Novel androgen receptor antagonist identified by structure-based virtual screening, structural optimization, and biological evaluation

Highlights

• A novel hit (C18) with IC₅₀ of 2.4 μM against AR transcriptional activity in LNCaP cell was identified through SBVS.

• Optimization of C18 resulted in the discovery of a more potent AR antagonist (AT2) with 16-fold improved anti-AR potency.

• AT2 could effectively inhibit the transcriptional function of AR and block the nuclear translocation of AR.

Abstract

Androgen receptor (AR) plays important roles in the development of prostate cancer (PCa), and therefore it has been regarded as the most important therapeutic target for both hormone-sensitive prostate cancer (HSPC) and advanced PCa. In this study, a novel hit (C18) with IC₅₀ of 2.4 μM against AR transcriptional activity in LNCaP cell was identified through structure-based virtual screening based on molecular docking and free energy calculations. The structure-activity relationship analysis and structural optimization of C18 resulted in the discovery of a structural analogue (AT2), a more potent AR antagonist with 16-fold improved anti-AR potency. Further assays indicated that AT2 was capable of effectively inhibiting the transcriptional function of AR and blocking the nuclear translocation of AR like the second-generation AR antagonists. The antagonists discovered in this study may be served as the promising lead compounds for the development of AR-driven PCa therapeutics.

Introduction

Prostate cancer (PCa) is the most diagnosed cancer among men worldwide [1]. Androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factor. Abnormal activation of the AR signaling pathway plays a pivotal role in the development and progression of PCa [2,3]. Currently, a vital approach to prevent the excessive activation of androgens is the treatment with AR antagonists to block the binding of androgens to AR [2,3]. Androgen deprivation therapy (ADT), the most important treatment for advanced PCa, reduces the levels of androgen production by surgical or pharmacological castration. Unfortunately, most patients usually develop into castration-resistant prostate cancer (CRPC) after 2 years of ADT treatment. The possible mechanisms of CRPC include AR mutations, AR amplifications, and active AR splice variants [4]. In general, existing studies show both PCa and CRPC are closely related to AR [[5], [6], [7]].

Current clinically used first-generation or second-generation AR antagonists, such as R-bicalutamide and Enzalutamide (Enz), have achieved great success against androgen-dependent PCa and improved the survival rate of PCa patients [2,8]. However, after the initially effective response, efficacy of these AR antagonists is suffered from the rapid emergence of drug resistance [[9], [10], [11], [12]]. One of the main reasons for drug resistance is the acquired point mutations at the AR ligand-binding pocket (LBP). An explanation for this reason is that some point mutations in the AR LBP, such as W741L, W741C, and T877 A, would create a more spacious LBP, thus converting these antagonists to agonists and inducing drug resistance [[13], [14], [15]]. A widely used approach to deal with this situation in drug design is to modify the original antagonists into larger chemical structures [[16], [17], [18], [19]]. Nevertheless, in general, larger molecules often confront several problems associated with unfavorable permeability and physiological distribution [20]. Therefore, it is still urgent to discover new AR antagonists with novel scaffolds to improve clinical outcomes.

A common approach to design antagonists with novel scaffolds is to use virtual screening (VS), which has also been used to identify novel hits of AR. For instance, the AR antagonist 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) was screened out by structure-based VS (SBVS) based on the crystal structure of the AR-metribolone complex [21]. Another AR antagonist 5,5a,6,10b-tetrahydroindeno[2,1-b]indole (VPC-12060) was discovered by SBVS and ligand-based virtual screening (LBVS) [22]. Wang et al. identified pyrazolopyramidine analogs as novel potent AR antagonists also by combining SBVS and LBVS [23]. Recently, we reported the identification of a series of novel AR ligands, including AR agonists and antagonists, through an integrated strategy by combining SBVS based on the crystal AR structures in complex with its agonists [24]. It has been proved that SBVS is potent to discover novel AR antagonists.

Molecular docking has been recognized as the most popular method for structure-based drug design [[25], [26], [27]]. It can predict the binding conformations of ligands to the target, and rank the ligands by scoring functions [[28], [29], [30]]. Compared with most scoring functions in molecular docking, the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method can achieve a better balance between identifying the binding poses and predicting the binding free energies, and therefore it has been widely used in SBVS and lead optimization [[31], [32], [33]]. In recent years, variable dielectric MM/GBSA (VD-MM/GBSA), a novel modified MM/GBSA algorithm, has attracted increasing attention [[34], [35], [36], [37]].

In this study, a multi-step SBVS strategy based on molecular docking and VD-MM/GBSA rescoring was employed to screen the Specs database, and 32 compounds were finally purchased for bioassay verification. Three of them exhibited strong bioactivities, and the competitive ligand binding assay showed that 1 compound (C18) was targeting the LBP of AR. Structural optimization based on molecular dynamics (MD) simulation and structure-activity relationship (SAR) analysis was then applied to the hit of C18, which resulted in the discovery of AT2, a more promising AR antagonist with 16 folds improved anti-AR potency relative to C18. Moreover, AT2 displayed much better anti-proliferative effects than Enz in three androgen independent cell lines, including PC3, C4-2, and DU145. The qPCR and immunofluorescence assays illustrated that AT2 can inhibit AR transcriptional activity and block nuclear translocation of AR. Our study provided valuable clues for the development of a novel class of AR therapeutic antagonists to combat PCa.