Structural basis for accommodation of nonsteroidal ligands in the androgen receptor

Abstract

The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissue-selective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact with the wild type AR. We have shown that changes induced to the positions of Trp-741, Thr-877, and Met-895 allow for ligand accommodation within the AR binding pocket and that a water-mediated hydrogen bond to the backbone oxygen of Leu-873 and the ketone of hydroxyflutamide is present when bound to the T877A AR variant. Additionally, we demonstrated that R-bicalutamide stimulates transcriptional activation in AR harboring the M895T point mutation. As a whole, these studies provide critical new insight for receptor-based drug design of nonsteroidal AR agonists and antagonists.

FIGURE 1. Structures and binding affinities of AR ligands

Binding affinity values were determined in our laboratory and previously reported (8, 9).

FIGURE 2. AR-mediated transcriptional activation by DHT, R-bicalutamide (R-bic), S-1, HF, and R-3 in the WT (a), T877A (b), W741L (c), and M895T (d) AR variants

Relative luciferase units (RLU) normalized with β-galactosidase (β-gal) activity. Inter-experiment variation was observed with RLU/β-galactosidase values due to transfection efficiency, but relative drug-induced transcriptional activation was found consistent and therefore more accurately represents changes in functional activity observed in AR mutations.

FIGURE 3. Stereo representations of AR interactions with R-3 and S-1

a, WT-R-3 complex fit into the 2F_o − F_c electron density maps contoured at the 2σ level (cyan) with R-3 shown in the F_o − F_c simulated annealing omit map contoured at the 4σ level (red). AR carbons, gray; R-3 carbons, gold; oxygen, red; nitrogen, blue; sulfur, orange; bromine, yellow. b, AR surface contacts with the R-3 binding pocket. Possible hydrogen bonds within 3.5 Å are represented by dashed lines. Notice that Gln-711 and Arg-752 hydrogen bond with the nitro group of R-3. Leu-704 and Asn-705 also form hydrogen bonds with R-3. Also notice the hydrophobic contacts from Trp-741, Met-742, and Met-895 with the R-3 bromine atom. Electron density for Trp-741 in the WT-R-3 structure cannot be visualized at the 2σ level, but electron density is more evident at lower σ levels. c, WT-S-1 complex fit into the 2F_o − F_c electron density maps contoured at the 2σ level (cyan) with S-1 (carbons in green) shown in the F_o − F_c simulated annealing omit map contoured at the 4σ level (red). d, AR surface contacts with the S-1 binding pocket. Similar hydrogen bonds to the AR are present in S-1 as with R-3. Notice the increased size of the S-1 binding pocket as compared with R-3 due to the S-1 B-ring. A water molecule hydrogen bonds to the His-874 side chain and backbone atoms of helices 4 and 5 within 3.0 Å of the B-ring fluorine atom, similar to that seen in the W741L-R-bicalutamide complex. Also notice the clear electron density for Trp-741 at the 2σ level as compared with the R-3-bound structure, likely because of decreased mobility of Trp-741 from binding of the S-1 B-ring.

FIGURE 4. Comparison of ligand interactions in the WT and the T877A mutant

a, wild type-DHT complex, slate (Protein Data Bank code 1I37). The 17β-OH group of DHT forms a hydrogen bond with Thr-877. b, WT-S-1 complex, green. The side chain of Thr-877 is rotated 180° in the WT-S-1 complex, situating the hydroxyl group of Thr-877 in close range to the S-1 carbon atoms bound to the chiral center. c, T877A-S-1 complex, cyan. In the T877A-S-1 complex, a water molecule is present that hydrogen bonds to the backbone oxygen of Leu-873 and the ketone of S-1. d, T877A-HF complex, orange. The water molecule bridging the ketone to Leu-873 is also present in the T877A-HF structure. e, 2F_o − F_c electron density maps from the T877A-HF complex contoured at the 1.5σ level with density shown for HF, Leu-873, and Ala-877 by using the carve function in PyMol with a radius of 2.0 Å demonstrate the clear electron density for this water molecule.

FIGURE 5. Ligand-induced changes to Trp-741 and Met-895 in stereo overlay and individually as space-fill representations

a, comparison of the changes induced by DHT (slate) (Protein Data Bank code 1I37), R1881 (ruby) (Protein Data Bank code 1XQ2), R-3 (gold), and S-1 (green) to the Trp-741, Met-745, Met-895 side chains. Notice the different location of Met-745 in the DHT-bound structure from displacement by the 19-methyl group, which causes the Trp-741 side chain to also move relative to its position in R1881. The Trp-741 indole ring is positioned similarly in R1881- and R-3-complexed structures; however, the bromine atom on R-3 displaces the Met-895 side chain. Also notice the change in position of the Trp-741 indole ring to allow accommodation of the S-1 B-ring. b, changes in the position of Met-895 in the W741L AR bound to S-1 (black) and R-bicalutamide (yellow). Compared with the WT-S-1 complex, Met-895 moves toward the Leu-741 side chain in the W741L-S-1 complex, compensating for the loss of bulk in this mutant. In the W741L-R-bicalutamide complex, the Met-895 side chain is wedged between the Leu-741 and the sulfonyl group of R-bicalutamide. Notice that the position of the Met-895 side chain in the WT-S-1 complex would be sterically precluded by the sulfonyl group of R-bicalutamide in the presence of Trp-741.

FIGURE 6. Ligand interactions with helices 3, 5, 11, and 12

a, WT AR LBD complexed to S-1 (green) rotated 180° about the y-axis relative to Fig. 3, c and d. Helix 3, blue; helices 4/5, Trp741, purple; helix 11, Thr-877, red; helix 12, Met-895, gold. b, surface contacts of the secondary structural elements of the WT AR LBD with S-1. Notice the contacts from Trp-741, Thr-877, and Met-895 on helices 5, 11, and 12, respectively. c, surface contacts of the secondary structural elements of the W741L AR LBD with R-bicalutamide. Notice the loss of contacts of helix 5 from the loss of bulk in the W741L mutant and that helix 12 increases contacts to the ligand as a result of Met-895 repositioning. Also notice the location of the sulfonyl group of R-bicalutamide, which increases the binding pocket size relative to S-1.