Abstract 3424: Template-assisted covalent modification of DCAF16 enables BRD4 molecular glue degraders

Abstract Molecular glue degraders have emerged as a powerful class of small-molecule therapeutics, as demonstrated by the clinical successes of thalidomide analogs in the treatment of hematological malignancies. These small molecules act by recruiting ubiquitin ligases to disease-relevant proteins, resulting in neosubstrate ubiquitination and degradation. To date, only a small number of ubiquitin ligase - neosubstrate interactions have been exploited by molecular glue degraders, limiting the targeting scope of this therapeutic modality. Covalent chemistry, which confers high binding affinity and the ability of targeting shallow binding sites, can expand the addressable protein interfaces for molecular glue degraders. In this study, we identified a new class of BRD4 covalent molecular glue degraders (GNE-0011, TMX-4137, MMH-252, MMH-272) by derivatizing electrophiles on the solvent-facing side of a non-degradative BRD4 inhibitor, JQ1. These degraders recruit the ubiquitin ligase CUL4DCAF16 to BRD4 via a novel, trans-labeling mechanism we term “template-assisted covalent modification.” These covalent small molecules have only weak reactivity to DCAF16 in the absence of BRD4. However, BRD4 binding allows its BD2 domain, in complex with DCAF16, to serve as a structural template to boost the covalent modification of these degraders to DCAF16. This leads to efficient BRD4-degrader-DCAF16 ternary complex formation and subsequent BRD4 degradation. We solved the cryo-electron microscopy structure of the DDB1-DCAF16 ligase complex bound to BRD4(BD2) and MMH-272, demonstrating that DCAF16 and BRD4(BD2) have pre-existing structural complementarity which optimally orients the reactive moiety of MMH-272 for DCAF16 covalent modification. We performed systematic mutagenesis screens of both DCAF16 and BRD4(BD2) to characterize the drug-induced interaction, discovering that Cys58 of DCAF16 is modified by covalent warheads and is critical for the activity of these degraders, and that His437 of BRD4(BD2) and its adjacent residues are important for a stable interaction with DCAF16. Analysis of a chemical series revealed that the covalent reactivity of these small molecules is required for degradation, and that the degree of reactivity can tune the potency and specificity of the degraders. Our work highlights a new class of BRD4 molecular glue degraders that are enabled by the template-assisted covalent modification of DCAF16. The templated reactivity is likely to exist in other covalent small molecules that engage complementary protein interfaces, and this trans-labeling mechanism can be exploited to develop covalent drugs with more precisely controlled reactivity. Moreover, our findings demonstrate that the decoration of solvent-exposed electrophiles to protein-binding small molecules has great potential to enable discovery and optimization of novel molecular glue degraders. Citation Format: Yen-Der Li, Michelle W. Ma, Muhammad Murtaza Hassan, Kedar Puvar, Mingxing Teng, Brittany Sandoval, Ryan Lumpkin, Scott B. Ficarro, Michelle Y. Wang, Shawn Xu, Brian J. Groendyke, Logan H. Sigua, Isidoro Tavares, Charles Zou, Jonathan M. Tsai, Paul M. Park, Hojong Yoon, Radosław P. Nowak, Jarrod A. Marto, Jun Qi, Katherine A. Donovan, Mikołaj Słabicki, Nathanael S. Gray, Eric S. Fischer, Benjamin L. Ebert. Template-assisted covalent modification of DCAF16 enables BRD4 molecular glue degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3424.