The TMPRSS2:ERG gene fusion, present in approximately 50% of prostate cancers in patients of European ancestry, drives oncogenesis through aberrant overexpression of the ERG transcription factor. Despite its role as a truncal oncogenic driver, ERG has been considered undruggable due to the absence of enzymatic activity and apparent lack of ligandable pockets. Here, we demonstrate continued dependency on ERG in metastatic prostate cancer and identify a druggable pocket within its N-terminal Pointed (PNT) domain. Using an inducible shRNA system in TMPRSS2:ERG-positive VCaP cells, we show that ERG depletion causes profound growth inhibition. To therapeutically exploit this vulnerability, we conducted a domain-focused differential scanning fluorimetry screen targeting the ERG PNT domain, followed by structure-activity relationship optimization. This approach yielded PBITE-1 (PNT-Binding Inhibitor of the Transcription factor ERG), a small molecule that selectively binds the ERG PNT domain. NMR chemical-shift perturbation mapping and molecular docking revealed that PBITE-1 engages a discrete, solvent-exposed surface comprising two α-helices and an adjacent flexible loop, defining a ligand-binding pocket within the PNT domain. In cellular models, PBITE-1 directly engaged ERG, selectively inhibited proliferation and invasion, and induced apoptosis in ERG-driven prostate and hematologic malignancies. PBITE-1 potently suppressed growth of ERG-positive mouse and human-derived prostate cancer organoids. Furthermore, PBITE-1 treatment significantly induced tumor cell apoptosis in VCaP xenograft models. These findings establish the ERG PNT domain as ligandable and provide preclinical evidence that ERG is directly targetable by small molecules, enabling future development of ERG-directed inhibitors and targeted protein degraders.

SMARCA2 is an attractive synthetic lethal target in human cancers with mutated, inactivated SMARCA4. We report herein the discovery of highly potent and selective SMARCA2 PROTAC degraders, as exemplified by SMD-3236, which was designed using a new, high-affinity SMARCA ligand and a potent VHL-1 ligand. SMD-3236 achieves DC50 < 1 nM and Dmax > 95% against SMARCA2 and >2000-fold degradation selectivity over SMARCA4. SMD-3236 potently inhibits cell growth in a panel of SMARCA4-deficient cell lines and displays minimal activity in SMARCA4 wild-type cell lines. SMD-3236 induces profound and persistent SMARCA2 depletion in tumor tissues for 1 week with a single administration, while sparing SMARCA4 protein. SMD-3236 effectively inhibits tumor growth with weekly administration in the H838 SMARCA4-deficient human cancer xenograft model at well-tolerated dose schedules. SMD-3236 represents a promising SMARCA2 degrader for extensive evaluation as a new therapy for the treatment of SMARCA4-deficient human cancers.

In the SWI/SNF chromatin-remodeling complex, the mutually exclusive catalytic ATPase subunits SMARCA2 and SMARCA4 proteins have a synthetic-lethal relationship. Selectively targeting SMARCA2 for degradation is a promising and new therapeutic strategy for human cancers harboring inactivated mutated SMARCA4. In this study, we report the design, synthesis, and biological evaluation of novel SMARCA2/4 ligands and our subsequent design of PROTAC degraders using high-affinity SMARCA ligands and VHL-1 ligands. Our efforts led to the discovery of high-affinity SMARCA2/4 bromodomain ligands and the development of a potent and selective SMARCA2 degrader and a highly potent SMARCA2/4 and PBRM1 degrader.

Purpose:Activating pyruvate kinase M2 (PKM2) has been shown to be neuroprotective in preclinical models of photoreceptor degeneration. We recently developed novel, small molecule activators for ocular delivery. Here, we sought to characterize the ocular pharmacology, toxicity, and efficacy of MCTI-566, a novel PKM2 activator, to translate this therapeutic strategy to the clinic. Methods:X-ray protein crystallography and isothermal titration calorimetry assessed the interaction of MCTI-566 with PKM2. PKM2 activation and tissue pharmacokinetics were examined after intravitreal or systemic administration of MCTI-566. Retinal toxicity was evaluated in rats after intravitreal injection. The effect of MCTI-566 on photoreceptor death was assessed using in vitro and in vivo models of outer retinal stress and on the inflammatory response in the rd10 retina using flow cytometry and quantitative real-time polymerase chain reaction. Results:The PKM2-MCTI-566 co-crystal structure demonstrated a binding pocket distinct from endogenous activators. MCTI-566 increases retinal PK activity 200% following intravitreal or systemic administration. MCTI-566 distributed to the retina after intravitreal or systemic administration, activated the target for ≥90 days and was specific for photoreceptor PKM2. No retinal toxicity was observed after repeated intravitreal administration. MCTI-566 reduced photoreceptor apoptosis in a model of retinal detachment, and delayed photoreceptor degeneration and altered the inflammatory response in the rd10 retina. Conclusions:MCTI-566 is a small molecule drug candidate for photoreceptor neuroprotection. Translational Relevance:MCTI-566, a long-acting and well-tolerated ocular PKM2 activator, may be a potential therapeutic to combat currently untreatable retinal degenerations.

SIRT5 is a sirtuin deacylase that removes negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal conditions, the phenotypes of SIRT5 deficiency are quite subtle. Here, we identify two homozygous SIRT5 variants in patients suspected to have mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generated a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross phenotypes. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, but are likely not by themselves the primary pathogenic cause of the neuropathology observed in the patients.

STAT6 is an attractive therapeutic target for human cancers and other human diseases. Starting from a STAT6 ligand with Ki = 3.5 μM binding affinity, we obtained AK-068 with Ki = 6 nM to STAT6 and at least >85-fold binding selectivity over STAT5. Using AK-068 and cereblon ligands, we discovered AK-1690 as the first, potent and selective PROTAC STAT6 degrader. AK-1690 effectively induces degradation of STAT6 protein in cells with DC50 values of as low as 1 nM while showing minimal effect on other STAT members up to 10 μM. A single dose of AK-1690 effectively depletes STAT6 in mouse tissues. Determination of the first cocrystal structure of STAT6 in complex with AK-1690 provides a structural basis for their interactions. AK-1690 is a powerful tool with which to investigate the roles of STAT6 in human diseases and biological processes and a promising lead compound for further optimization.

Melanoma is the deadliest form of skin cancer with a 5-year survival rate of less than 20%. While significant strides have been made in the field of kinase-targeted and immune-based therapies for melanoma, the development of resistance to these therapeutic agents has hindered the success of treatment. Drug-resistant melanoma is particularly reliant on enhanced cap-dependent translation to drive the production of oncoproteins that promote growth and survival. The m7GpppX cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor of cap-dependent translation initiation, and its overexpression in melanoma tumors has been shown to drive resistance to BRAFV600E kinase-targeted inhibitors. These findings point to eIF4E-targeted therapies as a promising strategy to overcome drug resistance in melanoma. Herein, we build upon our previous work of developing cell-permeable cap analogue inhibitors to design second-generation cap analogues that inhibit eIF4E-mediated cap-dependent translation in drug-resistant melanoma cells.

Abstract In human lung, melanoma and other types of human cancers, the mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated, which leads to inactivation of its functions. SMARCA2, a close homologous protein of SMARCA4, is an attractive synthetic lethality target for human cancers with SMARCA4 deficiency. Herein, we report the discovery and biological evaluation of potent, highly selective and efficacious SMARCA2 PROTAC degraders exemplified by UM-SMD-3236. UM-SMD-3236 has a DC50 value of <1 nM in inducing degradation of SMARCA2 in cells and demonstrates >400-fold selectivity over SMARCA4 protein. Of significance, while UM-SMD-3236 achieves a Dmax of >95% against SMARCA2, it shows a Dmax of 50% against SMARCA4 in cells. In vivo, a single intravenous dose of UM-SMD-3236 attains 85-93% of SMARCA2 depletion in tumor tissues for 7 days, while showing no reduction of the SMARCA4 protein. Weekly intravenous administration of UM-SMD-3236 is highly effective in inhibition of tumor growth in SMARCA4 deficient xenograft models of human cancer. Importantly, UM-SMD-3236 shows no signs of toxicity in mice at highly efficacious doses. UM-SMD-3236 represents a highly promising SMARCA2 degrader for extensive evaluation as a potential new therapy for the treatment of SMARCA4-deficient human cancers. References 1. Oike, T.; Ogiwara, H.; Tominaga, Y.; Ito, K.; Ando, O.; Tsuta, K.; Mizukami, T.; Shimada, Y.; Isomura, H.; Komachi, M.; Furuta, K.; Watanabe, S.-I.; Nakano, T.; Yokota, J.; Kohno, T. A Synthetic Lethality-Based Strategy to Treat Cancers Harboring a Genetic Deficiency in the Chromatin Remodeling Factor BRG1. Cancer Res. 2013, 73, 5508-5518. 2. Yang, L.; Tu, W.; Huang, L.; Miao, B.; Kaneshige, A.; Jiang, W.; Leng, L.; Wang, M.; Wen, B.; Sun, D.; Wang, S. Discovery of SMD-3040 as a Potent and Selective SMARCA2 PROTAC Degrader with Strong in vivo Antitumor Activity. J. Med. Chem. 2023, 66, 10761-10781. Citation Format: Lin Yang, Wenbin Tu, Liyue Huang, Lingying Leng, Wei Jiang, Meilin Wang, Bo Wen, Duxin Sun, Jeanne Stuckey, Shaomeng Wang. Development of potent, highly selective and efficacious SMARCA2 degraders [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 3879.

Dysregulation of translation is a hallmark of cancer that enables rapid changes in cellular protein production to shape oncogenic phenotypes. Translation initiation is governed by the m7GpppX cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), the rate-limiting factor of cap-dependent translation initiation. eIF4E is overexpressed in many cancers and drives the production of oncoproteins that promote tumor growth and survival. Accordingly, eIF4E has been established as an attractive albeit challenging therapeutic target. Building upon our previous work of developing cell-permeable cap analogue prodrugs that inhibit eIF4E binding to the m7GpppX cap, herein we disclose the design of second-generation cap analogues with alternative N-9-substituted linkers which exhibit anticancer activity in BRAFV600E mutant melanoma cell lines.

Abstract In human non-small cell lung cancer, melanoma and other types of human cancers, the mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated, which leads to inactivation of its functions. SMARCA2, a close homologous protein of SMARCA4, is an attractive synthetic lethality target for human cancers with SMARCA4 deficiency. Herein, we report the discovery and biological evaluation of potent, highly selective, orally efficacious SMARCA2 PROTAC degraders exemplified by UM-SMD-8801. UM-SMD-8801 has DC50 <10 nM and Dmax >90% against SMARCA2 and >1,000-fold degradation selectivity over SMARCA4. Consistently, UM-SMD-8801 potently inhibits cell growth in SMARCA4 mutated cancer cell lines with low nanomolar IC50 values and shows >100-fold weaker activity in SMARCA4 wild-type cancer cell lines. UM-SMD-8801 has a good overall pharmacokinetic profile and an excellent oral bioavailability in mice. Oral administration of UM-SMD-8801 is highly effective in reducing SMARCA2 protein by >90% in tumor tissues in mice, while having minimal effect on SMARCA4 protein. UM-SMD-8801 represents a very promising SMARCA2 degrader for extensive evaluation as a potential new therapy for the treatment of SMARCA4-deficient human cancers. Citation Format: Lingying Leng, Lin Yang, Wenbin Tu, Rohan Rej, Srinivasa Rao Allu, Liyue Huang, Wei Jiang, Yu Wang, Jeanne Stuckey, Farzad R Sarkari, Meilin Wang, Lu Wang, Bo Wen, Duxin Sun, Shaomeng Wang. Discovery of potent, highly selective and orally efficacious SMARCA2 degraders [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 4506.

Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1mut) acquires an altered 3' splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1wt) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1 mut , we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1 mut and human primary MDS cells carrying U2AF1 mut . RNA-seq analysis of K562-U2AF1mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1 mut cells by inhibiting the U2AF1wt protein.

STAT3 is an attractive therapeutic target for cancer and other human diseases. We have previously reported the discovery of potent, selective, and efficacious PROTAC STAT3 degraders SD-36 and SD-91. In this study, we have designed and synthesized a novel series of STAT3 degraders using a new, high-affinity STAT3 ligand with excellent chemical stability and cereblon ligands. Our efforts led to the discovery of SD-436, a highly potent and selective STAT3 degrader. A single intravenous administration of SD-436 at 5 mg/kg effectively induces rapid, complete, and durable depletion of STAT3 in mouse native and xenograft tumor tissues. SD-436 achieves complete and long-lasting tumor regression even with a weekly dosing schedule in leukemia and lymphoma xenograft models in mice. SD-436 represents a promising STAT3 degrader for advanced preclinical development as a new therapy for the treatment of human cancers and other human diseases.

Starting from a nonselective bromodomain and extraterminal (BET) inhibitor and a cereblon ligand, we have used precise conformational control for the development of two potent and highly selective BRD4 degraders, BD-7148 and BD-9136. These compounds induce rapid degradation of BRD4 protein in cells at concentrations as low as 1 nM and demonstrate ≥1000-fold degradation selectivity over BRD2 or BRD3 protein. Proteomic analysis of >5700 proteins confirmed their highly selective BRD4 degradation. A single dose of BD-9136 selectively and effectively depletes BRD4 protein in tumor tissues for >48 h. BD-9136 effectively inhibits tumor growth without adverse effects on mice and is more efficacious than the corresponding pan BET inhibitor. This study suggests selective degradation of BRD4 as a strategy for the treatment of human cancers and demonstrates a strategy for the design of highly selective PROTAC degraders.

Eukaryotic translation initiation factor 4E (eIF4E) is an RNA-binding protein that binds to the m 7 GpppX-cap at the 5' terminus of coding mRNAs to initiate cap-dependent translation. While all cells require cap-dependent translation, cancer cells become addicted to enhanced translational capacity, driving the production of oncogenic proteins involved in proliferation, evasion of apoptosis, metastasis, and angiogenesis among other cancerous phenotypes. eIF4E is the rate-limiting translation factor and its activation has been shown to drive cancer initiation, progression, metastasis, and drug resistance. These findings have established eIF4E as a translational oncogene and promising, albeit challenging, anti-cancer therapeutic target. Although significant effort has been put forth towards inhibiting eIF4E, the design of cell-permeable, cap-competitive inhibitors remains a challenge. Herein, we describe our work towards solving this long-standing challenge. By employing an acyclic nucleoside phosphonate prodrug strategy, we report the synthesis of cell-permeable inhibitors of eIF4E binding to capped mRNA to inhibit cap-dependent translation.

Supplementary Methods, Figures 1-15 from SM-164: A Novel, Bivalent Smac Mimetic That Induces Apoptosis and Tumor Regression by Concurrent Removal of the Blockade of cIAP-1/2 and XIAP

STAT5 is an attractive therapeutic target for human cancers. We report herein the discovery of a potent and selective STAT5 degrader with strong antitumor activity in vivo. We first obtained small-molecule ligands with sub-micromolar to low micromolar binding affinities to STAT5 and STAT6 SH2 domains and determined co-crystal structures of three such ligands in complex with STAT5A. We successfully transformed these ligands into potent and selective STAT5 degraders using the PROTAC technology with AK-2292 as the best compound. AK-2292 effectively induces degradation of STAT5A, STAT5B, and phosphorylated STAT5 proteins in a concentration- and time-dependent manner in acute myeloid leukemia (AML) cell lines and demonstrates excellent degradation selectivity for STAT5 over all other STAT members. It exerts potent and specific cell growth inhibitory activity in AML cell lines with high levels of phosphorylated STAT5. AK-2292 effectively reduces STAT5 protein in vivo and achieves strong antitumor activity in mice at well-tolerated dose schedules.

PDF - 3347KB, Synthesis and characterization of the lead compound 1 (UMI-59) and its analog 2 (UMI-77), additional details for used methods, and nine supplementary figures.

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2’s efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.