Ravi Shashi Nayana Munuganti

Targeting the Binding Function 3 (BF3) Site of the Androgen Receptor Through Virtual Screening. 2. Development of 2-((2-phenoxyethyl) thio)-1H-benzimidazole Derivatives

The human androgen receptor (AR) is a proven therapeutic target in prostate cancer. All current antiandrogens, such as Bicalutamide, Flutamide, Nilutamide, and Enzalutamide, target the buried hydrophobic androgen binding pocket of this protein. However, effective resistance mechanisms against these therapeutics exist such as mutations occurring at the target site. To overcome these limitations, the surface pocket of the AR called binding function 3 (BF3) was characterized as an alternative target for small molecule therapeutics. A number of AR inhibitors directly targeting the BF3 were previously identified by us ( J. Med. Chem. 2011 . 54 , 8563 ). In the current study, based on the prior results, we have developed structure-activity relationships that allowed designing a series of 2-((2-phenoxyethyl)thio)-1H-benzimidazole and 2-((2-phenoxyethyl)thio)-1H-indole as lead BF3 inhibitors. Some of the developed BF3 ligands demonstrated significant antiandrogen potency against LNCaP and Enzalutamide-resistant prostate cancer cell lines.

Identification of a Potent Antiandrogen that Targets the BF3 Site of the Androgen Receptor and Inhibits Enzalutamide-Resistant Prostate Cancer

There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1H-indol-2-yl)-1H-indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

Discovery of 1H-indole-2-carboxamides as novel inhibitors of the androgen receptor binding function 3 (BF3).

To overcome resistance to conventional anti-androgens of human androgen receptor (AR), the allosteric site of the AR binding function 3 (BF3) was investigated as an alternative target for small molecule therapeutics. A library of 1H-indole-2-carboxamides were discovered as BF3 inhibitors and exhibited strong antiproliferative activity against LNCaP and enzalutamide-resistant prostate cancer cell lines. Several of the lead compounds may prove of particular benefit as a novel alternative treatment for castration-resistant prostate cancers.

Targeting Binding Function-3 of the Androgen Receptor Blocks Its Co-Chaperone Interactions, Nuclear Translocation, and Activation

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3–directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936–45. ©2016 AACR.

Drug-Discovery Pipeline for Novel Inhibitors of the Androgen Receptor

The androgen receptor (AR) is an important regulator of genes responsible for the development and recurrence of prostate cancer. Current therapies for this disease rely on small-molecule inhibitors that block the transcriptional activity of the AR. Recently, major advances in the development of novel AR inhibitors resulted from X-ray crystallographic information on the receptor and utilization of in silico drug design synergized with rigorous experimental testing.Herein, we describe a drug-discovery pipeline for in silico screening for small molecules that target an allosteric region on the AR termed the binding-function 3 (BF3) site. Following the identification of potential candidates, the compounds are tested in cell culture and biochemical assays for their ability to interact with and inhibit the AR. The described pipeline is readily accessible and could be applied in drug design efforts toward any surface-exposed region on the AR or other related steroid nuclear receptor.

Benzothiophenone Derivatives Targeting Mutant Forms of Estrogen Receptor-α in Hormone-Resistant Breast Cancers

Estrogen receptor-α positive (ERα+) breast cancers represent 75% of all invasive breast cancer cases, while de novo or acquired resistance to ER-directed therapy is also on the rise. Numerous factors contribute to this phenomenon including the recently-reported ESR1 gene mutations such as Y537S, which amplifies co-activator interactions with ERα and promotes constitutive activation of ERα function. Herein, we propose that direct targeting of the activation function-2 (AF2) site on ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. A systematic computer-guided drug discovery approach was employed to develop a potent ERα inhibitor that was extensively evaluated by a series of experiments to confirm its AF2-specific activity. We demonstrate that the developed small-molecule inhibitor effectively prevents ERα-coactivator interactions and exhibits a strong anti-proliferative effect against tamoxifen-resistant cells, as well as downregulates ERα-dependent genes and effectively diminishes the receptor binding to chromatin. Notably, the identified lead compound successfully inhibits known constitutively-active, resistance-associated mutant forms of ERα observed in clinical settings. Overall, this study reports the development of a novel class of ERα AF2 inhibitors, which have the potential to effectively inhibit ERα activity by a unique mechanism and to circumvent the issue of mutation-driven resistance in breast cancer.

Abstract 3189: Neuronal transcription factor BRN2 is an androgen suppressed driver of neuroendocrine differentiation in prostate cancer

Neuroendocrine prostate cancer (NEPC) is an incurable, rapidly progressing and lethal disease. NEPC is increasingly recognized as a highly therapy resistant tumor variant that evolves from castration-resistant prostate cancer (CRPC) in a subset of patients treated with potent androgen receptor (AR) pathway inhibitors like enzalutamide (ENZ). Importantly, mechanisms by which the AR directly controls the emergence of NEPC from CRPC under the selective pressure of ENZ remain elusive. As the AR is the cornerstone therapeutic target in men with CRPC, understanding its contribution to the development of NEPC is critical to better implement current standard-of-care therapies such as ENZ, and to identify novel therapeutic targets for this incurable disease. Hallmarks of NEPC are resistance to ENZ and the loss or reduced activity of the AR. Thus, we hypothesized that a consequence of ENZ treatment and resistance in CRPC is relief of AR-mediated suppression of factors that drive NEPC. To investigate this, we developed a model of prostate cancer that mimics clinical progression to CRPC and ENZ resistance (ENZR CPRC). Mirroring what is observed in some patients who progress on ENZ, 25% of our ENZR CRPC tumors and derived cell lines showed strong reduction in classic activity of the AR and a NEPC phenotype. By interrogating NEPC-like ENZR CRPC and ENZ-treated CRPC tumors and cell lines with RNA-Seq and mechanistic in vitro and in vivo studies, as well as human tumors with RNA-Seq and IHC, we identified the master neural transcription factor BRN2 as a central and clinically relevant driver of NEPC differentiation. Specifically, we found AR binding in the BRN2 enhancer directly represses BRN2 transcription and this release drives NEPC marker expression and aggressive growth of ENZR CRPC. Our data also reveal a striking overlap of AR and SOX binding motifs in the enhancer region of BRN2 creating a competitive binding scenario between AR and SOX2, another cell-fate determining transcription factor associated with NEPC. We discovered that upregulation of BRN2 further enhances SOX2 expression and that a BRN2-SOX2 interaction contributes to NEPC differentiation. Importantly, we found BRN2 is highly expressed in human NEPC and metastatic CRPC, especially in patients with low AR activity, highlighting its clinical relevance to disease that is difficult to treat with mainstay therapies. These data underscore the consequences of potent AR inhibition in CRPC, revealing a novel mechanism of AR-dependent control of NEPC via direct suppression of BRN2. This work uncovers BRN2 as a clinically relevant potential therapeutic target to prevent emergence of NEPC from ENZR CRPC. Citation Format: Jennifer L. Bishop, Daksh Thaper, Sepideh Vahid, Ravi S. Munuganti, Paul Ahn, Alastair Davies, Kirsi Ketola, Ka Mun Nip, Dong Lin, Yuzhuo Wang, Himisha Beltran, Amina Zoubeidi. Neuronal transcription factor BRN2 is an androgen suppressed driver of neuroendocrine differentiation in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3189. doi:10.1158/1538-7445.AM2017-3189

Abstract 2508: The development of anti-androgens with a novel mechanism of action for treatment of castration-resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The androgen receptor (AR) is one of the most validated therapeutic targets in prostate cancer (PCa). Conventional anti-androgens lose effectiveness as cancer therapeutics because anti-androgen resistance usually develops after long term treatment. The challenge is that the current therapeutics bind to the same site of the AR (hormone binding pocket) and act via the same mode, to which the receptor has already developed effective resistance mechanisms. Hence, there is a pressing need for novel therapeutics that inhibit the AR through novel, alternative modes of action. Recent studies have identified a novel binding pocket on the surface of AR called binding function 3 (BF3) that is important for the AR transcriptional activity. In order to identify compounds that specifically bind to BF3 site and inhibit the AR, we conducted a systematic in silico screen (that included large-scale docking, in-site rescoring, and consensus voting procedures) followed by experimental validation of the identified hit molecules. As a result, we have discovered a novel chemical series of indoles as lead BF3 inhibitors. One of the most potent inhibitors identified, VPC-13163, demonstrated an IC50 of 0.31µM in AR eGFP transcriptional assay. Confirming it as a true BF3 binder, VPC-13163 neither displaced the co-activator from an alternative coactivator binding site, activation function 2 site, nor androgen from the hormone binding pocket. Additionally, the Biolayer Interferometry assay detected direct reversible interactions between the AR ligand binding domain and the inhibitor. VPC-13163 demonstrated strong anti-proliferative activity against LNCaP and Enzalutamide-resistant prostate cancer cell lines (MR49F) whereas it did not affect the growth of AR independent PC3 cell line. It also inhibits prostate specific antigen (PSA) in both LNCaP and MR49F and reduces expression of AR target genes, PSA and TMPRSS2. These findings suggest that VPC-13163 exhibits AR BF3 specific mechanism of action. Furthermore, VPC-13163 reduces AR-dependent growth of xenograft tumors in vivo. Based on these outcomes, it can be anticipated that such drug prototypes will lay a foundation for the development of alternative or supplementary small-molecule therapies capable of combating PCa even in its drug resistant forms. Because the emergence of castration resistance is the lethal end stage of the disease, we anticipate that the proposed research will eventually have a substantial impact on patient survival. Citation Format: Ravi Shashi Nayana Munuganti, Mohamed DH Hassona, Eric Leblanc, Fuqiang Ban, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. The development of anti-androgens with a novel mechanism of action for treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2014-2508

Abstract 2521: Small molecule inhibitors targeting the activation function-2 site of estrogen receptor-α

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Approximately 75% of Breast Cancers (BCas) are classified as Estrogen Receptor alpha (ERα) positive. Treatment with anti-estrogens such as Tamoxifen has been the main therapeutic approach for more than 30 years. However, one third of women treated with Tamoxifen for 5 years develop recurrent disease. Experimental and clinical observations have suggested that ERα signalling continues to play an important role even after the development of resistance. Moreover, biopsies from BCa patients who relapsed on Tamoxifen indicated that ERα expression was retained in more than 50% of the cases. Because of the emergence of hormone resistance, there is a clear need to develop entirely novel anti-ERα therapeutics, such as drugs that would directly disrupt the interaction between ERα and its coactivator proteins at the corresponding regulatory interfaces, exemplified by a well-characterized Activation Function-2 (AF-2) site.In the current study we have used state of artificial intelligence systems to rationally select new anti-ERα drug candidates. Using the power of modern computers, we performed large-scale docking using millions of existing chemicals from the ZINC database and identified several promising small molecules as candidate AF-2 binders. We then conducted biological screens to identify compounds that can bind to the AF-2 pocket and inhibit ERα transactivation. A reporter assay was developed using T47D-Kbluc breast cancer cells, a line which had been stably transfected with an estrogen responsive luciferase reporter gene construct consisting of three estrogen response elements (EREs) upstream of a TATA promoter, to evaluate the potential of these compounds to inhibit ERα transcriptional activity. Compounds that inhibited ERα-mediated transcription of the reporter gene in a concentration dependent manner were further analysed. These compounds do not displace estrogen, but block ERα-coactivator interaction, as measured by TR-FRET assay, thereby confirming that inhibition of coactivator recruitment is not by the allosteric mechanism of conventional antagonists. One of our best compounds, VPC-16046, shows direct reversible binding to the ERα ligand binding domain as detected by Biolayer Interferometry assay. This compound demonstrated a strong anti-proliferative effect on MCF7, T47D and Tamoxifen resistant cells without affecting the growth of ERα-negative HeLa cells, used as a control in MTS assay.In summary, our study has identified a novel class of ERα AF2 inhibitors that have the potential to effectively inhibit ERα transcriptional activity by a mechanism which does not target the estrogen binding site and thereby circumvents treatment resistance seen with conventional, clinically used anti-estrogens. Treatment with these inhibitors should lead to a substantial improvement in the survival rate of women with advanced Tamoxifen-resistant BCa. Citation Format: Kriti Singh, Ravi Shashi Nayana Munuganti, Eric Leblanc, Artem Cherkasov, Paul S. Rennie. Small molecule inhibitors targeting the activation function-2 site of estrogen receptor-α. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2521. doi:10.1158/1538-7445.AM2014-2521