Kriti Singh

Selectively Targeting the DNA-binding Domain of the Androgen Receptor as a Prospective Therapy for Prostate Cancer

Background: The androgen receptor (AR) is a transcription factor regulating progression of prostate cancer. Results: Developed compounds inhibit AR transcriptional activity in vitro and in vivo by selective targeting of the AR-DNA-binding domain (DBD). Conclusion: By targeting the DBD, the compounds differ from conventional anti-androgens. Significance: Anti-androgens with a novel mechanism of action have the potential to treat recurrent prostate cancer. The androgen receptor (AR) is a transcription factor that has a pivotal role in the occurrence and progression of prostate cancer. The AR is activated by androgens that bind to its ligand-binding domain (LBD), causing the transcription factor to enter the nucleus and interact with genes via its conserved DNA-binding domain (DBD). Treatment for prostate cancer involves reducing androgen production or using anti-androgen drugs to block the interaction of hormones with the AR-LBD. Eventually the disease changes into a castration-resistant form of PCa where LBD mutations render anti-androgens ineffective or where constitutively active AR splice variants, lacking the LBD, become overexpressed. Recently, we identified a surfaced exposed pocket on the AR-DBD as an alternative drug-target site for AR inhibition. Here, we demonstrate that small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length and splice variant AR forms at low to sub-micromolar concentrations. The inhibition is lost when residues involved in drug interactions are mutated. Furthermore, the compounds did not impede nuclear localization of the AR and blocked interactions with chromatin, indicating the interference of DNA binding with the nuclear form of the transcription factor. Finally, we demonstrate the inhibition of gene expression and tumor volume in mouse xenografts. Our results indicate that the AR-DBD has a surface site that can be targeted to inhibit all forms of the AR, including enzalutamide-resistant and constitutively active splice variants and thus may serve as a potential avenue for the treatment of recurrent and metastatic prostate cancer.

Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients

BackgroundThe 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.ResultsWe 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.ConclusionsThis 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.

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.

Characterization of a New Class of Androgen Receptor Antagonists with Potential Therapeutic Application in Advanced Prostate Cancer

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer. Mol Cancer Ther; 12(11); 2425–35. ©2013 AACR.

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.

Erratum: Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer. (American Society for Biochemistry and Molecular Biology Inc. (2014) 289 (26417-26429) DOI: 10.1074/jbc.M114.553818)

Based on misinterpretation of the NMR spectra provided by our chemical vendor, the structure of VPC-14449 should be corrected. VPC-14449 (4-(4-(4,5-bromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine) should be replaced with VPC-14449 (4-(4-(2,4-dibromo-1H-imidazol-1-yl)thiazol2-yl)morpholine). In Fig. 1A, the graphical representation of VPC-14449 should be changed to the correct structure. Our industry partner synthesized the published VPC-14449 structure (4,5-bromo) and noticed that its NMR spectrum was different from that of our VPC-14449 stock synthesized by our chemical vendor. Upon synthesizing the correct structure (2,4-bromo), it was found that the NMR spectrum of the newly synthesized 2,4-bromo compound superimposed with the NMR spectrum of the compound supplied by our chemical vendor, establishing that the original compound used in this article was the 2,4-bromo version. In conclusion, our stock of VPC-14449 supplied by our chemical vendor and used in this article is (4-(4-(2,4-dibromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine). This error does not affect the results or conclusions of this work, as we simply reported the incorrect structure of our compound. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 10, p. 4359, March 10, 2017

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 PD6-7: In-silico discovery of novel estrogen receptor-α inhibitors as potential therapeutics for tamoxifen resistant breast cancer

Estrogen receptor-α (ER) positive breast cancer (BCa) represents 75% of all invasive BCas. Conventional ER-directed drug Tamoxifen targets the estrogen binding pocket (EBP) of the receptor. However, over prolonged periods of treatment, the therapeutic efficacy of Tamoxifen declines due to development of resistance. Numerous factors are causative for this phenomenon, including recently reported mutations in the receptor (T537S). Therefore, there is an urgency to develop novel anti-ER therapeutics that exhibit entirely different mode of ER inhibition. A promising alternative strategy is to prevent receptor-coactivator interaction and block further crucial steps in ER activity. ER-coactivator interface should be less prone to adaptive mutations as any mutations at this site would also likely block the coactivator recruitment, we therefore targeted the Activation Function-2 (AF2) site, a coactivator binding pocket on ER, called to overcome the limitations of Tamoxifen. Although AF2 is a shallow surface pocket, the pharmacophore-rich features of this site make it a druggable target. To identify potential ER AF2 inhibitors, virtual screening was performed. Initial hits were subjected to lead optimization and more potent analogues were rationally designed by exploiting critical features of this site. Potential compounds were tested for their ability to inhibit ER transcriptional activity using the T47D-KBluc cell line stably transfected with an ER-specific luciferase reporter. Consequently, the lead compound VPC-16339 (IC50=8.24µM) was identified. The direct binding between VPC-16339 and the receptor was confirmed by Biolayer Interferometry assay. More importantly, VPC-16339 prevents coactivator recruitment at the AF2 pocket in a dose dependent manner as measured by TR-FRET coactivator recruitment assay. Increasing concentrations of estradiol did not affect the IC50 of the lead compound, thereby ruling out the possibility of VPC-16339 binding to EBP. VPC-16339 demonstrated a strong anti-proliferative effect on MCF7 and Tamoxifen resistant cells, with no effect on ER- HeLa cells, suggesting its selective ER-mediated action, as further validated by ER luciferase assay in Tamoxifen resistant cells. VPC-16339 effectively inhibits mRNA and protein expression levels of the estrogen dependent genes such as pS2,CathD and CDC2. Due to AF2-guided mechanism, VPC-16339 successfully overcomes Tamoxifen resistance and inhibits the constitutively active Tamoxifen resistant form of ER (T537S). In summary, we report VPC-16339 as an ER AF2 specific inhibitor with promising anti-proliferative effect in BCa cell lines including Tamoxifen resistant cell lines. VPC-16339 effectively inhibits the mutant form of the receptor (T537S) which is responsible for acquired endocrine resistance. It can be anticipated that ER AF2 inhibitors will provide an alternative therapeutic strategy that can be applied concurrently or simultaneously with current anti-ER treatments for BCa patients with advanced disease. Citation Format: Kriti Singh, Ravi SN Munuganti, Miriam Butler, Artem Cherkasov, Paul S Rennie. In-silico discovery of novel estrogen receptor-α inhibitors as potential therapeutics for tamoxifen resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-7.

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