Letter to the editor

RORγt inhibitors suppress T_H17 responses in inflammatory arthritis and inflammatory bowel disease

A variety of biological processes are regulated by posttranslational modifications. Posttranslational modifications including phosphorylation, ubiquitination, glycosylation, and proteolytic cleavage, control diverse physiological functions in the gastrointestinal tract. Therefore, a better understanding of their implications in intestinal diseases, including inflammatory bowel disease, irritable bowel syndrome, celiac disease, and colorectal cancer would provide a basis for the identification of novel biomarkers as well as attractive therapeutic targets. Posttranslational modifications can be common denominators, as well as distinct biomarkers, characterizing pathological differences of various intestinal diseases. This review provides experimental evidence that identifies changes in posttranslational modifications from patient samples, primary cells, or cell lines in intestinal disorders, and a summary of carefully selected information on the use of pharmacological modulators of protein modifications as therapeutic options.

Though macrophages and neutrophils are considered to be the principal immune cells involved in gout inflammation, recent studies highlight an emerging role of T cell subsets in the pathogenesis of gout. Some studies found that abnormal functions of several T cell subsets and aberrant expressions of their signature cytokines existed in gouty arthritis. Additionally, recent studies also suggested that therapeutic strategies by targeting pro-inflammatory T cell subsets or their related cytokines could ameliorate monosodium urate (MSU) crystals-induced arthritis in mice. The important role of T cells in gouty arthritis may provide some explanation for the absence of acute gout attacks among individuals with severe hyperuricemia or clinical evidence of MSU crystals deposition. Nevertheless, the molecular mechanisms underlying the role of those T cell subsets in gouty arthritis and their role in the initiation, progression and resolution of gouty arthritis are largely elusive, which need to be elaborated in future research. Uncovering the role of those T cell subsets in gout may transform our understanding of gout and facilitate new promising preventive or therapeutic strategies for gouty arthritis.

RORγt is the master regulator of the IL-23/IL-17 axis, a pathway that is clinically validated for the treatment of various immunological disorders. Over the last few years, our group has reported different chemotypes that potently act as inverse agonists of RORγt. One of them, the tricyclic pyrrolidine chemotype, has demonstrated biologic-like preclinical efficacy and has led to our clinical candidate BMS-986251. In this letter, we discuss the invention of an annulation reaction which enabled the synthesis of a tricyclic exocyclic amide chemotype and the identification of compounds with RORγt inverse agonist activity. Preliminary structure activity relationships are disclosed.

In an effort to discover oral inverse agonists of RORγt to treat inflammatory diseases, a new 2,6–difluorobenzyl ether series of cyclopentyl sulfones were found to be surprisingly more potent than the corresponding alcohol derivatives. When combined with a more optimized phenyl ((R)–3–phenylpyrrolidin–3–yl)sulfone template, the 2,6–difluorobenzyl ethers yielded a set of very potent RORγt inverse agonists (e.g., compound 26, RORγt Gal4 EC₅₀ 11 nM) that are highly selective against PXR, LXRα and LXRβ. After optimizing for stability in human and mouse liver microsomes, compounds 29 and 38 were evaluated in vivo and found to have good oral bioavailability (56% and 101%, respectively) in mice. X–ray co–crystal structure of compound 27 in RORγt revealed that the bulky benzyl ether group causes helix 11 of the protein to partially uncoil to create a new, enlarged binding site, which nicely accommodates the benzyl ether moiety, leading to net potency gain.

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-γt (RORγt) 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 RORγt 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 RORγt 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 RORγt, 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 RORγt suggests a high potential for subtype-selective targeting of this NR to more effectively treat patients with autoimmune diseases.

T-helper 17 (Th17) cells are involved in the occurrence and development of several inflammation-associated diseases. Interleukin (IL)-17, the main cytokine secreted by differentiated Th17 cells, mediates immunoreactions and plays important roles in immunological diseases, including psoriasis, rheumatic arthritis, and inflammatory bowel disease. The maturation and stabilization of the differentiated Th17 cell phenotype are associated with the expression of IL-17A, which is induced by the activation of signal transducer and activator of transcription 3 (STAT3). Prohibitin 1 (PHB1) also plays an essential role in T-cell activation. However, the molecular mechanisms underlying Th17 cell differentiation and the role of PHB1 have not yet been completely elucidated, and this information would greatly facilitate the development of therapeutic strategies to prevent or treat immune-associated diseases. Here, we found that STAT3 expression was correlated with PHB1 mRNA expression during Th17 cell differentiation. Double-labeling immunofluorescence assay results showed that exogenous PHB1 and STAT3 proteins were not only located primarily in the nucleus but also in the cytoplasm of human Th17 cells. Co-immunoprecipitation assays of Th17 cells revealed that PHB1 interacted with STAT3 and with activated STAT3 phosphorylated at Ser727 but not at Tyr705. Knocking down PHB1 with specific short hairpin RNAs attenuated both STAT3 and IL-17 expression levels as well as IL-17 secretion in Th17 cells. These results indicate that PHB1 and STAT3 interact to affect IL-17 secretion in Th17 cells and provide important insights for modulating Th17-mediated pathogenic immune responses.