Structural studies unravel the active conformation of apo RORγt nuclear receptor and a common inverse agonism of two diverse classes of RORγt inhibitors

Abstract

The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists.

Fi-score: a novel approach to characterise protein topology and aid in drug discovery studies

“…Finally, disordered regions or regions that combine several secondary structure elements might have sign value depending on the dominaVng structural moVf. All of the above findings are supported by earlier studies showing that B-factors can act as indicators of the relaVve vibraVonal moVon of atoms where low values belong to a well-ordered site, and the highest values come from the most flexible regions [10], [15], [33], [38], [40]. Table 2.…”

“…There are many studies that support these findings as it has been established that B-factors can be used to idenVfy flexibility in proteins and can also be linked to hydrophilicity as well as absolute net charge [12], [23], [25], [34]- [37]). Bfactors can also aid in idenVfying biologically acVve small molecules for a site of interest [24], [25], [36], [38]. Table 1.…”

HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

“…Mutant constructs were generated using a site-directed mutagenesis kit (Q5, New England Biolabs) using primers described in the supplement. Our RORγLBD-SRC2 cDNA was designed based on a previously published construct 20 , synthesized by Genscript, and cloned into the pESUMO vector using BsaI and XhoI (New England Biolabs). RORγHinge-LBD was cloned into the pBIND vector as previously described 24 .…”

“…While the intrinsic activity of the receptor has been presumed to be ligand-dependent, recent studies have called to question the basis of the receptor's activity in situ. These studies include the crystal structure and NMR solution of 'apo' RORγ 20 , and recent molecular dynamic simulations 21,22 . Collectively, these studies have identified that the gauche rotomeric state of W317 stabilizes H11, H11' and H12 through extensive hydrophobic interaction networks formed with F486, F506, Y502, and H479 as the key drivers of RORγ hyperactivation by stabilizing the required Y502-H479 hydrogen bond.…”

RORγ Structural Plasticity and Druggability

“…What is more, H11' is more accessible for a ligand sitting in this pocket than the orthosteric one. H11' is another unique feature only existing in the ROR family and is not conserved between RORα, RORβ, and RORγ [22,42]. Targeting the residues in H11' provides opportunities for increased drug selectivity.…”