Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo

Abstract

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.

Fig 1. Representative RORγt inhibitors (Compounds 1–3) and inverse agonist, SR2211.

Structures of Compounds 1–3 compared to inverse agonist, SR2211 (A) and chemical synthesis route for Compound 3 (B).

Fig 2. Single molecule X-ray crystallography of Compound 3.

Small molecule X-ray structure of Compound 3 indicates only the (R,R)-enantiomer is present in the crystal structure. Crystal conformation of compound is and depicted in ball and stick representation (carbon–green; hydrogen–white; oxygen–red; nitrogen–blue; fluorine–magenta; chlorine–orange).

Fig 3. RORγt-mediated inhibition of IL-17A production in human Th17 cell cultures.

Schematic of human Th17 differentiation (A) or Th17 maintenance (B) assays. Representative flow cytometry plots of intracellular cytokine staining from cultures from (A) including Th0 cell nonpolarizing condition (C). IL-17A cytokine production from enriched naïve CD4⁺ T cells from healthy donor PBMC cultured under Th17 cell conditions for 6 days (D) or from enriched human Th17 cells from healthy donor PBMC cultured with IL-23 and IL-1β, for 4 days (E), in the presence of the indicated RORγt inhibitors Compounds 1–3. Data are normalized to and represented as percent inhibition compared to DMSO controls. Error bars are representative of 4 individual donors, from 2 independent experiments.

Fig 4. Imiquimod-induced skin inflammation is attenuated by RORγt inhibitor Compound 1.

Ear thickness (mm) was measured in naïve or IMQ-treated animals on day 4 using digital micro-calipers (A). Th17 cytokine gene expression analysis was performed for Il17a (B), Il17f (C) and Il22 (D) on day 4. Expression is normalized to Gapdh and presented as fold change over naïve. Kinetic assessment of plasma concentration of Compound 1 (E). Each symbol represents an individual animal and error bars denote mean ± SEM. Statistical significance (*p ≤ 0.05) was determined using one-way ANOVA with Tukey’s multiple comparisons test, *significant over naïve; **significance over vehicle-treated group. Data are representative of 2 independent experiments with n = 3-8/group.

Fig 5. IMQ-induced skin inflammation and RORγt activity is inhibited by Compound 3.

Ear thickness (mm) was measured in naïve or IMQ-treated animals on day 4 using digital micro-calipers (A). Th17 cytokine gene expression analysis was performed for Il17a (B), Il17f (C) and Bclxl (D) on day 4. Expression is normalized to Gapdh and presented as fold change over naïve. Kinetic assessment of plasma concentration of Compound 3 (E). Each symbol represents an individual animal and error bars denote mean ± SEM. Statistical significance (*p ≤ 0.05) was determined using one-way ANOVA with Tukey’s multiple comparisons test, *significant over naïve; **significance over vehicle-treated group. Data are representative of 1–4 independent experiments with n = 6-24/group.

Fig 6. Compound 3 plasma concentration inversely correlates with Th17 cytokine gene expression.

Th17 cytokine gene expression analysis was performed for Il17a (A) and Il17f (B) from IMQ-treated animals dosed with 6 or 60 mg/kg Compound 3. Gene expression is normalized to Gapdh and presented as fold change over naïve and plotted relative to plasma concentrations for Compound 3. Each symbol represents an individual animal and a linear regression (blue line) coefficient was calculated. Statistical significance (*p ≤ 0.05) was determined using linear regression analysis. Data are from a single experiment with n = 8/group.

Fig 7. RORγt inhibitor protects in EAE immunization model.

C57BL/6 mice were immunized with MOG_35-55 peptide on day 1 and were treated orally, twice daily (from day 1-day 28) with vehicle (0.5% MC/0.25% Tween80) or RORγt inhibitors at indicated doses. FTY720 (Gilenya) was dosed once daily at 3 mg/kg starting at day 1 in MilliQ water. The clinical score (0–5) was determined in these mice from day 7–28. Scores were calculated as follows: 0- no clinical signs, 0.5- partial tail weakness, 1.0- complete tail paralysis, 1.5- flaccid tail & abnormal gait, 2.0- flaccid tail and clear weakness of hind legs, 2.5- partial paralysis in one hind limb, 3.0- complete paralysis in both hindlimbs, 4.0- partial weakness in forelimbs, 5.0- complete paralysis in both forelimbs and hindlimbs). Data are presented as mean ± SEM. Statistical significance of clinical scores were calculated by Wilcoxon’s non-parametric or 2-tailed Student’s t-test, respectively; *p < 0.05, compared with vehicle-treated EAE mice. Data are from a single experiment with n = 12/group.

Fig 8. In vitro RORγt inhibitor activity on PDO growth & formation.

PDOs T020P and T031P organoids were dissociated and treated with Compound 3 or SR2211 and either organoid growth or organoid formation defects examined. (A) Organoid growth assays, PDOs were treated with Compound 3 (red line) or SR2211 (blue line) beginning on day 3 after plating for the duration and dose indicated. All values were calculated as % relative to vehicle treated organoids in 3 replicate experiments. (B) Organoid formation assays, PDOs were treated with Compound 3 (red line) or SR2211 (blue line) starting on day 1 after plating for the duration and dose indicated. All values were calculated as % of vehicle treated organoids in 3 replicate experiments.

Fig 9. In vivo analysis of RORγt inhibitor activity on tumor growth.

Tumors were implanted in the flanks of C57Bl/6 mice and treatment began when average tumor volume reached 200 mm³. (A) Tumor volumes from animals treated with vehicle, Compound 3 alone, gemcitabine, Compound 3 plus gemcitabine or a positive control, cisplatin, were evaluated over 28 days of dosing. Averages were calculated from n = 8 mice per group. Error bars represent SEM. (B) Concentrations were measured by HPLC and plotted as mean ± SD. (C) qPCR expression analysis normalized to β-actin expression for vehicle, Compound 3, or Compound 3 plus gemcitabine treated tumor samples and plotted as mean ± SD.