The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors. We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants. This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.

Myc (avian myelocytomatosis viral oncogene homolog) represents one of the most sought after drug targets in cancer. Myc transcription factor is an essential regulator of cell growth, but in most cancers it is overexpressed and associated with treatment-resistance and lethal outcomes. Over 40 years of research and drug development efforts did not yield a clinically useful Myc inhibitor. Drugging the “undruggable” is problematic, as Myc inactivation may negatively impact its physiological functions. Moreover, Myc is a disordered protein that lacks effective binding pockets on its surface. It is well established that the Myc function is dependent on dimerization with its obligate partner, Max (Myc associated factor X), which together form a functional DNA-binding domain to activate genomic targets. Herein, we provide an overview of the knowledge accumulated to date on Myc regulation and function, its critical role in cancer, and summarize various strategies that are employed to tackle Myc-driven malignant transformation. We focus on important structure-function relationships of Myc with its interactome, elaborating structural determinants of Myc-Max dimer formation and DNA recognition exploited for therapeutic inhibition. Chronological development of small-molecule Myc-Max prototype inhibitors and corresponding binding sites are comprehensively reviewed and particular emphasis is placed on modern computational drug design methods. On the outlook, technological advancements may soon provide the so long-awaited Myc-Max clinical candidate.

Breast cancer (BCa) is one of the most predominantly diagnosed cancers in women. Notably, 70% of BCa diagnoses are Estrogen Receptor α positive (ERα+) making it a critical therapeutic target. With that, the two subtypes of ER, ERα and ERβ, have contrasting effects on BCa cells. While ERα promotes cancerous activities, ERβ isoform exhibits inhibitory effects on the same. ER-directed small molecule drug discovery for BCa has provided the FDA approved drugs tamoxifen, toremifene, raloxifene and fulvestrant that all bind to the estrogen binding site of the receptor. These ER-directed inhibitors are non-selective in nature and may eventually induce resistance in BCa cells as well as increase the risk of endometrial cancer development. Thus, there is an urgent need to develop novel drugs with alternative ERα targeting mechanisms that can overcome the limitations of conventional anti-ERα therapies. Several functional sites on ERα, such as Activation Function-2 (AF2), DNA binding domain (DBD), and F-domain, have been recently considered as potential targets in the context of drug research and discovery. In this review, we summarize methods of computer-aided drug design (CADD) that have been employed to analyze and explore potential targetable sites on ERα, discuss recent advancement of ERα inhibitor development, and highlight the potential opportunities and challenges of future ERα-directed drug discovery.

Recurrent, metastatic prostate cancer continues to be a leading cause of cancer-death in men. The androgen receptor (AR) is a modular, ligand-inducible transcription factor that regulates the expression of genes that can drive the progression of this disease, and as a consequence, this receptor is a key therapeutic target for controlling prostate cancer. The current drugs designed to directly inhibit the AR are called anti-androgens, and all act by competing with androgens for binding to the androgen/ligand binding site. Unfortunately, with the inevitable progression of the cancer to castration resistance, many of these drugs become ineffective. However, there are numerous other regulatory sites on this protein that have not been exploited therapeutically. The regulation of AR activity involves a cascade of complex interactions with numerous chaperones, co-factors and co-regulatory proteins, leading ultimately to direct binding of AR dimers to specific DNA androgen response elements within the promoter and enhancers of androgen-regulated genes. As part of the family of nuclear receptors, the AR is organized into modular structural and functional domains with specialized roles in facilitating their inter-molecular interactions. These regions of the AR present attractive, yet largely unexploited, drug target sites for reducing or eliminating androgen signaling in prostate cancers. The design of small molecule inhibitors targeting these specific AR domains is only now being realized and is the culmination of decades of work, including crystallographic and biochemistry approaches to map the shape and accessibility of the AR surfaces and cavities. Here, we review the structure of the AR protein and describe recent advancements in inhibiting its activity with small molecules specifically designed to target areas distinct from the receptor’s androgen binding site. It is anticipated that these new classes of anti-AR drugs will provide an additional arsenal to treat castration-resistant prostate cancer.

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.

The inhibition of the androgen receptor (AR) is an established strategy in prostate cancer (PCa) treatment until drug resistance develops either through mutations in the ligand-binding domain (LBD) portion of the receptor or its deletion. We previously identified a druggable pocket on the DNA binding domain (DBD) dimerization surface of the AR and reported several potent inhibitors that effectively disrupted DBD-DBD interactions and consequently demonstrated certain antineoplastic activity. Here we describe further development of small molecule inhibitors of AR DBD dimerization and provide their broad biological characterization. The developed compounds demonstrate improved activity in the mammalian two-hybrid assay, enhanced inhibition of AR-V7 transcriptional activity, and improved microsomal stability. These findings position us for the development of AR inhibitors with entirely novel mechanisms of action that would bypass most forms of PCa treatment resistance, including the truncation of the LBD of the AR.