The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a versatile RNA-binding protein playing a critical role in alternative pre-mRNA splicing regulation in cancer. Emerging data have implicated hnRNP A1 as a central player in a splicing regulatory circuit involving its direct transcriptional control by c-Myc oncoprotein and the production of the constitutively active ligand-independent alternative splice variant of androgen receptor, AR-V7, which promotes castration-resistant prostate cancer (CRPC). As there is an urgent need for effective CRPC drugs, targeting hnRNP A1 could, therefore, serve a dual purpose of preventing AR-V7 generation as well as reducing c-Myc transcriptional output. Herein, we report compound VPC-80051 as the first small molecule inhibitor of hnRNP A1 splicing activity discovered to date by using a computer-aided drug discovery approach. The inhibitor was developed to target the RNA-binding domain (RBD) of hnRNP A1. Further experimental evaluation demonstrated that VPC-80051 interacts directly with hnRNP A1 RBD and reduces AR-V7 messenger levels in 22Rv1 CRPC cell line. This study lays the groundwork for future structure-based development of more potent and selective small molecule inhibitors of hnRNP A1–RNA interactions aimed at altering the production of cancer-specific alternative splice isoforms.

While Myc is an essential regulator of growth in normal cells, it is also frequently associated with cancer progression, therapy-resistance and lethal outcomes in most human cancers. In prostate cancer (PCa), Myc transcription factors are implicated in the pathogenesis and progression of the full spectrum of PCa, from adenocarcinoma to advanced castration-resistant and neuroendocrine phenotypes. Though a high-value therapeutic target, clinically approved anti-Myc drugs have yet to be discovered. To elicit its oncogenic effects, Myc must form a heterodimer with its partner Max, which together bind DNA and activate transcription of a spectrum of target genes that promote cell growth, proliferation, metabolism, and apoptosis while blocking differentiation. In this study, we identified a binding site on the DNA-binding domain of the structurally ordered Myc-Max complex and employed a computer-aided rational drug discovery approach to identify small molecules that effectively inhibit Myc-Max functionality. A large-scale virtual screening protocol implementing structure-based methodologies was utilized to select a set of top-ranked compounds that were subsequently evaluated experimentally and characterized mechanistically for their ability to inhibit Myc-Max transcriptional activity and subsequent downstream functions, to reduce viability in PCa cell lines, disrupt protein-DNA interactions and to induce apoptosis as their mechanism of action. Among compounds identified that effectively inhibit Myc-Max activity with low to mid-micromolar range potency and no or minimal generic cytotoxicity, VPC-70067, a close analog of the previously identified Myc inhibitor 10058-F4, served as proof-of-concept that our in silico drug discovery strategy performed as expected. Compound VPC-70063, of a chemically different scaffold, was the best performer in a panel of in vitro assays, and the forerunner for future hit-to-lead optimization efforts. These findings lay a foundation for developing more potent, specific and clinically optimized Myc-Max inhibitors that may serve as promising therapeutics, alone or in combination with current anti-cancer treatments, for treatment of specific phenotypes or heterogeneous tumors.

Lin28 is a pluripotency factor that regulates cancer cell stem-like phenotypes to promote cancer development and therapy-resistant tumor progression. It acts through its cold shock domain and zinc knuckle domain (ZKD) to interact with the Let-7 pre-microRNA and block Let-7 biosynthesis. Chemical inhibition of Lin28 from interacting with Let-7 presents a therapeutic strategy for cancer therapy. Herein, we present the computer-aided development of small molecules by in silico screening 18 million compounds from the ZINC20 library, followed by the biological validation of 163 predicted compounds to confirm 15 new Lin28 inhibitors. We report three lead compounds, Ln7, Ln15, and Ln115, that target the ZKD of both Lin28A and Lin28B isoforms and block Lin28 from binding Let-7. They restore Let-7 expression and suppress tumor oncogenes such as SOX2 in cancer cells and show strong inhibitory effects on cancer cell stem-like phenotypes. However, minimal impacts of these compounds were observed on Lin28-negative cells, confirming the on-target effects of these compounds. We conclude from this study the discovery of several new Lin28 inhibitors as promising candidate compounds that warrant further drug development into potential anticancer therapies.

Resistance to drug treatments is common in prostate cancer (PCa), and the gain-of-function mutations in human androgen receptor (AR) represent one of the most dominant drivers of progression to resistance to AR pathway inhibitors (ARPI). Previously, we evaluated the in vitro response of 24 AR mutations, identified in men with castration-resistant PCa, to five AR antagonists. In the current work, we evaluated 44 additional PCa-associated AR mutants, reported in the literature, and thus expanded the study of the effect of darolutamide to a total of 68 AR mutants. Unlike other AR antagonists, we demonstrate that darolutamide exhibits consistent efficiency against all characterized gain-of-function mutations in a full-length AR. Additionally, the response of the AR mutants to clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesterone and hydrocortisone), was also investigated. As genomic profiling of PCa patients becomes increasingly feasible, the developed “AR functional encyclopedia” could provide decision-makers with a tool to guide the treatment choice for PCa patients based on their AR mutation status.