Identification of an allosteric binding site for RORγt inhibition

Abstract

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

Figure 1. Co-crystal structure of RORγt with MRL-871.

(a) Chemical structures of a representative orthosteric ligand T0901317 (ref. 27), binding to the canonical site of NRs, and indazoles MRL-871, MRL-058 and MRL-003. (b) Zoomed-in view of MRL-871 in the novel allosteric-binding pocket of RORγt formed by helices 4, 5, 11 and 12. MRL-871 shown as orange sticks in the electron density. The RORγt residues involved in hydrophobic interactions and hydrogen bonding are shown as white and green sticks, respectively. (c) Superposition of RORγt (blue) in complex with orthosteric ligand T0901317 shown in red (PDB ID 4NB6) and RORγt (green) in complex with MRL-871, shown in orange. MRL-871 (orange) sits in a new allosteric pocket in direct contact with H12 inducing its repositioning. Orthosteric ligand T0901317 destabilizes H12, which is therefore not visible in the blue structure. (d) A two-dimensional plot showing the interactions between MRL-871 and the surrounding amino acids.

Figure 2. Allosteric inhibition.

(a) TR-FRET assay showing the effect of the agonist cholesterol, the inverse agonist T0901317 and indazoles MRL-871, MRL-058 and MRL-003 on cofactor recruitment to the RORγt LBD in a dose-dependent manner. MRL-871, MRL-058, MRL-003 and T0901317 function as inhibitors, while cholesterol promotes cofactor binding to RORγt. Error bars are defined as s.d. (n=3). (b) IL17a mRNA expression in EL4 cells treated with MRL-871, MRL-058, MRL-003 (10 μM, 24 h) or DMSO. The level of IL17a mRNA expression was normalized to that of GAPDH expression. All data are expressed as the mean±s.e.m. (n=6). Statistical analysis was performed using an one-way analysis of variance comparing against the DMSO control following Dunnett post hoc test. Error bars denote s.e.m. **P<0.01. (c) Inhibition of cofactor binding by the allosteric modulator MRL-871 is independent of cholesterol concentration (IC₅₀ 4.7±1.4 nM; 2.5±0.9 nM; 1.8±1.5 nM; 2.3±1.8 nM at 0, 1, 10 and 25 μM cholesterol, respectively). Error bars are defined as s.d. (n=3). (d) Inhibition of cofactor binding by ligand T0901317 depends on cholesterol concentration (IC₅₀ 11±3.4 nM; 18±4.9 nM; 195±59 nM; 548±152 nM at 0, 1, 10 and 25 μM cholesterol, respectively). Error bars are defined as s.d. (n=3).

Figure 3. Co-crystal structure of RORγt with MRL-871, MRL-299, MRL-367 and MRL-673.

(a) Overlay of RORγt (green) co-crystallized with MRL-871, MRL-299, MRL-367 and MRL-673. For an overall structural comparison of RORγt in complex with MRL-299 and MRL-367 see Supplementary Fig. 3. (b) Magnified view of the overlay of MRL-871 (orange) and MRL-367 (cyan) in the novel allosteric binding pocket of RORγt (overall root mean squared deviation (r.m.s.d) 0.219). (c) Magnified view of overlay of MRL-367 (cyan) and MRL-673 (yellow) in the novel allosteric binding pocket of RORγt (overall r.m.s.d 0.38). MRL-673 is a racemic mixture and the two isomers are depicted. Arrows highlight repositioning of the side chains of residues Tyr502 and Phe498 and the accompanying shift in the backbone (for example, Ala497) on binding MRL-673.

Figure 4. Molecular mechanism of allosteric RORγt modulation.

(a) Agonistic conformation of RORγt (PDB entry 4NIE) showing the position of the agonistic ligand (red) in the classical orthosteric ligand binding site. Helices 11′ and 12 (yellow) of the LBD are folded in a stable agonistic conformation, supporting binding of the LXXLL cofactor peptide (blue). (b) Antagonistic conformation of RORγt (PDB entry 4QM0) induced by a classical inverse agonist (purple) in the classical orthosteric ligand-binding site. Helices 11 and 12 of the LBD are unstructured (arrow). (c) Superposition of a and b. When H12 is disordered, the LXXLL cofactor peptides lack a complementary surface for binding to RORγt (d) Antagonistic conformation of RORγt, induced by allosteric modulator MRL-871 (orange). MRL-871 makes direct contact with H12 (brown) at the allosteric site in the RORγt LBD and stabilizes the folding of H12. (e) Superposition of a and d. The binding of the allosteric modulator MRL-871 results in unfolding of helix 11′ and shifts the position of H12 to directly block cofactor peptide binding. For a structural comparison with agonist hydroxycholesterol liganded RORγt see Supplementary Fig. 5.

Figure 5. Schematic model of the novel molecular mechanism of allosteric RORγt inhibition regarding H12 folding and positioning.

In the apo form, H12 is dynamic and thus limits LXXLL cofactor binding. An inverse agonist destabilizes H12 and lowers LXXLL binding. Agonist binding stabilizes H12 in a optimal conformation for LXXLL binding. Binding at the novel allosteric site, reorients the H12 of RORγt and actively blocks LXXLL binding.