Design and Synthesis of Novel Cereblon Binders for Use in Targeted Protein Degradation.

Modulating the chemical composition of cereblon (CRBN) binders is a critical step in the optimization process of protein degraders that seek to hijack the function of this E3 ligase. Small structural changes can have profound impacts on the overall profile of these compounds, including depth of on-target degradation, neosubstrate degradation selectivity, as well as other drug-like properties. Herein, we report the design and synthesis of a series of novel CRBN binding moieties. These CRBN binders were evaluated for CRBN binding and degradation of common neosubstrates Aiolos and GSPT1. A selection of these binders was employed for an exploratory matrix of heterobifunctional molecules, targeting CRBN-mediated degradation of the androgen receptor.

Identification of Small Molecule Inhibitors and Ligand Directed Degraders of Calcium/Calmodulin Dependent Protein Kinase Kinase 1 and 2 (CaMKK1/2).

CaMKK2 signals through AMPK-dependent and AMPK-independent pathways to trigger cellular outputs including proliferation, differentiation, and migration, resulting in changes to metabolism, bone mass accrual, neuronal function, hematopoiesis, and immunity. CAMKK2 is upregulated in tumors including hepatocellular carcinoma, prostate, breast, and gastric cancer, and genetic deletion in myeloid cells results in increased antitumor immunity in several syngeneic models. Validation of the biological roles of CaMKK2 has relied on genetic deletion or small molecule inhibitors with activity against several biological targets. We sought to generate selective inhibitors and degraders to understand the biological impact of inhibiting catalytic activity and scaffolding and the potential therapeutic benefits of targeting CaMKK2. We report herein selective, ligand-efficient inhibitors and ligand-directed degraders of CaMKK2 that were used to probe immune and tumor intrinsic biology. These molecules provide two distinct strategies for ablating CaMKK2 signaling in vitro and in vivo.

Profiling CELMoD-Mediated Degradation of Cereblon Neosubstrates.

Targeted protein degradation is garnering increased attention as a therapeutic modality due in part to its promise of modulating targets previously considered undruggable. Cereblon E3 Ligase Modulating Drugs (CELMoDs) are one of the most well-characterized therapeutics employing this modality. CELMoDs hijack Cereblon E3 ligase activity causing neosubstrates to be ubiquitinated and degraded in the proteasome. Here, we describe a suite of assays-cellular substrate degradation, confirmation of CELMoD mechanism of action, in vitro ubiquitination, and Cereblon binding-that can be used to characterize CELMoD-mediated degradation of Cereblon neosubstrates. While the assays presented herein can be run independently, when combined they provide a strong platform to support the discovery and optimization of CELMoDs and fuel validation of targets degraded by this drug modality.

Discovery of CRBN E3 Ligase Modulator CC-92480 for the Treatment of Relapsed and Refractory Multiple Myeloma.

Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.

CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL.

Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -ß, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.

Use of core modification in the discovery of CC214-2, an orally available, selective inhibitor of mTOR kinase.

We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate cancer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were discovered through a core modification of our original compound series. Analogs from this series have excellent mTOR potency and maintain selectivity over the related PI3Kα lipid kinase. Compounds such as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells, in vitro and in vivo.

Discovery and SAR exploration of a novel series of imidazo[4,5-b]pyrazin-2-ones as potent and selective mTOR kinase inhibitors.

We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show >1000-fold selectivity over the related PI3Kα lipid kinase. Further, compounds such as 2 achieve mTOR pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by inhibition of pS6 and pAkt (S473).

Immunomodulatory drugs reorganize cytoskeleton by modulating Rho GTPases.

IMiDs immunomodulatory drugs, including lenalidomide and pomalidomide represent a novel class of small molecule anticancer and anti-inflammatory drugs with broad biologic activities. However, the molecular mechanism through which these drugs exert their effects is largely undefined. Using pomalidomide and primary human monocytes, we report that pomalidomide rapidly and selectively activated RhoA and Rac1, but not Cdc42 or Ras, in the absence of any costimulation. Consistent with the activation of Rho GTPases, we found that pomalidomide enhanced F-actin formation, stabilized microtubules, and increased cell migration, all of which were blocked by selective inhibitors of ROCK1 and Rac1. Further, we showed that in Swiss 3T3 cells, pomalidomide only activated RhoA, not Rac1 or Cdc42, and potently induced stress fiber formation. The pomalidomide effect on actin cytoskeleton was blocked by the ROCK1 inhibitor, but not Rac1 inhibitor. Finally, we demonstrated that pomalidomide was able to regulate the activity of Rho GTPases and the formation of F-actin in primary human T cells as it did in monocytes and showed that the activation of RhoA was essential for pomalidomide-induced interleukin-2 expression in T cells. These novel activities provide what we believe a critical mechanism by which IMiDs drugs function as therapeutic immunomodulatory agents.

Mechanism of action of NB2001 and NB2030, novel antibacterial agents activated by beta-lactamases.

Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit beta-lactamases to release triclosan through hydrolysis of the beta-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A beta-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C beta-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k(cat)/K(m)). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the beta-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.