PROTAC Research Articles

Abstract P05: Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2

Abstract Overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance, and poor survival outcomes. Previously, we have established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes in NKTL. As EZH2 enzymatic inhibitors have proven ineffective against NKTL, we embarked on the development of novel EZH2 Proteolysis Targeting Chimera (PROTAC) compounds. EZH2 PROTACs consist of an EZH2- and E3-ligase binding domain which promotes targeted ubiquitination of the EZH2 protein thereby marking it for proteasomal degradation. Our initial efforts focused on the iterative synthesis of EZH2 PROTAC candidates optimizing factors such as permeability, solubility and stable ternary complex formation. Through extensive screening, we identified C2911 and C1311, which successfully trigger the dose-dependent degradation of EZH2 and PRC2 components concomitant with a significant loss of survival and induction of apoptosis in both NKTL and DLBCL. These effects were rescued by pre-treatment with proteasome inhibitor Bortezomib or when E3 ligase activity was disrupted through siRNA knockdown. In NKTL, we show that EZH2 PROTAC treatment decreased JAK3 and phospho-STAT5 protein expression and identified the suppression of the JAK-STAT-MYC axis as a potential mechanism of action. EZH2 PROTAC treatment effectively reactivates transcription of canonical EZH2 target genes such as MYT1 and ANPEP and downregulates expression of oncogenes c-Myc and E2F1. Pharmacokinetic studies in vivo demonstrate that these PROTACs exhibit good plasma stability, half-life (>4h) and low clearance thereby establishing potential for future clinical development. The efficacy of EZH2 PROTACs in xenograft mice are currently being evaluated. Collectively, our data supports the discovery of novel EZH2 PROTACs and the critical role it plays in successfully targeting the oncogenic role of EZH2 in NKTL or other non-canonical EZH2-driven cancers through protein depletion where enzymatic-only inhibition was ineffective. We are further investigating the mechanisms stabilizing EZH2 protein levels to enhance PROTAC-mediated cytotoxicity. Citation Format: Nurulhuda Mustafa, Zubaida Talal Alnaseri, Darrel Tan Egk Jian, David Quach Thanh Truong, Wan Hsin Lim, Wee Joo Chng. Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2 [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P05.

Targeted protein degradation in CNS disorders: a promising route to novel therapeutics?

Targeted protein degradation (TPD) is a rapidly expanding field, with various PROTACs (proteolysis-targeting chimeras) in clinical trials and molecular glues such as immunomodulatory imide drugs (IMiDs) already well established in the treatment of certain blood cancers. Many current approaches are focused on oncology targets, leaving numerous potential applications underexplored. Targeting proteins for degradation offers a novel therapeutic route for targets whose inhibition remains challenging, such as protein aggregates in neurodegenerative diseases. This mini review focuses on the prospect of utilizing TPD for neurodegenerative disease targets, particularly PROTAC and molecular glue formats and opportunities for novel CNS E3 ligases. Some key challenges of utilizing such modalities including molecular design of degrader molecules, drug delivery and blood brain barrier penetrance will be discussed.

Tumor-targeted PROTAC prodrug nanoplatform enables precise protein degradation and combination cancer therapy.

Proteolysis targeting chimeras (PROTACs) have emerged as revolutionary anticancer therapeutics that degrade disease-causing proteins. However, the anticancer performance of PROTACs is often impaired by their insufficient bioavailability, unsatisfactory tumor specificity and ability to induce acquired drug resistance. Herein, we propose a polymer-conjugated PROTAC prodrug platform for the tumor-targeted delivery of the most prevalent von Hippel-Lindau (VHL)- and cereblon (CRBN)-based PROTACs, as well as for the precise codelivery of a degrader and conventional small-molecule drugs. The self-assembling PROTAC prodrug nanoparticles (NPs) can specifically target and be activated inside tumor cells to release the free PROTAC for precise protein degradation. The PROTAC prodrug NPs caused more efficient regression of MDA-MB-231 breast tumors in a mouse model by degrading bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinase 9 (CDK9) with decreased systemic toxicity. In addition, we demonstrated that the PROTAC prodrug NPs can serve as a versatile platform for the codelivery of a PROTAC and chemotherapeutics for enhanced anticancer efficiency and combination benefits. This study paves the way for utilizing tumor-targeted protein degradation for precise anticancer therapy and the effective combination treatment of complex diseases.

Orally Bioavailable Proteolysis-Targeting Chimeras: An Innovative Approach in the Golden Era of Discovering Small-Molecule Cancer Drugs

Proteolysis-targeting chimeras (PROTACs) are an emerging therapeutic modality that show promise to open a target space not accessible to conventional small molecules via a degradation-based mechanism. PROTAC degraders, due to their bifunctional nature, which is categorized as ‘beyond the Rule of Five’, have gained attention as a distinctive therapeutic approach for oral administration in clinical settings. However, the development of PROTACs with adequate oral bioavailability remains a significant hurdle, largely due to their large size and less than ideal physical and chemical properties. This review encapsulates the latest advancements in orally delivered PROTACs that have entered clinical evaluation as well as developments highlighted in recent scholarly articles. The insights and methodologies elaborated upon in this review could be instrumental in supporting the discovery and refinement of novel PROTAC degraders aimed at the treatment of various human cancers.

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Use of ionic liquids in amidation reactions for proteolysis targeting chimera synthesis.

Selective degradation of disease-causing proteins using proteolysis targeting chimeras (PROTACs) has gained great attention, thanks to its several advantages over traditional therapeutic modalities. Despite the advances made so far, the structural chemical complexity of PROTACs poses challenges in their synthetic approaches. PROTACs are typically prepared through a convergent approach, first synthesizing two fragments separately (target protein and E3 ligase ligands) and then coupling them to produce a fully assembled PROTAC. The amidation reaction represents the most common coupling exploited in PROTACs synthesis. Unfortunately, the overall isolated yields of such synthetic procedures are usually low due to one or more purification steps to obtain the final PROTAC with acceptable purity. In this work, we focused our attention on the optimization of the final amidation step for the synthesis of an anti-SARS-CoV-2 PROTAC by investigating different amidation coupling reagents and a range of alternative solvents, including ionic liquids (ILs). Among the ILs screened, [OMIM][ClO4] emerged as a successful replacement for the commonly used DMF within the HATU-mediated amidation reaction, thus allowing the synthesis of the target PROTAC under mild and sustainable conditions in very high isolated yields. With the optimised conditions in hand, we explored the scalability of the synthetic approach and the substrate scope of the reaction by employing different E3 ligase ligand (VHL and CRBN)-based intermediates containing linkers of different lengths and compositions or by using different target protein ligands. Interestingly, in all cases, we obtained high isolated yields and complete conversion in short reaction times.

Discovery of the First-in-Class G9a/GLP PROTAC Degrader.

Aberrantly expressed lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9), have been implicated in numerous cancers. Recent studies have uncovered both catalytic and noncatalytic oncogenic functions of G9a/GLP. As such, G9a/GLP catalytic inhibitors have displayed limited anticancer activity. Here, we report the discovery of the first-in-class G9a/GLP proteolysis targeting chimera (PROTAC) degrader 10 (MS8709), as a potential anticancer therapeutic. 10 induces G9a/GLP degradation in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Futhermore, 10 does not alter the mRNA expression of G9a/GLP and is selective for G9a/GLP over other methyltransferases. Moreover, 10 displays superior cell growth inhibition to the parent G9a/GLP inhibitor UNC0642 in prostate, leukemia, and lung cancer cells and has suitable mouse pharmacokinetic properties for in vivo efficacy studies. Overall, 10 is a valuable chemical biology tool to further investigate the functions of G9a/GLP and a potential therapeutic for treating G9a/GLP-dependent cancers.

Liposomal STAT3-Degrading PROTAC Prodrugs Promote Anti-Hepatocellular Carcinoma Immunity via Chemically Reprogramming Cancer Stem Cells.

Cancer stem cells (CSCs) with hyperactivated signal transducer and activator of transcription 3 (STAT3) are a major driver of hepatocellular carcinoma (HCC). Herein, we report a nanointegrative proteolysis-targeting chimera (PROTAC)-based STAT3 degradation strategy that enables efficient chemical reprogramming of HCC-associated CSCs, which potently inhibits CSC growth while evoking anti-HCC immune responses. The PROTAC prodrug was synthesized by conjugating the STAT3 binding domain (inS3) with a thioketal-caged E3 ligase ligand (VL-TK) via an oligo(ethylene glycol) linker (OEG) with tuned length and flexibility and encapsulating it in cRGD-modified cationic liposomes for CSC-targeted delivery while facilitating their lysosomal escape. The PROTAC prodrugs were activated by the upregulated ROS levels in CSCs and efficiently degraded STAT3 for chemical reprogramming, which would not only impair their stemness features but also remodel the immunosuppressive TME into an immunosupportive state to boost anti-HCC immunity. This strategy provides an approach for improving HCC treatment in clinics.

Targeting BRD4 with PROTAC degrader ameliorates LPS-induced acute lung injury by inhibiting M1 alveolar macrophage polarization

ObjectivesAcute lung injury (ALI) is a highly inflammatory condition with the involvement of M1 alveolar macrophages (AMs) polarization, eventually leading to the development of non-cardiogenic edema in alveolar and interstitial regions, accompanied by persistent hypoxemia. Given the significant mortality rate associated with ALI, it is imperative to investigate the underlying mechanisms of this condition so as to identify potential therapeutic targets. The therapeutic effects of the inhibition of bromodomain containing protein 4 (BRD4), an epigenetic reader, has been proven with high efficacy in ameliorating various inflammatory diseases through mediating immune cell activation. However, little is known about the therapeutic potential of BRD4 degradation in acute lung injury. MethodsThis study aimed to assess the protective efficacy of ARV-825, a novel BRD4-targeted proteolysis targeting chimera (PROTAC), against ALI through histopathological examination in lung tissues and biochemical analysis in bronchoalveolar lavage fluid (BALF). Additionally, the underlying mechanism by which BRD4 regulated M1 AMs was elucidated by using CUT & Tag assay. ResultsIn this study, we found the upregulation of BRD4 in a lipopolysaccharide (LPS)-induced ALI model. Furthermore, we observed that intraperitoneal administration of ARV-825, significantly alleviated LPS-induced pulmonary pathological changes and inflammatory responses. These effects were accompanied by the suppression of M1 AMs. In addition, our findings revealed that the administration of ARV-825 effectively suppressed M1 AMs by inhibiting the expression of IRF7, a crucial transcriptional factor involved in M1 macrophages. ConclusionOur study suggested that targeting BRD4 using ARV-825 is a potential therapeutic approach for ALI.

Abstract LB433: Targeting of ABI1 via DNA-based PROTACs

Abstract Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women. Although current therapies reduce initial tumor burden, patients often relapse with aggressive tumors which can metastasize. Abelson Interactor 1 (ABI1) is an adaptor protein which is traditionally seen as a WAVE complex component- regulating actin polymerization, cell migration and adhesion. In this role, ABI1 serves as a driver of breast cancer metastasis. Recent studies have discovered that ABI1 contains a homeobox homology region (HHR) which is able to bind DNA and regulate transcription. In order to specifically target and degrade ABI1, we designed a novel PROteolysis-TArgeting Chimera (PROTAC) system using DNA as the bait for ABI1. We hypothesized that by linking ABI1 DNA targets to the ubiquitin system, we would be able to effectively target ABI1 for degradation. Lenalidomide, an E3 ubiquitin ligase ligand, was attached to an oligonucleotide via click chemistry. Cells were then treated with PROTAC systems via direct addition to media or via lipofection reagents, and levels of ABI1 were detected using western blotting. Using this DNA-based PROTAC system in cell-culture models of both breast and ABI1-elevated prostate cancer suggested that the system is able to target and degrade ABI1. Usage of lipofection reagents were able to improve drug delivery and effectiveness. Further research is needed to identify off-target effects and effectiveness in vivo models of disease to reduce metastasis. In conclusion, PROTACs targeting ABI1 may prove to be an effective and novel method to reduce metastasis and improve outcomes for cancer treatment. Citation Format: Kevin M. Lin, Xiang Li, Leszek Kotula. Targeting of ABI1 via DNA-based PROTACs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB433.

Sulfatase-Induced In Situ Formulation of Antineoplastic Supra-PROTACs.

Proteolysis targeting chimera (PROTAC) technology is an innovative strategy for cancer therapy, which, however, suffers from poor targeting delivery and limited capability for protein of interest (POI) degradation. Here, we report a strategy for the in situ formulation of antineoplastic Supra-PROTACs via intracellular sulfatase-responsive assembly of peptides. Coassembling a sulfated peptide with two ligands binding to ubiquitin VHL and Bcl-xL leads to the formation of a pro-Supra-PROTAC, in which the ratio of the two ligands is rationally optimized based on their protein binding affinity. The resulting pro-Supra-PROTAC precisely undergoes enzyme-responsive assembly into nanofibrous Supra-PROTACs in cancer cells overexpressing sulfatase. Mechanistic studies reveal that the pro-Supra-PROTACs selectively cause apparent cytotoxicity to cancer cells through the degradation of Bcl-xL and the activation of caspase-dependent apoptosis, during which the rationally optimized ligand ratio improves the bioactivity for POI degradation and cell death. In vivo studies show that in situ formulation enhanced the tumor accumulation and retention of the pro-Supra-PROTACs, as well as the capability for inhibiting tumor growth with excellent biosafety when coadministrating with chemodrugs. Our findings provide a new approach for enzyme-regulated assembly of peptides in living cells and the development of PROTACs with high targeting delivering and POI degradation efficiency.

Abstract LB178: HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of all breast carcinomas and has poor prognosis. Chemotherapy is the mainstay treatment for TNBC patients, yet with limited clinical benefit. Androgen receptor (AR) is expressed in approximately 50% of TNBC tumors and thus the anti-androgen therapy could be a promising solution for the treatment of AR+ TNBC. Nevertheless, AR inhibitors including enzalutamide and bicalutamide or the androgen biosynthesis inhibitor abiraterone have failed to demonstrate satisfying anti-tumor activity in clinical trials. We have discovered HP518, an AR-targeting PROTAC (proteolysis targeting chimera) that induces potent degradation of both the wild-type AR and AR mutants. HP518 is currently under the clinical development for the treatment of mCRPC. Here we report the preclinical data on HP518 for the treatment of AR+ TNBC. HP518 robustly degrades AR in multiple TNBC cell lines with a half-maximal degradation concentration (DC50) < 1 nM. HP518 strongly inhibits AR+ TNBC cell growth, whereas the AR inhibitor enzalutamide does not show significant anti-proliferative effect on these cells in the presence or absence of androgen. Consistent with the in vitro results, HP518 significantly and dose-dependently inhibits tumor growth in MDA-MB-453 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models. PI3KCA activating mutations are frequently expressed in AR+ TNBC. HP518 in combination with alpelisib, a PI3Kα inhibitor shows a synergistic anti-tumor activity. Mechanistically, HP518, but not AR inhibitor enzalutamide downregulates the mRNA expression levels of a set of DNA replication-related genes in AR+ TNBC cells, indicating a novel and ligand-independent role for AR in regulating growth of AR+ TNBC cells. Therefore, HP518, an AR-targeting PROTAC degrader holds great potential to fulfill the unmet clinical needs for the AR+ TNBC therapy. Selected pre-clinical data will be presented. HP518 structure will not be disclosed. Citation Format: Jing Li, Hongwei Yuan, Kefan Chen, Yongxu Huo, Guoqing Liu, Wu Du, Xinghai Li. HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB178.

Abstract LB179: Identification of the highly potent and orally available ER-targeting PROTAC degrader HP568 for the treatment of breast cancer

Abstract Breast cancer remains an unmet medical need. It has become the most occurring and deadliest cancer for women globally. Estrogen receptor (ER) is expressed in 75% of breast cancers and has the key role in tumorigenesis and progression of ER+ breast cancers. Endocrine treatments that target ER are the mainstay therapies for the ER+ breast cancers, yet drug resistance unavoidably develops due to mutations in the ER gene. The ER-targeting proteolysis targeting chimera (PROTAC) degrader is an emerging new modality that interrupts the ER signaling through degradation of ER and offers unprecedented potential for solving the endocrine resistance. We have discovered HP568, an ER-targeting PROTAC that is highly potent against both the wild-type ER and ER mutants. HP568 has a favorable ADME profile that is superior to known competitor ER-targeting PROTAC. In the head-to-head comparison efficacy studies, HP568 has demonstrated the dose-dependent and superior efficacy that is well consistent with the drug exposure profile and PD biomarkers. HP568 also shows a synergetic effect in combination with a CDK4/6 inhibitor. HP568 is clean in the secondary pharmacology profiling, and well tolerated in animal tox studies. HP568 is under the preclinical development and has the potential to be the best-in-class ER-targeting degrader. HP568 structure will not be disclosed. Citation Format: Wu Du, Zhilin Tu, Dekun Qin, Haibo Li, Jingyi Duan, Shijuan Liu, Meng Zhang, Zhenyan Nie, Zhihui Yuan, Jing Li, Xinghai Li. Identification of the highly potent and orally available ER-targeting PROTAC degrader HP568 for the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB179.

Abstract LB177: Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC

Abstract Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men worldwide. Androgens play an important role in the development and progression of prostate cancers and targeting androgen receptor (AR) signaling axis over decades has remained a mainstay of prostate cancer therapy. Next generation hormonal agents (NHAs) enzalutamide and abiraterone have brought clinical benefits for metastatic castrate-resistant prostate cancer (mCRPC) patients, AR axis-targeted therapy resistance is inevitable due to AR gene amplification or mutations. AR-V7 is the most clinically relevant AR splice variant associated with endocrine resistance and poor prognosis. AR-V7 constitutively activates AR signaling and lacks the ligand binding domain (LBD) where hormones and AR antagonists interact, resulting in resistance to AR signaling inhibitors (ARSi). Thus, AR-V7 is a promising therapeutic target to address ARSi resistance in mCRPC. We have discovered a highly potent AR-V7 proteolysis targeting chimera (PROTAC), HC-4955 that degrades both the wild-type AR (WT-AR) and AR-V7 mutant. HC-4955 degrades AR-V7 with DC50 < 5 nM. In silico molecular docking analysis predicts the binding of HC-4955 to the N-terminal domain of AR and then the essential amino acids contributed to HC-4955 binding are validated by mutational analysis studies. HC-4955 also potently degrades mutants of AR-LBD mutations including L702H and T878A and these mutations are also associated with ARSi resistance. HC-4955 strongly inhibits proliferation of ARSi-resistant prostate cancer cell lines with IC50 < 10 nM. HC-4955 shows a robust anti-tumor activity in the AR-V7-bearing 22Rv1 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models, with majority of the tumors completely regressed and > 90% AR-V7 expression decreased at 1-5 mg/kg (QD, PO) in these models. The pre-clinical results support the clinical development of HC-4955 for the treatment of mCRPC. Selected pre-clinical data along with the chemical structure of HC-4955 will be presented. Citation Format: Jing Li, Zhilin Tu, Wu Du, Xi Xiang, Zeyu Wen, Yang You, Lijuan Zhao, Yin Chen, Chengcheng Yang, Hongwei Yuan, Xinghai Li. Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB177.

Abstract LB210: Identification of histone deacetylase 10 as a repressor of cancer cell migration

Abstract Histone deacetylases (HDACs) are a family of 18 enzymes with the potential to be epigenetic modifiers. Of the 4 classes of HDACs, HDAC6 and HDAC10 belong to the class IIb subgroup. Unlike HDAC6, HDAC10 contains two deacetylase-like domains, with one active deacetylase domain and one inactive leucine-rich domain, and a preference for a specific acetylated polyamine over other substrates. Aberrant expression of HDAC10 has been implicated in cancer and other diseases. However, the deacetylation targets and biological functions of HDAC10 have not been well characterized due to a lack of specific and potent HDAC10 inhibitors with cellular activity. We previously established CRISPR/Cas9-mediated HDAC10 knockout (KO) cell lines in HCT116 and HeLa S3 cells. In the current study, we used the HCT116 wildtype and KO pair for bulk RNA sequencing, differential gene expression, and gene set enrichment analyses (GSEA), which revealed an upregulation of gene markers and pathways potentially associated with metastasis and invasion in the HDAC10-KO cells, including S100A14, LAMA4, and TGF-beta. As the role of HDAC10 expression on cell migration and invasion has not been previously reported, we performed cell migration assays using our HDAC10 parental and knockout cell line pairs. In both HCT116 and HeLa systems, HDAC10 KO cells exhibited dramatically higher rates of cell migration in Boyden chamber and scratch assays. These same assays were performed in HDAC10-expressing cells in the presence of the HDAC10-specific inhibitor DKFZ-728, which similarly caused a dose-dependent increase in migration, strongly supporting a catalytic role for HDAC10 in repressing migration. In addition to these results, we present data investigating the role of HDAC10 in cell migration/invasion in representative prostate cancer cell lines (LNCAP and LAPC4), as the prostate tumor microenvironment is particularly rich in polyamines, including N 8-acetylated spermidine, the substrate of HDAC10. Finally, using our panel of HDAC10-KO cell lines and inhibitors, we analyze the contribution of commonly perturbed genes to the migratory phenotype with the goal of defining the precise mechanism of action. Overall, our studies provide functional evidence that loss of HDAC10 can increase cell migration, and thus, may lead to increased metastatic potential. These results emphasize the need for isoform-specific inhibitors and may have important clinical implications, as commonly utilized HDAC inhibitors, including vorinostat (SAHA), tubastatin A, and trichostatin A (TSA), also inhibit HDAC10. Our ongoing studies include incorporating HDAC10 proteolysis targeting chimeras (PROTACs) into our functional assays to examine differences between HDAC10 inhibition and PROTAC-mediated degradation, providing further rationale for modulating HDAC10 activity as an antineoplastic strategy. Citation Format: Ting-Ann Liu, Ashley Nwafor, Rajendra Kumar, Laura Sena, Raphael R. Steimbach, Aubry K. Miller, Robert A. Casero, Tracy Murray Stewart. Identification of histone deacetylase 10 as a repressor of cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB210.

Abstract ND05: The discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma

Abstract Discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma (NHL)Dan Sherman, Sheryl M. Gough, Lynn DeCarr, Sarah Eaton, Mark Bookbinder, Jennifer Pizzano, Elizabeth Bortolon, John Corradi, Rashaun Wilson, Daniel Rogoz, Ram Lingamaneni, Wei Zhang, Michelle Zhang, Xin Chen, Gan Wang, Hanqing Dong, Michael Berlin, Keith R. Hornberger, Lawrence Snyder, Achal Pashine and Ian Taylor. Arvinas, Inc., 5 Science Park, New Haven, CT, USA. Approximately 18,000 cases of Diffuse large B-cell lymphomas (DLBCL) are diagnosed worldwide per year making this cancer the most prevalent (30%) of the non-Hodgkin’s Lymphomas (NHL). Despite the success of R-CHOP treatment, approximately 40% of patients are refractory or relapse. While much progress has been made with CAR-Ts and bi-specific antibodies following frontline therapy in recent years, there remains a need for an orally bioavailable small-molecule that targets molecular drivers of NHL. The BCL6 transcription factor has long been known as an oncogenic driver of B-cell malignancy. The BCL6 gene has been found to be translocated and/or mutated resulting in deregulation of its expression, predominantly in DLBCL and Burkitt’s lymphoma (BL). The deregulated expression and scaffolding role of BCL6 in the transcriptional repressor complex make it an ideal target for the PROteolysis Targeting Chimera (PROTAC®) technology with one BCL6 degrader recently entering Phase 1 trials. We undertook a medicinal chemistry campaign to identify a BCL6-targeting PROTAC®. This effort identified ARV-393, an orally bioavailable PROTAC® that recruits BCL6 and the E3 ligase cereblon to rapidly degrade BCL6 via the cell’s natural ubiquitin proteasome system. ARV-393 is potent, demonstrating DC50 and GI50 values <1 nM in numerous DLBCL and BL cell lines. This agent demonstrates deep BCL6 degradation in vitro and in vivo, resulting in excellent TGI in several tumor xenograft models supporting a first-in-human trial in 1H 2024. We will present the chemical structure of ARV-393 and selected pre-clinical data. Citation Format: Dan Sherman. The discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND05.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

SuFEx-based chemical diversification for the systematic discovery of CRBN molecular glues

Molecular glues are small molecules that stabilize protein–protein interactions, enabling new molecular pharmacologies, such as targeted protein degradation. They offer advantages over proteolysis targeting chimeras (PROTACs), which present challenges associated with the size and properties of heterobifunctional constructions, but glues lack the rational design principles analogous to PROTACs. One notable exception is the ability to alter the structure of Cereblon (CRBN)-based molecular glues and redirect their activity toward new neo-substrate proteins. We took a focused approach toward modifying the CRBN ligand, 5′-amino lenalidomide, to alter its neo-substrate specificity using high-throughput chemical diversification by parallelized sulfur(VI)-fluoride exchange (SuFEx) transformations. We synthesized over 3,000 analogs of 5′-amino lenalidomide using this approach and screened the crude products using a phenotypic screen for cell viability, identifying dozens of analogs with differentiated activity. We characterized four compounds that degrade G-to-S phase transition 1 (GSPT1) protein, providing a proof-of-concept model for SuFEx-based discovery of CRBN molecular glues.

The 2024 IUPAC-Richter Award Goes to Craig M. Crews

Abstract Craig M. Crews has been awarded the 2024 IUPAC-Richter Prize in Medicinal Chemistry. Crews is a Professor in the Department of Molecular, Cellular and Developmental Biology and Professor of Chemistry and Pharmacology at Yale University. His discovery and implementation of the PROteolysis TArgeting Chimeras (PROTACs) technology for removing specific unwanted proteins revolutionized the field by identifying a new method for affecting protein function. The fact that this technology has been widely adapted and investigated not only within the pharmaceutical industry, but also in academia, demonstrates the significance and impact of this work. While no drugs have yet been approved, several are in development and these molecules are progressing, because they are effective and safe. He also played a key role in the discovery of carfilzomib, an approved proteasome inhibitor for the treatment of multiple myeloma. The acceptance lecture will be held in Rome, Italy (1-5 September 2024) at the XXVIII EFMC International Symposium on Medicinal Chemistry.

Breaking Bad Proteins-Discovery Approaches and the Road to Clinic for Degraders.

Proteolysis-targeting chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD), with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches. This review focuses on drug discovery with respect to bifunctional and molecular glue degraders within the ubiquitin proteasome system, including analysis of mechanistic concepts and discovery approaches, with an overview of current clinical and pre-clinical degrader status in oncology, neurodegenerative and inflammatory disease.