Eric Leblanc

Targeting the Binding Function 3 (BF3) Site of the Human Androgen Receptor Through Virtual Screening

The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called binding function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and X-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its antiandrogen resistant forms.

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short‐hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small‐interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2‐dilinoleyl‐4‐(2‐dimethylaminoethyl)‐[1,3]‐dioxolane (DLin‐KC2‐DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin‐KC2‐DMA‐containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin‐KC2‐DMA LNPs were also effective in silencing the AR in a wild‐type AR expressing cell line, LAPC‐4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR‐siRNA systems containing DLin‐KC2‐DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.

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.

Inhibitors of androgen receptor activation function-2 (AF2) site identified through virtual screening.

The androgen receptor (AR) is one of the most studied drug targets for the treatment of prostate cancer. However, all current anti-androgens directly interact with the AR at the androgen binding site, which is prone to resistant mutations, calling for new strategies of the AR inhibition. The current study represents the first attempt to use virtual screening to identify inhibitors of activation function-2 (AF2) of the human AR. By combining large-scale docking with experimental approaches, we were able to identify several small molecules that interact with the AF2 and effectively prevent the transcriptional activation of the AR. The crystallographic structure of one of these inhibitors in complex with the AR provides critical insight into the corresponding protein-ligand interactions and suitable for future hit optimization. Taken together, our results provide a promising ground for development of novel anti-androgens that can help to address the problem of drug resistance in prostate cancer.

Discovery of small-molecule inhibitors selectively targeting the DNA-binding domain of the human androgen receptor.

The human androgen receptor (AR) is considered as a master regulator in the development and progression of prostate cancer (PCa). As resistance to clinically used anti-AR drugs remains a major challenge for the treatment of advanced PCa, there is a pressing need for new anti-AR therapeutic avenues. In this study, we identified a binding site on the DNA binding domain (DBD) of the receptor and utilized virtual screening to discover a set of micromolar hits for the target. Through further exploration of the most potent hit (1), a structural analogue (6) was identified demonstrating 10-fold improved anti-AR potency. Further optimization resulted in a more potent synthetic analogue (25) with anti-AR potency comparable to a newly FDA-approved drug Enzalutamide. Site-directed mutagenesis demonstrated that the developed inhibitors do interact with the intended target site. Importantly, the AR DBD inhibitors could effectively inhibit the growth of Enzalutamide-resistant cells as well as block the transcriptional activity of constitutively active AR splice variants, such as V7.

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.

Identification of novel androgen receptor antagonists using structure- and ligand-based methods.

Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PCa). The AR hormone-binding site (HBS) is intensively studied and represents the target area for current antiandrogens including Bicalutamide and structurally related Enzalutamide. As resistance to antiandrogens invariably emerges in advanced prostate cancer, there exists a high medical need for the identification and development of novel AR antagonists of different chemotypes. Given the wealth of structural information on the AR in complex with a variety of ligands, we have applied an integrated structure- and ligand-based virtual screening methodology to identify novel AR antagonists. Virtual hits generated by a consensus voting approach were experimentally evaluated and resulted in the discovery of a number of structurally diverse submicromolar antagonists of the AR. In particular, one identified compound demonstrated anti-AR potency in vitro that is comparable to the clinically used Bicalutamide. These results set a ground for the development of novel classes of PCa drugs that are structurally different from current AR antagonists.