Discovery of Novel Androgen Receptor Ligands by Structure-based Virtual Screening and Bioassays

Abstract

Androgen receptor (AR) is a ligand-activated transcription factor that plays a pivotal role in the development and progression of many severe diseases such as prostate cancer, muscle atrophy, and osteoporosis. Binding of ligands to AR triggers the conformational changes in AR that may affect the recruitment of coactivators and downstream response of AR signaling pathway. Therefore, AR ligands have great potential to treat these diseases. In this study, we searched for novel AR ligands by performing a docking-based virtual screening (VS) on the basis of the crystal structure of the AR ligand binding domain (LBD) in complex with its agonist. A total of 58 structurally diverse compounds were selected and subjected to LBD affinity assay, with five of them (HBP1-3, HBP1-17, HBP1-38, HBP1-51, and HBP1-58) exhibiting strong binding to AR-LBD. The IC₅₀ values of HBP1-51 and HBP1-58 are 3.96 µM and 4.92 µM, respectively, which are even lower than that of enzalutamide (Enz, IC₅₀ = 13.87 µM), a marketed second-generation AR antagonist. Further bioactivity assays suggest that HBP1-51 is an AR agonist, whereas HBP1-58 is an AR antagonist. In addition, molecular dynamics (MD) simulations and principal components analysis (PCA) were carried out to reveal the binding principle of the newly-identified AR ligands toward AR. Our modeling results indicate that the conformational changes of helix 12 induced by the bindings of antagonist and agonist are visibly different. In summary, the current study provides a highly efficient way to discover novel AR ligands, which could serve as the starting point for development of new therapeutics for AR-related diseases.

Keywords: AR agonist; AR antagonist; AR ligand; Androgen receptor; Virtual screening.

Figure 1

The workflow of the docking-based virtual screening and bioassay verification Based on the structures of AR-LBD in complex with testosterone (PDB ID: 2Q71) and EM-5744 (PDB ID: 2PNU), respectively, 58 compounds were finally selected from the ChemBridge database sequentially by Glide HTVS docking, Glide SP, ADMET criteria, as well as clustering, and then subjected to bioassays. AR-LBD, ligand binding domain of androgen receptor; HTVS, high-throughput virtual screening; SP, standard precision; ADMET, absorption, distribution, metabolism, excretion, and toxicity.

Figure 2

Competitive binding assay of the 58 compounds identified through VS A. Relative AR binding affinity of the 58 compounds (10 µM) analyzed using PolarScreen™ AR Competitor Assay using DHT as control. The numbers at X axis represent the numerical codes of the identified compounds; e.g., 10 represents HBP1-10. The AR binding affinity of DHT was set as 100%. B. Four AR favorable compounds HBP1-3, HBP1-38, HBP1-51, and HBP1-58 were evaluated under various concentrations for their precise binding affinity with Enz as control. Standard errors were derived from at least triplicate assays. C. Structures of HBP1-3, HBP1-17, HBP1-38, HBP1-51, and HBP1-58, and four known AR ligands tested in this study. These include testosterone (endogenous androgen), EM-5744 (reported AR agonist), Bic (marketed AR antagonist), and Enz (marketed AR antagonist). DHT, dihydrotestosterone; Bic, bicalutamide; Enz, enzalutamide.

Figure 3

Cell viability assay of the selected AR ligands Five AR ligands HBP1-3, HBP1-17, HBP1-38, HBP1-51, and HBP1-58 were analyzed with various prostate cancer cell lines including LNCaP (A), C4-2 (B), and DU145 (C). Enz and Bic were taken as controls. The cell viability assay was repeated at least 3 times for each compound at each concentration. Viability of cells without treatment of compound was set as 100%.

Figure 4

The efficacy of the selected AR ligands on transcriptional activity assay of AR A. Compounds HBP1-3, HBP1-17, HBP1-38, HBP1-51, and HBP1-58 were subjected to LNCaP-ARR₂PB-eGFP based transcriptional activity assay to assess their antagonist activities. Enz was used as a control and incubated with cells at concentrations from 0.0032 µM to 2 µM, while the five tested compounds were incubated with the cells at 10 µM. 1 nM DHT was added for background fluorescence. In addition, cells receiving equal volume of DMSO solution with (dark gray bar) or without (light gray bar) DHT served as controls too. B. Transcriptional activity assay of HBP1-58, using Enz and Bic as positive controls. C. Androgen like activities of compounds HBP1-3, HBP1-17, HBP1-38, and HBP1-51 were evaluated using LNCaP-ARR2PB-eGFP based transcriptional activity assay without preincubation of DHT. The test concentrations range from 0.001 nM to 10 µM. DHT served as the positive control. Assays were performed in triplicate. Multiple group comparisons were analyzed with 1-way ANOVA (**, P < 0.05 were considered statistically significant).

Figure 5

The binding poses of the AR ligands in the AR-LBD The binding poses of testosterone (A), HBP1-51 (B), HBP1-3 (C), HBP1-38 (D), EM-5744 (E), and HBP1-58 (F) in the binding pocket of AR-LBD were predicted using molecular docking. The binding poses of testosterone and EM-5744 were directly captured from the crystal structures, and poses of the four HBP ligands were generated using the Glide docking. Residues identified by the molecular mechanics/generalized born surface area (MM/GBSA) free energy decomposition were highlighted in pink.

Figure 6

Principal components analysis of the Cα atoms of the AR conformational motions during simulation The cloud represents the last 10 ns of trajectories projected onto the first two eigenvectors for the AR/testosterone (A), AR/HBP1-51 (B), AR/Enz (C), and AR/HBP1-58 (D). The distribution of dots indicates the conformational changes in the system, with each dot representing a snapshot from MD simulation trajectory. MD, molecular dynamics.

Figure 7

The agonistic and antagonistic state of AR-LBD complexed with the selected AR ligands revealed by MD simulations Shown are the conformational changes of helix 12 after 30 ns MD simulations for testosterone (A), HBP1-51 (B), Enz (C), and HBP1-58 (D). The initial structures of the receptor/ligand complexes were colored in light gray, and the conformations produced by the MD simulations were colored in pink.

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