Search for: All records

Abstract Targeted protein degradation (TPD) is a powerful strategy for targeting and eliminating disease-causing proteins. While heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) are more modular, the rational design of monovalent or molecular glue degraders remains challenging. In this study, we generated a small library of BET-domain inhibitor JQ1 analogs bearing elaborated electrophilic handles to identify permissive covalent degradative handles and E3 ligase pairs. We identified an elaborated fumaramide handle that, when appended onto JQ1, led to the proteasome-dependent degradation of BRD4. Further characterization revealed that the E3 ubiquitin ligase CUL4^DCAF16—a common E3 ligase target of electrophilic degraders—was responsible for BRD4 loss by covalently targeting C173 on DCAF16. While this original fumaramide handle, when appended onto other protein-targeting ligands, did not accommodate the degradation of other neo-substrates, a truncated version of this handle attached to JQ1 was still capable of degrading BRD4, now through targeting both C173 and C178. This truncated fumaramide handle, when appended on various protein targeting ligands, and was also more permissive in degrading other neo-substrates, including CDK4/6, SMARCA2/4, and the androgen receptor (AR). We further demonstrated that this optimized truncated fumaramide handle, when transplanted onto an AR DNA binding domain-targeting ligand, could degrade both AR and the undruggable truncation variant of AR, AR-V7, in androgen-independent prostate cancer cells in a DCAF16-dependent manner. Overall, we have identified a unique DCAF16-targeting covalent degradative handle that can be transplanted across several protein-targeting ligands to induce the degradation of their respective targets for the modular design of monovalent or bifunctional degraders. 

Abstract While MYC is a significant oncogenic transcription factor driver of cancer, directly targeting MYC has remained challenging due to its intrinsic disorder and poorly defined structure, deeming it “undruggable.” Whether transient pockets formed within intrinsically disordered and unstructured regions of proteins can be selectively targeted with small molecules remains an outstanding challenge. Here, we developed a bespoke stereochemically-paired spirocyclic oxindole aziridine covalent library and screened this library for degradation of MYC. Through this screen, we identified a hit covalent ligand KL2-236, bearing a unique sulfinyl aziridine warhead, that engaged MYCin vitroas pure MYC/MAX protein complex andin situin cancer cells to destabilize MYC, inhibit MYC transcriptional activity and degrade MYC in a proteasome-dependent manner through targeting intrinsically disordered C203 and D205 residues. Notably, this reactivity was most pronounced for specific stereoisomers of KL2-236 with a diastereomer KL4-019 that was largely inactive. Mutagenesis of both C203 and D205 completely attenuated KL2-236-mediated MYC degradation. We have also optimized our initial KL2-236 hit compound to generate a more durable MYC degrader KL4-219A in cancer cells. Our results reveal a novel ligandable site within MYC and indicate that certain intrinsically disordered regions within high-value protein targets, such as MYC, can be interrogated by isomerically unique chiral small molecules, leading to destabilization and degradation. 

Abstract Androgen-independent prostate cancers, correlated with heightened aggressiveness and poor prognosis, are caused by mutations or deletions in the androgen receptor (AR) or expression of truncated variants of AR that are constitutively activated. Currently, drugs and drug candidates against AR target the steroid-binding domain to antagonize or degrade AR. However, these compounds cannot therapeutically access largely intrinsically disordered truncated splice variants of AR, such as AR-V7, that only possess the DNA binding domain and are missing the ligand binding domain. Targeting intrinsically disordered regions within transcription factors has remained challenging and is considered “undruggable”. Herein, we leveraged a cysteine-reactive covalent ligand library in a cellular screen to identify degraders of AR and AR-V7 in androgen-independent prostate cancer cells. We identified a covalent compound EN1441 that selectively degrades AR and AR-V7 in a proteasome-dependent manner through direct covalent targeting of an intrinsically disordered cysteine C125 in AR and AR-V7. EN1441 causes significant and selective destabilization of AR and AR-V7, leading to aggregation of AR/AR-V7 and subsequent proteasome-mediated degradation. Consistent with targeting both AR and AR-V7, we find that EN1441 completely inhibits total AR transcriptional activity in androgen-independent prostate cancer cells expressing both AR and AR-V7 compared to AR antagonists or degraders that only target the ligand binding domain of full-length AR, such as enzalutamide and ARV-110. Our results put forth a pathfinder molecule EN1441 that targets an intrinsically disordered cysteine within AR to destabilize, degrade, and inhibit both AR and AR-V7 in androgen-independent prostate cancer cells and highlights the utility of covalent ligand discovery approaches in directly targeting, destabilizing, inhibiting, and degrading classically undruggable transcription factor targets.