225 (PB105) - Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4

Targeted protein degradation using the endogenous Ubiquitin Proteasome System (UPS) represents a fundamentally new approach to drug discovery that potentially allows proteins that cannot be modulated by conventional small molecule inhibitors to be brought under therapeutic control. Plexium has developed the DELPhe platform, which combines solid phase synthesis of DNA encoded libraries with high-throughput ultra-miniaturized cell-based assays, to readily and cost-effectively identify both bifunctional (PROTAC) and monovalent degraders for traditionally undruggable targets including scaffolding proteins, protein-protein interactions and transcription factors. In addition to modulating previously “undruggable” proteins, degradation can lead to greater efficacy and a prolonged downstream signaling response, thereby addressing common obstacles seen with small molecule inhibitors. The bromodomain extra-terminal (BET) protein family are epigenetic readers that have been targeted using small molecule inhibitors. Compounds currently in development typically inhibit multiple BET family members. The bromodomain protein BRD4 is a transcriptional and epigenetic regulator that associates at super-enhancers, driving the expression of oncogenic proteins such as MYC that are critical for the pathogenesis of cancer. We describe here the use of Plexium's DELPhe platform to sample extensive chemical space and discover small molecule monovalent degraders that demonstrate selective and sustained degradation of BRD4. Near complete degradation was observed within 4 hours and DC50 potency of <10 nM was achieved for BRD4 without any appreciable degradation of the highly homologous BRD2 and BRD3 proteins. Degradation of BRD4 resulted in down regulation of MYC protein levels and potent anti-proliferative activity (20-200 nM) against a panel of tumor cell lines with activity superior to inhibition with the pan-BET inhibitor JQ1. Co-treatment with either proteosome or neddylation inhibitors blocked BRD4 degradation, suggesting that protein turnover is regulated by a Cullin-RING Ligase (CRL). Biochemical studies verified a direct interaction between the small molecule and BRD4, suggesting that binding promotes a conformational change that exposes key protein motifs to ubiquitination and degradation. Mutational analysis and a ubiquitin ligase-focused CRISPR screen were used to provide insights into defining the principles of degradation. Collectively, these data demonstrate that the Plexium DELPhe platform enables the discovery of selective and potent monovalent degraders of BRD4. Citation Format: Gregory S. Parker, Julia A. Toth, Geoffray Leriche, Simon Bailey, Kenneth Chng, Sara Fish, Aleks Jamborcic, Elizabeth Daniele, Erika Green, Michael Hocker, Adam Kallel, Peggy A. Thompson, Steven D. Brown, Kandaswamy Vijayan. Discovery of selective and potent BRD4 protein degraders using Plexium's DELPhe platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1283.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent “direct” degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein–protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

<div>Abstract<p>Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent “direct” degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity <i>in vitro</i> and <i>in vivo</i>. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein–protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity <i>in vivo</i>.</p></div>

Background: First-generation targeted protein degraders (TPDs) known as Proteolysis-Targeting Chimeras (PROTACs) are hetero-bifunctional molecules that simultaneously bind both a target protein and an E3 ubiquitin ligase, marking the target for proteasomal degradation. PROTACS have shown promise in Phase I studies but are limited by their large size, long development timelines, and emergence of resistance. We sought to overcome these limitations with next-generation TPDs known as CURE-PROs. These small molecule monomers self-assemble and dimerize inside the cancer cell in the presence of the target oncoprotein and E3 ligase to form a quaternary complex that facilitates ubiquitination of the target protein. Due to their small size, the individual CURE-PRO monomer ligands have superior oral and tumor cell absorption and can be rapidly developed or modified utilizing a large library of monomers and linker components. We selected BRD4 as our initial oncoprotein target to validate in vitro and in vivo activity of CURE-PROs. Methods: Twenty-seven CURE-PRO monomers consisting of derivatives of the BRD4 protein ligands, JQ1 and OXT-015, and ligand monomers that bind E3 ligases (Cereblon, VHL, and MDM2) were combined to create 170 unique CURE-PRO dimers. These combinations were tested in cancer cell lines, in the presence/absence of proteasome inhibitors, and BRD4 protein degradation was assessed by Western Blot. CURE-PRO-mediated degradation of BRD4 was also evaluated in nude mice with bilateral human cancer xenografts using three doses over eight hours. A parallel arm using a BRD4 PROTAC (ARV-825) served as a positive control. The tumors were excised 1, 2, 4, 16 and 40 hours after the last dose and protein expression was analyzed by Western Blot. Results: Cell screening identified 49 CURE-PRO combinations, consisting of different E3 ligase partners and BRD4 ligands, that induced greater than 50% BRD4 degradation. BRD4 degradation occurred only in the presence of both CURE-PRO monomers and was dependent on the pairs forming a reversible dimer. Five CURE-PRO pairs reduced protein levels by more than 95%, with a half-maximal degradation concentrations (DC50) of 30-100 nM. Consistent with a PROTAC mechanism-of-action, CURE-PRO-induced degradation of BRD4 was inhibited by proteasome inhibitors. In the xenograft models, BRD4 protein levels were reduced to ~40% at 2-4 hours after exposure to a CURE-PRO pair, with full recovery by 40 hours. Conclusions: Several CURE-PRO pairs, using different E3 ligase partners, achieved BRD4 protein degradation in vitro comparable to PROTACs, with mechanism-of-action through the proteasome system. Our lead CURE-PRO pair significantly degraded BRD4 in a cancer xenograft mouse model. This work constitutes the first proof-of-mechanism of reversible self-assembling drug dimers for targeted protein degradation in vitro and in vivo. Future studies are planned to optimize degradation efficacy and identify if resistance emerges. Citation Format: Sarah F Giardina, Elena Valdambrini, Micheal Peel, Manny D Bacolod, Mace L Rothenberg, Richard B Lanman, J David Warren, Francis Barany. CURE-PROs: Proof-of-mechanism for the first reversible self-assembling targeted protein degraders [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B072.

e15101 Background: Many proteins, including transcription factors and scaffolding proteins, are not amenable to targeting by traditional small molecule inhibitors due to the lack of a well-defined binding pocket or active site. Proteolysis-Targeting Chimeras (PROTACs) are a new class of hetero-bifunctional molecules that bind both a target protein and an E3 ubiquitin ligase, bringing the two into proximity for appending ubiquitin, and subsequently marking the target protein for proteasomal degradation. Currently, thirteen PROTACs are in clinical trials for oncology indications. However, the clinical utility of PROTACs is challenged by their large size and long development timelines. Also, resistance mutations in the E3 ligase or transporter overexpression inevitably evolve. Thus, a new platform for small-molecule degraders that enables ultra-rapid drug development timelines, efficient cellular uptake, and can be developed to overcome innate and acquired drug resistance is needed. Methods: Coferons, developed in our laboratory, are small molecules that self-assemble upon binding to a target, where they form reversible covalent dimers through bio-orthogonal linker chemistries. We have combined features of the Coferon platform and PROTACs to generate CURE-PROs (Combinatorial Ubiquitination REal-time PROteolysis), consisting of one target protein ligand and one E3 ligase ligand that form reversible heterodimers that lead to targeted protein degradation within cells. By modifying known ligands for BRD4, and the E3 ubiquitin ligases Cereblon, VHL, and MDM2, with linkers able to reversibly join the BRD4 to the ligase ligands, we synthesized libraries of CURE-PRO monomers that can be combined to create thousands of CURE-PRO dimer combinations. We explored whether this platform could yield meaningful BRD4 degradation in vitro and in vivo. Results: Rapid combinatorial cell-based screening identified several BRD4-E3 ligase CURE-PRO combinations that induced greater than 50% BRD4 degradation, with the most promising CURE-PRO pairs achieving more than 95% protein degradation. Consistent with a PROTAC mechanism-of-action, successful CURE-PRO combinations confirmed significant protein degradation which was inhibited by proteasome inhibitors or competition with parent ligands. Significant BRD4 degradation was also observed in mice bearing bilateral human xenograft tumors, confirming CURE-PRO proof-of-mechanism in vivo. Conclusions: The combinatorial nature of our platform has the potential to significantly reduce synthesis time and effort to identify the optimal linker length and E3 ligase for each target protein. The CURE-PRO platform consists of expanding libraries of monomers for both additional oncoprotein targets as well as E3 ligases, which can be redeployed to shorten lead development timelines.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

AML-399: A Pan-BET Degrader Shows Broad Pre-Clinical Activity in a Large Acute Myeloid Leukemia Cohort

Targeted protein degradation is a powerful induced-proximity tool to control cellular protein concentrations using small molecules. However, the design of selective degraders remains empirical. Among bromodomain and extra-terminal (BET) family proteins, BRD4 is the primary therapeutic target over family members BRD2/3/T. Existing strategies for selective BRD4 degradation use pan-BET inhibitors optimized for BRD4:E3 ubiquitin ligase (E3) ternary complex formation, but these result in residual inhibition of undegraded BET-bromodomains by the pan-BET ligand, obscuring BRD4-degradation phenotypes. Using our selective inhibitor of the first BRD4 bromodomain, iBRD4-BD1 (IC50 = 12 nM, 23- to 6200-fold intra-BET selectivity), we developed dBRD4-BD1 to selectively degrade BRD4 (DC50 = 280 nM). Notably, dBRD4-BD1 upregulates BRD2/3, a result not observed with degraders using pan-BET ligands. Designing BRD4 selectivity up front enables analysis of BRD4 biology without wider BET-inhibition and simplifies designing BRD4-selective heterobifunctional molecules, such as degraders with new E3 recruiting ligands or for additional probes beyond degraders.

molecule Z363 co-regulates TAF10 and MYC via the E3 ligase TRIP12 to suppress tumour growth.

Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation-an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

Advances and opportunities in targeted protein degradation

Proteolysis Targeting Chimeras (PROTACs) are promising therapeutic modalities for eliminating disease-causing proteins, particularly in oncology. However, the widespread applicability of PROTAC technology is limited by the paucity of suitable E3 ubiquitin ligases available for targeted protein degradation (TPD) despite the existence of over 600 such ligases in the human genome. Many E3 ligases lack small-molecule ligands, rendering them inaccessible to recruitment by traditional PROTAC design methodology. To overcome this challenge, we instead leverage a high-affinity binder of the histone methyltransferase GLP, an endogenous substrate of the yet unharnessed E3 ligase SPOP, to facilitate PROTAC discovery and targeted degradation of the epigenetic regulator BRD4. Through the application of this novel substrate-bridged PROTAC strategy we discovered the compound MS479, which represents the first molecule of its class capable of recruiting SPOP for TPD and the first to do so by taking advantage of the endogenous interaction between an E3 ligase and one of its protein substrates. This approach successfully facilitates the polyubiquitination of BET-family proteins (i.e., BRD4/3/2) and their subsequent degradation of by the 26S proteasome. In particular, MS479 demonstrates significant efficacy in reducing the post-translational expression of BRD4 short isoform in HT29 cells in a time-, concentration-, GLP-, SPOP-, and ubiquitin-proteasome system- (UPS) dependent manner. Additionally, MS479 exhibits potent inhibition of colorectal cancer (CRC) cell proliferation. Our substrate-bridged PROTAC strategy will ultimately increase the available repertoire of E3 ligases that are tractable for TPD, serving as proof-of-concept for a novel and generalizable platform to target the large suite of E3 ubiquitin ligases that lack pharmacological ligands. Citation Format: Jerrel Lewis Catlett, Zhijie Deng, Youngeun Lee, Yan Xiong, Husnu Ü. Kaniskan, Jian Jin. Discovery of a bridged proteolysis targeting chimera (PROTAC) recruiting the SPOP E3 ubiquitin ligase for targeted protein degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3758.

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.g. glutarimide ligands for CUL4CRBN), offer the potential to target undruggable proteins. Yet, their rational design is largely unattainable due to the unpredictability of the ‘gain-of-function’ nature of the glue interaction upon chemical modification of ligands. We recently reported a covalent trans-labelling glue mechanism which we named ‘Template-assisted covalent modification’, where an electrophile decorated BRD4 inhibitor was effectively delivered to a cysteine residue on DCAF16 due to an electrophile-induced BRD4-DCAF16 interaction. Herein, we report our efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 recruitment and subsequent BRD4 degradation efficiency. We discovered a moderate correlation between the electrophile-induced BRD4-DCAF16 ternary complex formation and BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation optimally recruits DCAF16 and promotes BRD4 degradation. The diversity of covalent attachments in this class of BRD4 degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a new avenue for rational glue design by introducing covalent warheads to known binders.

Superior Pre-Clinical Efficacy of Novel Protac Based BET Degrader in a Large Acute Myeloid Leukemia Cohort