Delineation of the molecular determinants of the unique allosteric binding site of the orphan nuclear receptor ROR?t

Nuclear receptors (NRs) are high-interest targets in drug discovery because of their involvement in numerous biological processes and diseases. Classically, NRs are targeted via their hydrophobic, orthosteric pocket. Although successful, this approach comes with challenges, including off-target effects due to lack of selectivity. Allosteric modulation of NR activity constitutes a promising pharmacological strategy. The retinoic acid receptor-related orphan receptor-?t (ROR?t) is a constitutively active NR that positively regulates the expression of interleukin-17 in T helper 17 cells. Inhibiting this process is an emerging strategy for managing autoimmune diseases. Recently, an allosteric binding pocket in the C-terminal region of the ligand-binding domain (LBD) of ROR?t was discovered that is amenable to small-molecule drug discovery. Compounds binding this pocket induce a reorientation of helix 12, thereby preventing coactivator recruitment. Therefore, inverse agonists binding this site with high affinity are actively being pursued. To elucidate the pocket formation mechanism, verify the uniqueness of this pocket, and substantiate the relevance of targeting this site, here we identified the key characteristics of the ROR?t allosteric region. We evaluated the effects of substitutions in the LBD on coactivator, orthosteric, and allosteric ligand binding. We found that two molecular elements unique to ROR?t, the length of helix 11? and a Gln-487 residue, are crucial for the formation of the allosteric pocket. The unique combination of elements present in ROR?t suggests a high potential for subtype-selective targeting of this NR to more effectively treat patients with autoimmune diseases.