CRL4 as the target of lidomide and pomalidomide, is by induced substrate recog- the IMiD drugs CRBN nition by the CRL4 E3 ligase has provided clinical Nothing was known about the mechanism of action of tha- validation for targeted protein degradation as a new paradigm lidomide when it was brought to market in the 1950’s. in drug discovery [9]. These low molecular weight degraders Despite the lack of knowledge thalidomide was shown to remain the only known target protein degraders that have have numerous cellular and clinical effects. Its sedative and made it through clinical development (thalidomide, lenali- anti-nausea activity [15], its remarkable activity in treating domide, and pomalidomide, Fig. 2). The surprising versatility ENL associated with leprosy [16], its anti-wasting activity and 30 www.drugdiscoverytoday.com Vol. 31, 2019 Drug Discovery Today: Technologies | Protein degradation for drug discovery O O O O O O NH NH NH N O N O N O O O ThalidomideNH2 Lenalidomide NH2 Pomalidomide NH O 2 O O O O NH NH N N O N O N O O Avadomide (CC-122) Iberdomide(CC -220) Cl O O S H N N 2 H NH S O O Ind isulam Drug Discovery Today: Technologies Fig. 2. Structures of clinical stage, low molecular weight targeted protein degraders that redirect E3 ubiquitin ligases. effects on Kaposi sarcoma associated with AIDS [17–19], and degradation [10]. This finding was of clinical significance as ultimately its activity in multiple myeloma were all discov- it provided an explanation for the efficacy of lenalidomide ered without understanding the underlying molecular mech- in del(5q) myelodysplastic syndrome, as the gene for CK1a anism. resides in the 5q chromosomal region, and haploinsuffi- In seemingly unrelated research, Higgins et al. published a ciency potentiates susceptibility to further degradation paper describing the association of mild mental retardation [26]. The discovery of CK1a provided evidence that further in children with a truncating mutation in a gene of unknown protein substrates could be found for next-generation cere- function [20]. Because of the cognitive effects associated with blon modulators. Furthermore, the discovery of CK1a pro- this mutation and the sequence similarity with a structural vided a demonstration that small differences in ligand domain from bacterial lon protease, the new gene was called chemical structure could drive substantial differences in cereblon. In March of 2010, over fifty years after thalidomide substrate ubiquitination: pomalidomide, thalidomide and was first developed, cereblon was discovered by the laborato- CC-122 do not induce the recruitment of CK1a. It also ry of Hiroshi Handa to be involved in the mechanism of provided clinical validation for targeted protein degradation thalidomide teratogenicity and was proposed to be part of a as an important new mechanism for drug discovery and Cul4 E3 ligase [9]. Subsequently, cereblon was demonstrated demonstrated the potential for this approach to target pro- to be necessary for the immunomodulatory and anti-myelo- teins otherwise deemed ‘‘undruggable’’. However, the mech- CRBN ma effects of lenalidomide and pomalidomide [21,22]. In anism for how the IMiD drugs were directing the CRL4 2014, the molecular and structural mechanism of the IMiD ligase to target