Molecular Mechanism of Action of RORγt Agonists and Inverse Agonists: Insights from Molecular Dynamics Simulation

Abstract

As an attractive drug-target, retinoic acid receptor-related orphan receptor-gamma-t (RORγt) has been employed widely to develop clinically relevant small molecular modulators as potent therapy for autoimmune disease and cancer, but its molecular mechanism of action (MOA) remains unclear. In the present study, we designed and discovered two novel RORγt ligands that are similar in structure, but different in efficacy. Using fluorescence resonance energy transfer (FRET) assay, compound 1 was identified as an agonist with an EC50 of 3.7 μM (max. act.: 78%), while compound 2 as an inverse agonist with an IC50 value of 2.0 μM (max. inh.: 61%). We performed molecular dynamics (MD) simulations, and elucidated the MOA of RORγt agonist and inverse agonist. Through the analyses of our MD results, we found that, after RORγt is bound with the agonist 1, the side chain of Trp317 stays in the gauche- conformation, and thus helps to form the hydrogen bond, His479-Trp502, and a large hydrophobic network among H11, H11′, and H12. All these interactions stabilize the H12, and helps the receptor recruit the coactivator. When the RORγt is bound with the inverse agonist 2, the side chain of Trp317 is forced to adopt the trans conformation, and these presumed interactions are partially destroyed. Taken together, the critical role of residue Trp317 could be viewed as the driving force for the activation of RORγt.

Structural change of retinoic-acid receptor-related orphan receptor induced by binding of inverse-agonist: Molecular dynamics and ab initio molecular orbital simulations

“…Molecular dynamics (MD) simulations [14] are a very useful tool for elucidating changes in the structure of protein and its ligand-bound structures. To investigate the effect on the stability of the H12 conformation caused by the binding of a ligand to RORγt, atomistic MD simulations have been previously conducted [15] . In this study, the change in conformations of Trp317 residue as well as H12 was simulated for RORγt with an agonist or an inverse agonist.…”

“…It was also predicted that this change in the conformation of Trp317 is related to the stability of H12 and the reduction of the transcriptional activity of the inverse-agonist-bound RORγt. However, in these MD simulations [15] , the RORγt structure without a H12 domain was used, because the H12 conformation of the inverse-agonist-bound RORγt was not yet determined by experiment. Therefore, any changes in the H12 conformation for the inverse-agonist-bound RORγt could not be investigated.…”

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

“…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

“…Another in silico method, MD, has aided the study of the H11, H11,' H12 flexibility affected by different types of ligands and the atomic mechanisms on activating or inactivating RORγ and other NRs [29,53,54]. It is also an alternative method to study the SAR for a protein-ligand complex that lacks a co-crystal structure.…”