Degradation Of BRD4 Research Articles

BRD4 Degradation Enhanced Glioma Sensitivity to Temozolomide by Regulating Notch1 via Glu-Modified GSH-Responsive Nanoparticles.

Temozolomide (TMZ) serves as the principal chemotherapeutic agent for glioma; nonetheless, its therapeutic efficacy is compromised by the rapid emergence of drug resistance, the inadequate targeting of glioma cells, and significant systemic toxicity. ARV-825 may play a role in modulating drug resistance by degrading the BRD4 protein, thereby exerting anti-glioma effects. Therefore, to surmount TMZ resistance and achieve efficient and specific drug delivery, a dual-targeted glutathione (GSH)-responsive nanoparticle system (T+A@Glu-NP) is designed and synthesized for the co-delivery of ARV-825 and TMZ. As anticipated, T+A@Glu-NPs significantly enhanced penetration of the blood-brain barrier (BBB), facilitated drug uptake by glioma cells, and exhibited efficient accumulation in brain tissue. Additionally, T+A@Glu-NPs exhibited augmented efficacy against glioma both in vitro and in vivo through the induction of apoptosis, inhibition of proliferation, and cell cycle arrest. Furthermore, mechanistic exploration revealed that T+A@Glu-NPs degraded the BRD4 protein, leading to the downregulation of Notch1 gene transcription and the inhibition of the Notch1 signaling pathway, thereby augmenting the therapeutic efficacy of glioma chemotherapy. Taken together, the findings suggest that T+A@Glu-NPs represents a novel and promising therapeutic strategy for glioma chemotherapy.

MicroRNA-Triggered Programmable DNA-Encoded Pre-PROTACs for Cell-Selective and Controlled Protein Degradation.

Proteolysis-targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on-demand protein degradation. Here, we developed a miRNA-initiated assembled pre-PROTAC (miRiaTAC) platform that enables the on-demand activation and termination of target degradation in a cell type-specific manner. Using miRNA-21 as a model, we engineered DNA hairpins labeled with JQ-1 and pomalidomide and facilitated the modular assembly of DNA-encoded pre-PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR-21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre-PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre-PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near-infrared light-mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

MicroRNA‐Triggered Programmable DNA‐Encoded Pre‐PROTACs for Cell‐Selective and Controlled Protein Degradation

AbstractProteolysis‐targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on‐demand protein degradation. Here, we developed a miRNA‐initiated assembled pre‐PROTAC (miRiaTAC) platform that enables the on‐demand activation and termination of target degradation in a cell type‐specific manner. Using miRNA‐21 as a model, we engineered DNA hairpins labeled with JQ‐1 and pomalidomide and facilitated the modular assembly of DNA‐encoded pre‐PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR‐21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre‐PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre‐PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near‐infrared light‐mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

Design, Synthesis, and Activity Evaluation of BRD4 PROTAC Based on Alkenyl Oxindole-DCAF11 Pair.

Proteolytic targeting chimera (PROTAC) represent an advanced strategy for targeting undruggable proteins, and the molecular warheads targeting E3 ligases play a crucial role. Recently, we explored an alkenyl oxindole warhead targeting the E3 ligase DCAF11 and sought to validate its potential. In this study, we synthesized a range of BRD4 PROTACs (8a-8o, 14a-14f, 22a-22m) with modified alkenyl oxindole warheads and developed a high-throughput screening system based on high-content imaging. We identified L134 (22a) as a potent BRD4 degrader, achieving BRD4 degradation (Dmax > 98%, DC50 = 7.36 nM) and demonstrating antitumor activity. Mechanically, BRD4 degradation by L134 was mediated through the ubiquitin-proteasome system in a DCAF11-dependent manner. Therefore, this study provides a rapid screening method for effective PROTACs and highlights the PROTAC L134 based on alkenyl oxindole-DCAF11 pair as a promising candidate for treating BRD4-driven cancers.

Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues

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.

Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

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.

Progestogen-driven B7-H4 contributes to onco-fetal immune tolerance

Immune tolerance mechanisms are shared in cancer and pregnancy. Through cross-analyzing single-cell RNA-sequencing data from multiple human cancer types and the maternal-fetal interface, we found B7-H4 (VTCN1) is an onco-fetal immune tolerance checkpoint. We showed that genetic deficiency of B7-H4 resulted in immune activation and fetal resorption in allogeneic pregnancy models. Analogously, B7-H4 contributed to MPA/DMBA-induced breast cancer progression, accompanied by CD8+ Tcell exhaustion. Female hormone screening revealed that progesterone stimulated B7-H4 expression in placental and breast cancer cells. Mechanistically, progesterone receptor (PR) bound to a newly identified -58 kb enhancer, thereby mediating B7-H4 transcription via the PR-P300-BRD4 axis. PR antagonist or BRD4 degrader potentiated immunotherapy in a murine B7-H4+ breast cancer model. Thus, our work unravels a mechanistic and biological connection of a female sex hormone (progesterone) to onco-fetal immune tolerance via B7-H4 and suggests that the PR-P300-BRD4 axis is targetable for treating B7-H4+ cancer.

Intrinsic signaling pathways modulate targeted protein degradation

Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4–PROTAC–CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.

Ultrasound-Activated PROTAC Prodrugs Overcome Immunosuppression to Actuate Efficient Deep-Tissue Sono-Immunotherapy in Orthotopic Pancreatic Tumor Mouse Models.

The degradation of oncoproteins mediated by proteolysis-targeting chimera (PROTAC) has emerged as a potent strategy in cancer therapy. However, the clinical application of PROTACs is hampered by challenges such as poor water solubility and off-target adverse effects. Herein, we present an ultrasound (US)-activatable PROTAC prodrug termed NPCe6+PRO for actuating efficient sono-immunotherapy in a spatiotemporally controllable manner. Specifically, US irradiation, which exhibits deep-tissue penetration capability, results in Ce6-mediated generation of ROS, facilitating sonodynamic therapy (SDT) and inducing immunogenic cell death (ICD). Simultaneously, the generated ROS cleaves the thioketal (TK) linker through a ROS-responsive mechanism, realizing the on-demand activation of the PROTAC prodrug in deep tissues. This prodrug activation results in the degradation of the target protein BRD4, while simultaneously reversing the upregulation of PD-L1 expression associated with the SDT process. In the orthotopic mouse model of pancreatic tumors, NPCe6+PRO effectively suppressed tumor growth in conjunction with US stimulation.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

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.

DCAF16-Based Covalent Handle for the Rational Design of Monovalent Degraders.

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.

Photo-regulated PROTACs: A novel tool for temporal control of targeted protein degradation

PROTACs (Proteolysis targeting chimeras) are chimeric molecules designed to induce targeted protein degradation via the ubiquitin–proteasome system. These molecules catalytically degrade target proteins and sustainably inhibit their function. Therefore, PROTAC's unique mechanism of action is not only beneficial in medicine but also serves as a valuable tool for molecular biological analysis in fields like chemical biology, biochemistry, and drug discovery. This study presents a novel turn-off (ON-OFF) type PROTAC development strategy utilizing a photocleavable linker. The inclusion of this linker enables temporal control of the degradation activity targeting BRD4 protein upon UV light exposure. PROTAC-2 demonstrated the most potent degradation activity against BRD4 among the other synthesized PROTACs with varying linker lengths. The UV light-induced cleavage of PROTAC-2 was confirmed, leading to a reduction in its BRD4 degradation activity. Notably, this study introduces a novel linker capable of nullifying degradation activity of PROTACs which is activated by light irradiation. These findings offer a promising strategy for the development of turn-off type PROTACs, providing enhanced temporal control over protein degradation. The approach holds significant potential for applications in molecular function studies and drug discovery.

131I Induced In Vivo Proteolysis by Photoswitchable azoPROTAC Reinforces Internal Radiotherapy.

Photopharmacology, incorporating photoswitches such as azobenezes into drugs, is an emerging therapeutic method to realize spatiotemporal control of pharmacological activity by light. However, most photoswitchable molecules are triggered by UV light with limited tissue penetration, which greatly restricts the in vivo application. Here, this study proves that 131I can trigger the trans-cis photoisomerization of a reported azobenezen incorporating PROTACs (azoPROTAC). With the presence of 50µCimL-1 131I, the azoPROTAC can effectively down-regulate BRD4 and c-Myc levels in 4T1 cells at a similar level as it does under light irradiation (405nm, 60mWcm-2). What's more, the degradation of BRD4 can further benefit the 131I-based radiotherapy. The in vivo experiment proves that intratumoral co-adminstration of 131I (300 µCi) and azoPROTC (25mgkg-1) via hydrogel not only successfully induce protein degradation in 4T1 tumor bearing-mice but also efficiently inhibit tumor growth with enhanced radiotherapeutic effect and anti-tumor immunological effect. This is the first time that a radioisotope is successfully used as a trigger in photopharmacology in a mouse model. It believes that this study will benefit photopharmacology in deep tissue.

Abstract 6049: Cell-based PROTAC screening for cancer drug discovery

Abstract Proteolysis-Targeting Chimeras (PROTACs) have emerged as a promising strategy for selectively degrading specific target proteins, thereby offering a novel approach to precision cancer therapy. Among the most notable oncogenes, KRAS with the G12C mutation is responsible for 45%-50% of KRAS mutations in non-small cell lung carcinomas. Additionally, BRD4, a well-studied member of the Bromo- and Extra-Terminal (BET) protein family, is implicated in various hematological and solid tumors. The implementation of cell-based screening for PROTACs targeting either KRAS(G12C) or BRD4 represents a highly valuable platform for advancing cancer drug discovery. We utilized the NanoBRETTM Ternary Complex Assay, NanoBiTTM Assay, and Western Blot assay to evaluate the efficacy of PROTACs. The NanoBRETTM Ternary Complex Assay revealed that KRAS G12C degrader-1 and LC-2 effectively degrade KRAS(G12C), while dBET6 and MZ-1 demonstrated the ability to degrade BRD4. Notably, the NanoBiTTM assay showed that LC-2 dose-dependently degrades KRAS(G12C) in HEK293 cells co-transfected with KRAS(G12C)-LgBiT and KRAS(G12C)-SmBiT. Moreover, BET6 exhibited a dose-dependent degradation of endogenous BRD4 in HepG2 cells, and LC-2 demonstrated the degradation of endogenous KRAS(G12C) in MiaPaCa2 cells. In summary, the development of a robust cell-based assay platform for high-throughput screening of PROTACs marks a substantial advancement in cancer drug discovery. This approach not only enables the identification of promising PROTACs but also holds the potential for more effective and targeted interventions in the ongoing battle against cancer. Citation Format: Yong Wan, Haiqing Ma, Jianghong Wu. Cell-based PROTAC screening for cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6049.

Abstract 6042: CBP-8008: A first-in-class targeted pan-Bet protein degradation therapy using bi-specific XDC (Bi-XDC) technology for TNBC and mCRPC

Abstract Bi-XDC is a breakthrough cellular targeting technology using multi-targeting ligands to effectively deliver the payload to and into specific cells. Several anticancer Bi-ligand drug conjugates including CBP-1008, CBP-1018, and CBP-1019 have been brought into various clinical stages in China and US. Targeted protein degradation molecules can degrade previously “undruggable” target proteins through their catalytic mechanism of action. The field target protein degradation has evolved since 2001, and more than 20 PROTACs are under clinical trials at different stages. To increase the efficacy vs. tolerability window, we combine Bi-XDC technology and the catalytic approach of targeted protein degradation, to form C-PROTAC, by taking advantage of our privileged Bi-XDC technology and PROTAC’s degradation property. Further medicinal chemistry optimization, spacer optimization, and evaluation of many linker payloads led to the identification of our preclinical candidate CBP-8008, a highly potent pan-Bet protein degrader conjugated to bi-ligand via a cleavable linker. A small panel of cell lines was screened to identify which tumors were sensitive to the pan-Bet protein degradation. All the tested breast cancer and prostate cell lines were the most sensitive to the payload. CBP-8008 had a high affinity to the Prostate Specific Membrane Antigen (PSMA) and the folate receptor alpha (FRα). Much more CBP-8008 were binding the 293T-FP which overexpress both FR-α and PSMA than the cell lines which only express one target (293T-F or 293T-P) or negative cell line (293T). CBP-8008 can quickly enter cells through endocytosis after treatment for 15 min, and degradation of BRD2, BRD3, and BRD4 was observed in MDA-MB-231 after 6 h, which was similar to its payload. The compound was very stable in mouse and human plasma in vitro, and limited free payload was detected which potentially avoids hematotoxicity in clinical. In the tumor-beaning mice DMPK study, fewer free payload was detected in 6 hours while much more free payload was detected in the tumor than in the blood and the time was extended to 72 hours after a single dose treatment. We evaluated CBP-8008 in TNBC and mCRPC xenograft model and robust efficacy was observed as lower as 3 MPK, significantly better than BMS-986158, a potent Bet inhibitor in clinical. In conclusion: CBP-8008 is currently in preclinical development as a potential first-in-class pan-Bet protein degradation therapy using Bi-XDC technology for TNBC and mCRPC Citation Format: Tao Wang, Zhiguang Zhang, Guitao Wang, Fan Pan, Robert Huang. CBP-8008: A first-in-class targeted pan-Bet protein degradation therapy using bi-specific XDC (Bi-XDC) technology for TNBC and mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6042.

Abstract 6054: UPPGRADER: a bioinformatics-based novel E3 ligase discovery platform

Abstract In Target Protein Degradation (TPD) field, conventional E3 ligases utilized in the clinic are limited to CRBN and VHL, which opens the need for exploration of novel E3 ligases for the advancement of TPD. To address this, we have developed a proprietary platform, UPPGRADER, for systematic discovery of novel E3 ligases based on -omics data including single cell RNA sequencing (scRNA-seq) and whole genome sequencing (WGS), in conjunction with proteomics data and bioinformatics tools.UPPGRADER enables systematic analyses on alterations occurred in all genomic areas including E3 ligase genes and potential target genes. For example, analyzing Pan-Cancer Analysis of Whole Genomes (PCAWG) data identified amino acid changes into Cysteine or Lysine, which can be targeted by covalent binders with cancer specificity. Through such analysis, we discovered several novel E3 ligases, including but not limited to FBXW7, with the categorization of the findings based on specific cancer types.At the single cell expression level, UPPGRADER provides the capability to assess gene expression levels of E3 ligases and target proteins by proprietary measurements based on gene detection rate. This system also allows assessment of E3 ligase-Target gene co-expression per single cell. In colorectal cancer, UPPGRADER has shown that the co-expression of E3 ligase FBXW7 and well-known colorectal cancer target (KRAS) increases from 1% (4 out of 334) in normal epithelial cells to 17% (2,967 out of 17,458) in cancerous epithelial cells, highlighting a significant change in their simultaneous expression patterns during cancer progression.UPPGRADER also identified E3 ligase X which showed high co-expression level with KRAS gene alongside universal expression properties and high cancer cell dependencies. For E3 ligase X, we developed novel small molecule binders (nM binding affinity) with bioavailability > 20% in animal pharmacokinetics. Bifunctional degraders utilizing these binders displayed BRD4 degradation in more than 20 cell lines with double to triple digit nM DC50 and DC90 values. Other targets (AURKA, CRBN and others) were potently degraded with anticipated mechanism of action (assessed by E3 ligase KO cell line and proteasomal inhibitors).To conclude, UPPGRADER is a comprehensive bioinformatics tool for discovering novel E3 ligases with biological validations. Through recent explosion of scRNA-seq and WGS datasets in different diseases and mechanism-based identification of additional molecular targets, UPPGRADER is poised to advance the field of TPD. Citation Format: Kyungsik Ha, Woojeung Song, Woochang Choi, Gi Bbeum Lee, Soohee Ryu, Jun Kyu Lee, Hwajin Lee. UPPGRADER: a bioinformatics-based novel E3 ligase discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6054.

Optimization of Potent Ligands for the E3 Ligase DCAF15 and Evaluation of Their Use in Heterobifunctional Degraders.

Unlocking novel E3 ligases for use in heterobifunctional PROTAC degraders is of high importance to the pharmaceutical industry. Over-reliance on the current suite of ligands used to recruit E3 ligases could limit the potential of their application. To address this, potent ligands for DCAF15 were optimized using cryo-EM supported, structure-based design to improve on micromolar starting points. A potent binder, compound 24, was identified and subsequently conjugated into PROTACs against multiple targets. Following attempts on degrading a number of proteins using DCAF15 recruiting PROTACs, only degradation of BRD4 was observed. Deconvolution of the mechanism of action showed that this degradation was not mediated by DCAF15, thereby highlighting both the challenges faced when trying to expand the toolbox of validated E3 ligase ligands for use in PROTAC degraders and the pitfalls of using BRD4 as a model substrate.

Structure-Guided Design and Optimization of Covalent VHL-Targeted Sulfonyl Fluoride PROTACs.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest; however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report the structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1α binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or the androgen receptor without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design, expanding the substrate scope of covalent E3 ligase PROTACs.

Targeted protein degradation via intramolecular bivalent glues

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)—bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1–4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change ‘glues’ BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target–ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4–IBG1–DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

MZ1, a BRD4 inhibitor, exerted its anti-cancer effects by suppressing SDC1 in glioblastoma

BackgroundGlioblastoma (GBM) is a relatively prevalent primary tumor of the central nervous system in children, characterized by its high malignancy and mortality rates, along with the intricate challenges of achieving complete surgical resection. Recently, an increasing number of studies have focused on the crucial role of super-enhancers (SEs) in the occurrence and development of GBM. This study embarks on the task of evaluating the effectiveness of MZ1, an inhibitor of BRD4 meticulously designed to specifically target SEs, within the intricate framework of GBM.MethodsThe clinical data of GBM patients was sourced from the Chinese Glioma Genome Atlas (CGGA) and the Gene Expression Profiling Interactive Analysis 2 (GEPIA2), and the gene expression data of tumor cell lines was derived from the Cancer Cell Line Encyclopedia (CCLE). The impact of MZ1 on GBM was assessed through CCK-8, colony formation assays, EdU incorporation analysis, flow cytometry, and xenograft mouse models. The underlying mechanism was investigated through RNA-seq and ChIP-seq analyses.ResultsIn this investigation, we made a noteworthy observation that MZ1 exhibited a substantial reduction in the proliferation of GBM cells by effectively degrading BRD4. Additionally, MZ1 displayed a notable capability in inducing significant cell cycle arrest and apoptosis in GBM cells. These findings were in line with our in vitro outcomes. Notably, MZ1 administration resulted in a remarkable decrease in tumor size within the xenograft model with diminished toxicity. Furthermore, on a mechanistic level, the administration of MZ1 resulted in a significant suppression of pivotal genes closely associated with cell cycle regulation and epithelial-mesenchymal transition (EMT). Interestingly, our analysis of RNA-seq and ChIP-seq data unveiled the discovery of a novel prospective oncogene, SDC1, which assumed a pivotal role in the tumorigenesis and progression of GBM.ConclusionIn summary, our findings revealed that MZ1 effectively disrupted the aberrant transcriptional regulation of oncogenes in GBM by degradation of BRD4. This positions MZ1 as a promising candidate in the realm of therapeutic options for GBM treatment.