Structural basis for antagonism and resistance of bicalutamide in prostate cancer

Abstract

Carcinoma of the prostate is the most commonly diagnosed cancer in men. The current pharmacological treatment of choice for progressive androgen-dependent prostate cancer is the nonsteroidal antiandrogen, bicalutamide, either as monotherapy or with adjuvant castration or luteinizing hormone-releasing hormone superagonists to block the synthesis of endogenous testosterone. To date, no nonsteroidal or antagonist-bound androgen receptor (AR) structure is available. We solved the x-ray crystal structure of the mutant W741L AR ligand-binding domain bound to R-bicalutamide at 1.8-A resolution. This mutation confers agonist activity to bicalutamide and is likely involved in bicalutamide withdrawal syndrome. The three-dimensional structure demonstrates that the B ring of R-bicalutamide in the W741L mutant is accommodated at the location of the indole ring of Trp-741 in the WT AR bound to dihydrotestosterone. Knowledge of the binding mechanism for R-bicalutamide will provide molecular rationale for the development of new antiandrogens and selective AR modulators.

Fig. 1.

Purification of W741L AR LBD with R-bicalutamide shown by silver stained 12% SDS/PAGE Molecular Weight Marker (MWM). After PreScission Protease cleavage from the glutathione Sepharose, LBD, groEL, and dnak were observed in the supernatant (lane 1). Only LBD was present in the cation-exchange eluent (lane 2).

Fig. 2.

Structure and binding conformation of R-bicalutamide. (a) W741L AR LBD-R-bicalutamide complex. (b) Structure and numbering scheme of R-bicalutamide (c) R-bicalutamide within the Fo-Fc simulated annealing omit map with R-bicalutamide omitted. Intramolecular hydrogen bonds are denoted by distances in Å. Nitrogen, blue; oxygen, red; carbon, black; sulfur, yellow; fluorine, white.

Fig. 3.

R-bicalutamide interactions with the W741L binding pocket in multiple orientations. (a) Side chains fit into the 2Fo-Fc electron density maps contoured at the 2σ level (blue) with R-bicalutamide shown in the Fo-Fc simulated annealing omit map contoured at the 4σ level (red). (b) Possible hydrogen bonds depicted between R-bicalutamide and the W741L AR LBD in a similar orientation as Fig. 1a. (c) View from the bottom relative to Fig. 1a to emphasize the van der Waals interactions with the A ring of R-bicalutamide. (d) View from the back relative to Fig. 1a portrays the B ring binding pocket formed by residues of helices 5 and 12 in relation to AF-2 charge clamp residues K720 and E897. (e) View from the top relative to Fig. 1a demonstrates the location of the para position of the R-bicalutamide B ring in a hydrophilic environment.

Fig. 4.

Overlay of the R-bicalutamide-W741L complex (green) and DHT-WT complex (magenta). (a) Overview of the steroidal plane. Notice the similar positioning of the cyano group of R-bicalutamide to the 3-keto group of DHT and the differences in the location of bulk from these ligands. (b) Side view of the steroidal plane. The R-bicalutamide B ring in the W741L AR binds in the region occupied by the Trp-741 indole ring in the WT AR bound to DHT. Also notice differences in the locations of Met-895, Met-745, and Thr-877.