Crystal Structure of the T877A Human Androgen Receptor Ligand-binding Domain Complexed to Cyproterone Acetate Provides Insight for Ligand-induced Conformational Changes and Structure-based Drug Design

Casey E Bohl,Zengru Wu,Duane D Miller,Charles E Bell,James T Dalton

doi:10.1074/jbc.m611711200

Abstract

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8A in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor.drug complex. The structure demonstrates that bulk from the 17alpha-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

Highlights

Molecular modeling studies have offered a number of potential docking solutions to explain how drugs more bulky than steroidal agonists can be accommodated within the androgen receptor (AR) (10 – 14)
We presented the crystal structure of Cyproterone acetate (CPA) bound to the T877A AR ligand-binding domain (LBD)
Based on the structural findings observed in the T877A1⁄7CPA complex, we predicted that the L701A AR mutant would increase agonist activity of CPA due to the relief of the strain on helix 3 and recovery of the packing of the loop between helices 11 and 12 into the groove observed in other AR LBD structures

Summary

Introduction

Molecular modeling studies have offered a number of potential docking solutions to explain how drugs more bulky than steroidal agonists can be accommodated within the AR (10 – 14). Our recent report of the crystal structure of the AR LBD complexed to S-3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N[4-nitro-3-(trifluoromethyl)phenyl] propionamide (hereafter referred to as S-1 as in Ref. 15), an aryl propionamide selective androgen receptor modulator that acts as an agonist for the AR, demonstrated that movement of Trp-741 provides the additional space necessary for the compound to bind (8). We report structural evidence that CPA binding to the T877A AR induces unraveling of the C terminus of helix 11 and displacement of the loop between helices 11 and 12 Based on these findings, we show that the AR mutation, L701A, confers agonist activity to CPA similar to T877A and that a similar mutation in the GR converts CPA into a potent agonist. Identification of the expanded binding pocket occupied by CPA provides insight for exploiting receptor interactions using structure-based drug design

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Conclusion