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

Abstract

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism 1 to inflammation 2 and cancer 3 . As ligand-gated transcription factors, synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology 4 . However, nearly half of NRs do not have specific cognate ligands or its unclear if they possess ligand dependent activities and these receptors are called orphans. Here we demonstrate that ligand-dependent action of the orphan nuclear receptor RORγ can be defined by selectively disrupting putative endogenous-but not synthetic-ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings are corroborated with X-ray co-crystallography and sitedirected mutagenesis to collectively reveal the structural determinants of RORγ liganddependent activation, critical for designing full agonists for application in cancer immunotherapy.Combined these observations support a model of receptor activation to more accurately describe RORγ pharmacology. Likewise, this 'bump-and-hole' inspired approach could be extended to other orphan NRs to explore the ligand-dependent activities that are important for defining pharmacology.

α-Synuclein kinetically regulates the nascent fusion pore dynamics

“…We used Glomelt dye that yields enhanced fluorescence upon binding to the exposed hydrophobic regions of proteins. When a protein folds into a compact structure, the dye is expelled out of the hydrophobic region, producing a decrease in the Glomelt fluorescence (46). Here, we used this assay to probe for the impact of membrane curvature on trans complex formation.…”

Agonist Lock Touched and Untouched Retinoic Acid Receptor-Related Orphan Receptor-γt (RORγt) Inverse Agonists: Classification Based on the Molecular Mechanisms of Action

“…Later, scientists in Scripps deeply explored the structural basis for ligand-mediated hyperactivation of RORγ-LBD with various RORγt inverse agonists using hydrogen−deuterium exchange coupled with mass spectrometry (HDX-MS) and RORγ-LBD-ligand cocrystal structures. 107 Compound 31 (SR19355, Figure 16) is one of these investigated inverse agonists, which contains a 2-chloro-6-fluorobenzamide moiety. In the cocrystal structure (Figure 17C, PDB 6NWS), the amide of 31 forms a hydrogen bond with His479, which disrupts the His479-Tyr502 hydrogen bond and leads to destabilization of H12, like 29 and 30.…”

RORγ Structural Plasticity and Druggability

“…This was reflected in the difficulty of crystallizing the apo LBD in the absence of the coactivator [ 22 , 23 ]. HDX-MS results also demonstrated reduced dynamic properties of H12 when the RORγ LBD transitioned from the apo state to the agonistic state, supporting a disorder-to-order transition [ 24 ]. However, the H12 dynamic properties in the apo and inverse-agonist states are similar, suggesting that H12 may not have a discrete conformation when an inverse agonist binds in the orthosteric site.…”