Rational discovery of novel nuclear hormone receptor antagonists

Abstract

Nuclear hormone receptors (NRs) are potential targets for therapeutic approaches to many clinical conditions, including cancer, diabetes, and neurological diseases. The crystal structure of the ligand binding domain of agonist-bound NRs enables the design of compounds with agonist activity. However, with the exception of the human estrogen receptor-alpha, the lack of antagonist-bound "inactive" receptor structures hinders the rational design of receptor antagonists. In this study, we present a strategy for designing such antagonists. We constructed a model of the inactive conformation of human retinoic acid receptor-alpha by using information derived from antagonist-bound estrogen receptor-alpha and applied a computer-based virtual screening algorithm to identify retinoic acid receptor antagonists. Thus, the currently available crystal structures of NRs may be used for the rational design of antagonists, which could lead to the development of novel drugs for a variety of diseases.

Figure 1

Modeling of the antagonist-bound structure of RAR. Agonist (white) and antagonist (cyan) superimpose in the binding pocket of ERα, but the antagonist presents an additional protruding arm that pushes helix 12 (H12, green) away (A). As a result, H12 relocates in the coactivator binding pocket of the receptor (H12, red) (B). Based on the ERα structure, helix H12 of RARγ (red) was docked to the coactivator binding pocket of the RARγ-LBD (critical hydrophobic residues are displayed in magenta) (C), and the C terminus of the protein was remodeled from its agonist-bound conformation (green) to its antagonist-bound conformation (red) (D).

Figure 2

RAR antagonists. Two known antagonists (A and B) and two novel antagonists (C and D). (Left) Chemical structure. (Right) Conformation docked into the receptor (part of the receptor is displayed as a ribbon representation, and the binding pocket boundary is displayed in yellow). Cyan, carbons; red, oxygen; blue, nitrogen; magenta, fluorine; yellow, sulfur. Hydrogens are not represented for clarity.

Figure 3

Functional assays of the novel antagonists. HeLa cells were transfected with a Gal4-hRARα-LBD expression vector and a Gal4-CAT reporter gene (results were similar in studies using the three hRAR isoforms). The cells were incubated with 5 nM all-trans RA to stimulate CAT activity, and the effect of each antagonist on inhibiting CAT was examined at 2 and 20 μM concentration (the known antagonist RO-41–5253 was used as a positive control).

Figure 4

Novel RAR antagonists. (A and B) Stereo representation of antagonists 1 and 2 docked into the binding site of the receptor. The ligands make extensive hydrophobic interactions with residues from helix 3, helix 5, and helix 11. Antagonist 2 (B) is engaged in an additional hydrogen bond with Ser-234 of helix 3 and contacts the remodeled C terminus (red) at Pro-405. (C and D) The fit of antagonists 1 and 2 into the receptor binding pocket is shown.