Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity.

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.

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

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.

Identification of N-phenyl-2-(N-phenylphenylsulfonamido)acetamides as new RORγ inverse agonists: Virtual screening, structure-based optimization, and biological evaluation.

Retinoic acid receptor-related orphan receptors (RORs) are ligand-dependent transcriptional factors and members of the nuclear receptor superfamily. RORs regulate inflammation, metabolic disorders and circadian rhythm. RORγ is a promising therapeutic drug target for treating Th17-mediated autoimmune diseases. In our study, we performed structure-based virtual screening and ligand-based virtual screening targeting the RORγ ligand-binding domain and successfully identified N-phenyl-2-(N-phenylphenylsulfonamido) acetamides as a type of RORγ inverse agonist. Among the 28 purchased compounds, C11 was confirmed to be active with micromolar IC50 values in both an AlphaScreen assay (62.58 µM) and a cell-based reporter gene assay (4.54 µM). Structure-guided optimization of the compound C11 led to the identification of compound 39, which significantly enhanced RORγ inhibition with an IC50 value of 630 nM. The RORγ antagonism of 39 was 7-fold higher than that of hit compound C11. These results represent a promising starting point for developing potent small molecule RORγ inverse agonists for the treatment of autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

Cutting edge: Ubiquitin-specific protease 4 promotes Th17 cell function under inflammation by deubiquitinating and stabilizing RORγt.

RORγt is a key transcription factor that controls the development and function of inflammatory Th17. The mechanisms that regulate RORγt stability remain unclear. We report that Th17 cells highly express the deubiquitinase ubiquitin-specific protease (USP)4, which is essential for maintaining RORγt and Th17 cell function. Inhibition of the catalytic activity of USP4 with vialinin A, a compound derived from Chinese traditional medicine, dampened Th17 differentiation. USP4 interacted and deubiquitinated K48-linked polyubiquitination of RORγt, thereby promoting RORγt function and IL-17A transcription. Interestingly, TGF-ß plus IL-6 enhanced USP4-mediated deubiquitination of RORγt. Moreover, USP4 and IL-17 mRNA, but not RORγt mRNA, were significantly elevated in CD4(+) T cells from patients with rheumatic heart disease. Thus, USP4 could be a novel therapeutic target for the treatment of Th17-modulated autoimmune diseases.