Pharmacological Properties of JTE-151; A Novel Orally Available RORγ Antagonist That Suppresses Th17 Cell-Related Responses in Vitro and in Vivo

Abstract

Retinoid-related orphan receptor-γ (RORγ) is a nuclear receptor that plays important roles in the development and activation of T helper type-17 (Th17) cells. In this study, we characterized the pharmacological profile of JTE-151 ((4S)-6-[(2-chloro-4-methylphenyl)amino]-4-{4-cyclopropyl-5-[cis-3-(2,2-dimethylpropyl)cyclobutyl]isoxazol-3yl}-6-oxohexanoic acid), which is a novel RORγ antagonist identified by our group. JTE-151 dissociated co-activator peptide from the human RORγ-ligand binding domain (LBD) and recruited co-repressor peptide into human RORγ-LBD, and potently inhibited the transcriptional activity of RORγ of human, mouse and rat. JTE-151 also demonstrated high selectivity against other receptors in nuclear receptor family. JTE-151 suppressed the differentiation of mouse naïve CD4⁺ T cells into Th17 cells without affecting the differentiation of those cells into other CD4⁺ T cell subsets in vitro. In addition, JTE-151 inhibited the production of interleukin-17 (IL-17) but not interferon-γ (IFN-γ) and IL-4 from activated human helper T cells in vitro. Furthermore, treatment with JTE-151 suppressed the production of IL-17 in antigen-sensitized mice and ameliorated the severity of arthritis in mice with collagen-induced arthritis regardless of treatment start date. Based on these results, we reasoned that JTE-151 could serve as a novel therapeutic compound for various autoimmune diseases linked to Th17 cells, such as psoriasis and rheumatoid arthritis.

INTRODUCTION

CD4⁺ T cells play a pivotal role in acquired immune systems and include subsets such as T helper type-1 (Th1) cells, T helper type-2 (Th2) cells, T helper type-17 (Th17) cells, regulatory T (Treg) cells, and follicular helper T (Tfh) cells.^1–3) Th1 cells producing interferon-γ (IFN-γ) and Th2 cells producing interleukin-4 (IL-4) and IL-13 were identified as the first CD4⁺ T cell subsets. These two subsets cross-regulate each other’s signaling pathways and are the drivers of “cellular immunity” and “humoral immunity,” respectively.⁴⁾ Th17 cells producing IL-17 and Treg cells expressing Foxp3 were identified as alternative subsets of CD4⁺ T cells that regulate various immune responses,^5–7) and Tfh cells were defined as another CD4⁺ T cell subset with the ability to mediate B cell development in the follicular areas of lymphoid tissue.⁸⁾ The discovery of these CD4⁺ T cell subsets has contributed, in the past two decades, to our understanding of the intricate mechanisms of various autoimmune diseases.

Th17 cells are differentiated from naïve CD4⁺ T cells in the presence of transforming growth factor-β (TGF-β) and IL-6 in mice, and produce IL-17A, IL-17F, and IL-22.^9,10) In addition to those cytokines, IL-23 has an important role in the development of Th17 cells and is required for their maintenance and effector functions.^11–13) Previous reports showed presence of Th17 cells in the lesions of various autoimmune diseases such as rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease.^14–16) Therefore, much attention has been given to the role of Th17 cells and Th17-related cytokines in the pathogenesis of autoimmune diseases, leading to the development of various biologics targeting Th17-related cytokines as new therapeutic options for several autoimmune diseases as described above.¹⁷⁾ However, no small molecule compounds targeting Th17 cell functions have shown efficacy in the treatment of those diseases.

Nuclear receptors are transcriptional factors that regulate various biological functions such as metabolism and inflammation. The retinoid-related orphan receptor (ROR) is a family of nuclear receptors consisting of three subtypes: RORα, RORβ, and RORγ.¹⁸⁾ Each subtype has a distinct function, and RORγ is well known as a key master regulator of the differentiation and activation of Th17 cells.¹⁹⁾ Although several small molecules have been identified as RORγ modulators to date,²⁰⁾ the pharmacological consequences of RORγ inhibition have not been fully elucidated. Since nuclear receptors can exhibit distinct cellular functions based on conformational changes induced by ligand binding to the receptor,²¹⁾ it is important to pharmacologically characterize RORγ modulators with varied chemical structures in order to understand the roles of RORγ in Th17 cell function.

Recently, our group reported a novel orally available RORγ antagonist, JTE-151.²²⁾ Here, we aimed to characterize the pharmacological profiles of JTE-151 both in vitro and in vivo, and investigated its potential as a novel treatment option for Th17-related autoimmune diseases.

MATERIALS AND METHODS

Drugs and Animals

JTE-151, (4S)-6-[(2-chloro-4-methylphenyl)amino]-4-{4-cyclopropyl-5-[cis-3-(2,2-dimethylpropyl)cyclobutyl]isoxazol-3yl}-6-oxohexanoic acid (Fig. 1), was chemically synthesized at the Central Pharmaceutical Research Institute, Japan Tobacco Inc. (Osaka, Japan). For in vitro experiments, JTE-151 dissolved in dimethyl sulfoxide (DMSO) was diluted 50-500-fold to the indicated concentration with either the culture medium or fluorescence resonance energy transfer (FRET) assay buffer, while vehicle control consisted of DMSO diluted in a similar manner. For in vivo experiments, JTE-151 was suspended in 0.5% methyl cellulose (0.5% MC).

Fig. 1. Chemical Structure of JTE-151

(4S)-6-[(2-Chloro-4-methylphenyl)amino]-4-{4-cyclopropyl-5-[cis-3-(2,2-dimethylpropyl) cyclobutyl]isoxazol-3yl}-6-oxohexanoic acid.

Female 7-week-old C57BL/6J mice and male 6-week-old DBA/1J mice were purchased from the Jackson Laboratory (Kanagawa, Japan). Animals were maintained under specific pathogen-free conditions in a room with temperature of 23.0 ± 3.0 °C, air humidity of 55 ± 15%, and 12-h/12-h light/dark cycle. All procedures in animal study protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Central Pharmaceutical Research Institute, Japan Tobacco Inc.

FRET Assay for RORγ Binding Co-activator and Co-repressor Peptides, and Mammalian Luciferase Reporter Assay

Detailed information about the dual FRET assay protocol has been previously described.^23,24) Briefly, the human RORγ-ligand binding domain (LBD) (residues 253–518) was expressed as a fusion protein with glutathione-S-transferase (GST) in Sf9 cells. The lysates of human RORγ-LBD-expressing Sf9 cells were incubated with a biotinylated co-activator peptide (derived from peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α); biotinyl-NH-EEPSLLKKLLLAPA-CONH₂) or co-repressor peptide (derived from nuclear receptor corepressor 2 (NCOR2); biotinyl-NH-NLGLEDIIRKALMGS-CONH₂) in the presence or absence of JTE-151. Ligand-induced conformational change in the receptor LBD dissociates the binding of co-activator peptide or recruits co-repressor peptide to human RORγ-LBD, and the FRET signal between receptor and peptide was detected using allophycocyanin-linked anti-GST antibody and R-phycoerythrin-linked streptavidin. JTE-151 concentration required to produce 50% of the maximum possible effect (EC₅₀) was calculated using non-linear sigmoidal regression for each of three independent experiments.

The transcriptional activities of RORγ were measured by means of the following luciferase (Luc) reporter assay. A cDNA encoding human RORγ-LBD (GenBank, NM_005060.3, aa 253–518), mouse RORγ-LBD (GenBank, NM_011281.2, aa 251-516), or rat RORγ-LBD (GenBank, BC169105.1 and XM_347322.3, aa 243–508) was inserted into pFA-CMV vector (Agilent Technologies, Inc., CA, U.S.A.). The vector expresses GAL4-DNA binding domain fusion protein. The construct, GAL4-RORγ plasmid, was transiently co-transfected into Chinese Hamster Ovary (CHO) cells with pG5-Luc plasmid (Promega, WI, U.S.A.) using TransIT^® CHO transfection reagent I (Mirus Bio LLC, WI, U.S.A.). Four hours after transfection, cells were harvested into 384-well plates, applied with pre-diluted JTE-151, and plates were incubated at 37 °C cell culture incubator for two days. Cell viability was checked using resazurin (Life Technologies, CA, U.S.A.), and then transcriptional activity of RORγ was measured using SteadyLite Plus Reporter Gene Assay System (PerkinElmer, Inc., MA, U.S.A.). JTE-151 concentrations required to inhibit the transcriptional activities of human, rat, and mouse RORγ by 50% (IC₅₀) were calculated for each of three independent experiments, respectively.

T Cell Differentiation Assay

The 24-well plates were coated with 500 µL of 5 µg/mL anti-CD3 antibody (Thermo Fisher Scientific, Waltham, MA, U.S.A.) over night at 4 °C, washed twice with D-phosphate buffered saline (PBS) (−) and used as assay plates. Mouse naїve CD4⁺ T cells were prepared from the spleen by immunomagnetic separation. The naїve CD4⁺ T cells were seeded at 4.0 × 10⁵ cells into assay plates containing vehicle solution or JTE-151 solution. Then, 2 µg/mL anti-CD28 antibody solution (Becton Dickinson and Company, CA, U.S.A.) was added together with the following cytokines and antibodies: 0.5 µg/mL TGF-β (PeproTech, NJ, U.S.A.), 20 ng/mL IL-6 (PeproTech), 10 µg/mL anti-IFN-γ antibody (Thermo Fisher Scientific), and 10 µg/mL anti-IL-4 antibody (Thermo Fisher Scientific) for Th17 cells; 0.1 ng/mL IL-12 (PeproTech) and 10 µg/mL anti-IL-4 antibody for Th1 cells; 1 ng/mL IL-4 (PeproTech) for Th2 cells; 0.3 ng/mL TGF-β, 10 µg/mL anti-IFN-γ antibody and 10 µg/mL anti-IL-4 antibody for Treg cells. After culturing for 72 h, the cells differentiating into Th17, Th1, or Th2 cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, MO, U.S.A.) and 1 µg/mL ionomycin (Sigma-Aldrich) in the presence of 3 µg/mL brefeldin A for four hours, and then labeled with fluorescent antibodies against IL-17A, IFN-γ and IL-4 (Thermo Fisher Scientific), respectively. The cells differentiating into Treg cells without the PMA and ionomycin stimulation were labeled with a fluorescent antibody against Foxp3 (Thermo Fisher Scientific). All the cells were also labeled with fluorescent antibody against CD4 (Thermo Fisher Scientific). The ratio of each T cell subset in the CD4⁺ T cells was determined by flow cytometry. The JTE-151 concentration required to decrease the ratio of T cell subsets in the CD4⁺ cells by 50% compared with that in the vehicle group (IC₅₀) was calculated for each of three independent experiments. For gene expression assay, total RNA was extracted from the differentiated cells using an RNeasy Lipid Tissue Mini Kit (Qiagen, Tokyo, Japan). cDNA was generated from total RNA using a High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, U.S.A.), and real-time PCR was performed on an ABI PRISM 7900 system using the following Taqman Gene Expression Assays (Applied Biosystems): IL-17A Mm00439618_m1; IL-17F Mm00521423_m1; IL-22 Mm00444241_m1; IL-23R Mm00519943_m1; RORC Mm01261022_m1; GAPDH Mm99999915_g1. GAPDH transcripts were used as an internal control to normalize mRNA levels of each gene.

Cytokine Assay

Peripheral blood mononuclear cells were isolated from human peripheral blood from healthy volunteers by density-gradient centrifugation. Human helper T cells were isolated from peripheral blood mononuclear cells using a Memory CD4⁺ T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human helper T cells were seeded at 1.0 × 10⁵ cells into anti-CD3 antibody-coated plates containing vehicle solution or JTE-151 solution. Then, 3 µg/mL anti-CD28 antibody solution was added. The culture supernatants were collected after 72 h stimulation, and the IL-17, IFN-γ, and IL-4 concentrations in the supernatants were measured using respective Quantikine^® enzyme-linked immunosorbent assay (ELISA) Kits (R&D Systems, MN, U.S.A.) and a microplate reader. The JTE-151 concentration required to decrease cytokine concentration by 50% compared with that in the vehicle group, the (IC₅₀) value, was calculated for each of three independent experiments. All procedures in this assay protocol were approved by the Ethics Committee for the Use of Human-Derived Samples at Japan Tobacco Inc. (Approval Number: R-46), and according to the Declaration of Helsinki.

IL-17 Production in Antigen-Sensitized Mice

Detailed information about this experimental protocol has been previously described.²²⁾ Briefly, female C57BL/6J mice were immunized by subcutaneous injection of 150 µg of myelin oligodendrocyte glycoprotein 35–55 (MOG_35-55) peptide (AnaSpec, CA, U.S.A.) emulsified with 200 µg of complete Freund’s adjuvant containing M. tuberculosis H37 RA (Chondrex,, WA, U.S.A.) into the back on day 1. Then, 200 ng of pertussis toxin (PTX) was injected intraperitoneally on days 1 and 3.

JTE-151 or 0.5% MC (vehicle) was orally administered once a day from day 1 to day 8 at a volume of 10 mL/kg. Mice were anesthetized with isoflurane, and blood was collected from the orbital veins of each mouse on day 8. The plasma concentrations of IL-17 were measured by using Quantikine^® Mouse IL-17 ELISA kit (R&D Systems) and a microplate reader.

Collagen-Induced Arthritis in Mice

Detailed information about this experimental protocol has been previously described.²⁵⁾ Briefly, type II collagen (Collagen Research Center, Tokyo, Japan) was dissolved in 0.01 mol/L aqueous acetic acid at 4 mg/mL and emulsified with an equal volume of Freund’s complete adjuvant (CFA) containing 2 mg/mL heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, MI, U.S.A.). Male DBA/1J mice were immunized by intradermal injection of 100 µL of 2 mg/mL type II collagen emulsion at the base of the tail on day 1 (1st immunization) and day 22 (booster immunization).

JTE-151 or 0.5% MC (vehicle) was orally administered once a day from day 19, 22, or 25 to day 36 at a volume of 10 mL/kg.

The clinical symptoms of arthritis in each paw were evaluated with a visual scoring system as below. The limb score was graded on a scale of 0–2, where 0 = no change, 1 = mild swelling, 2 = severe swelling. The digit score was graded on a scale of 0–2, where 0 = no change, 1 = swelling of 1 digit, 2 = swelling of 2 or more digits. The score of each paw was the sum of the limb score and the digit score, and the severity of arthritis for each mouse was expressed as the average of the scores for the four paws. Scoring was done in a blinded manner.

Statistical Analysis

In IL-17 production assay in antigen-sensitized mice, the significance of differences between two groups was assessed by the Aspin-Welch t-test after the data were judged to be heteroscedastic by the F-test. The significance of differences between multiple groups was assessed by the Dunnett’s test after the data were judged to be homoscedastic by the Bartlett’s test.

In the murine collagen-induced arthritis (CIA) model, the following statistical analyses were performed in accordance with a closed testing procedure to evaluate the arthritis scores at day 36. When a statistically significant difference was confirmed between the normal group and the vehicle group using the Wilcoxon rank-sum test, the Steel test was used to compare the JTE-151-treated groups to the vehicle group. A two-tailed p value <0.05 was considered statistically significant.

All statistical analysis was performed using EXSUS ver. 8.0.0 or later (EPS Corporation, Tokyo, Japan) and SAS system ver. 9.2 or later (SAS Institute, NC, U.S.A.).

RESULTS

JTE-151 Induced Co-repressor Recruitment and Co-activator Dissociation, and Inhibited the Transcriptional Activity of RORγ with High Selectivity

To characterize the pharmacological profiles of JTE-151, we carried out two types of orthogonal in vitro assays.

Recruitment of conserved peptide derived from coregulators is widely used for nuclear receptor ligand binding assay. The interaction can be measured by FRET between receptor and a peptide derived from the corrugated protein. JTE-151 dissociated co-activator peptides from RORγ-LBD in a concentration-related manner with a EC₅₀ values of 18.6 ± 1.2 nmol/L (mean ± standard error of the mean (S.E.M.)) (Fig. 2A). Conversely, JTE-151 recruited co-repressor peptides to RORγ-LBD with a EC₅₀ values of 18.1 ± 1.8 nmol/L (mean ± S.E.M.), indicating that the binding of JTE-151 changes RORγ-LBD from an active into an inactive conformation.

Fig. 2. Effects of JTE-151 on the Co-peptide Binding Status and the Transcriptional Activity of RORγ

(A) The lysate of human RORγ-LBD expressing Sf9 cells were incubated with co-activator or co-repressor peptide in the presence or absence of the indicated concentrations of JTE-151. Data are representative of three independent experiments. (B) CHO-cell transfected cDNAs encoding human/rat/mouse RORγ-LBD were incubated in the presence or absence of the indicated concentrations of JTE-151. Results are expressed as the mean percentage (± S.E.M.) of the control value in three independent experiments.

The effect of JTE-151 on the transcriptional activity of RORγ was evaluated in the luciferase reporter gene assay. As shown in Fig. 2B, JTE-151 inhibited the transcriptional activity of human, rat, and mouse RORγ in a concentration-related manner, and the IC₅₀ values were 20.6 ± 2.4, 41.8 ± 3.9 and 32.2 ± 2.5 nmol/L (mean ± S.E.M.), respectively. These data suggested that there are no differences in the inhibitory effect of JTE-151 of RORγ transcriptional activity among humans, mice, and rats. Using similar reporter gene assay systems, assessments of the effect of JTE-151 on various nuclear receptors were carried out and the results showed the negligible and weak effect on the transcriptional activities of 15 other nuclear receptors including RORα and RORβ, all of which had IC₅₀ values of >8 µmol/L, as previously described.²²⁾

Collectively, these results indicated that JTE-151 is a potent and selective RORγ antagonist.

JTE-151 Specifically Suppressed the Differentiation of Naïve T Cells into Th17 Cells

First, we examined the effects of JTE-151 on the differentiation of mouse naïve CD4+ T cells into Th17, Th1, Th2, or Treg cells.

The mean ratios of IL-17A⁺, IFN-γ⁺, IL-4⁺, and Foxp3⁺ cells in the CD4⁺ cells, as an index of Th17, Th1, Th2, and Treg cells, respectively, were 11.27, 38.60, 4.16, and 48.89% in the vehicle group, respectively. JTE-151 inhibited the differentiation of naïve CD4⁺ T cells into Th17 cells in a concentration-related manner (Fig. 3A) and the IC₅₀ value was 32.4 ± 3.0 nmol/L. In contrast, JTE-151 had no effect on the differentiation into Th1, Th2 and Treg cells up to the highest concentration (3 µmol/L) (Fig. 3A). The gene expression levels of Th17-related molecules were also examined in this assay. JTE-151 reduced mRNA levels of various Th17-related genes including Il17a, Il17f, Il22, and Il23r at 0.3 µmol/L or higher concentration compared with the vehicle group (Fig. 3B), but the Rorc gene expression was unaffected in the presence of JTE-151 even at the highest concentration (Fig. 3B).

Fig. 3. Effect of JTE-151 on the Differentiation of Naïve CD4⁺ T Cells

Naïve CD4⁺ T cells were incubated with anti-CD3/anti-CD28 antibodies in addition to respective cytokines and antibodies in the presence or absence of the indicated concentrations of JTE-151. (A) The effects of JTE-151 on the differentiation of naive CD4⁺ T cells into Th17, Th1, Th2, or Treg cells. Results are expressed as the mean percentage (± S.E.M.) relative to the control group from three independent experiments. (B) mRNA expressions of Th17-related genes are presented as mean relative values normalized to the expression of GAPDH, obtained from duplicate samples of a separate experiment conducted under the same condition for the differentiation of naïve CD4⁺ T cells into Th17 cells. Data of “(−)” group were obtained from naïve CD4⁺ T cells which were incubated with anti-CD3/anti-CD28 antibodies alone, without other cytokines and antibodies.

These results suggested that JTE-151 does not affect the gene expression of RORγ itself and selectively suppresses the differentiation of naïve CD4⁺ T cells into Th17 cells.

JTE-151 Specifically Inhibited the IL-17 Production in Activated Helper T Cells

Next, we investigated the effect of JTE-151 on the cytokine production in the activated helper T cells. When human helper T cells were treated with anti-CD3/anti-CD28 antibodies, the IFN-γ, IL-4, and IL-17 concentrations were remarkably higher in the supernatants of the culture media compared with untreated samples (data not shown). JTE-151 inhibited this increase in the IL-17 concentration in a concentration-related manner (Fig. 4), and the IC₅₀ value was 27.7 ± 7.1 nmol/L. On the other hand, JTE-151 had no effect on the increases in the IFN-γ and IL-4 concentrations even at the highest concentration (Fig. 4). Similar results were obtained in mouse and rat helper T cells (data not shown).

Fig. 4. Effect of JTE-151 on the Activation of Human Helper T Cells

Human helper T cells isolated from peripheral blood mononuclear cells are incubated with anti-CD3/anti-CD28 antibodies. Results are expressed as the mean percentage (± S.E.M.) of the control value in three independent experiments.

These results suggested that JTE-151 inhibited cytokine production in Th17 cells but not Th1 and Th2 cells.

JTE-151 Inhibited the IL-17 Production in Antigen-Sensitized Mice

As shown above, JTE-151 specifically inhibited the differentiation and activation of Th17 cells in in vitro assays. Therefore, we evaluated the effect of JTE-151 on IL-17 production in antigen-sensitized mice.

The plasma IL-17 concentration in the antigen-sensitized vehicle-treated group was significantly higher than that in the normal group (Fig. 5). JTE-151 significantly inhibited the increase in the plasma IL-17 concentration at doses of 1 mg/kg or higher (Fig. 5). This result suggested that JTE-151 inhibited the differentiation and activation of Th17 cells in vivo as well as in vitro.

Fig. 5. Effect of JTE-151 on the IL-17 Production in Antigen-Sensitized Mice

The mice were treated as described in the Materials and Methods. JTE-151 or vehicle (0.5% MC) was orally administered at the indicated doses once a day for 7 d. Results are expressed as the mean + standard deviation (n = 12). ‡ p < 0.01 (vs. Normal group, Aspin–Welch t-test), $$ p < 0.01 (vs. Vehicle group, Dunnett’s test).

JTE-151 Ameliorated Collagen-Induced Arthritis in Mice

Previous reports showed that IL-17 plays a crucial role in collagen-induced arthritis in mice.²⁶⁾ Therefore, we investigated the anti-arthritic effect of JTE-151 in this model.

The arthritis score in the vehicle group on day 36 was significantly higher than that in the normal group (Fig. 6A). Oral administration of JTE-151 at doses of 3 mg/kg or higher from day 22 intended to prevent the development of arthritis symptoms, and the mean severity score in the JTE-151 30 mg/kg group was significantly lower than that in the vehicle group (Fig. 6A).

Fig. 6. Effect of JTE-151 in Collagen-Induced Arthritis Mice

The mice were treated as described in the Materials and Methods. JTE-151 or vehicle (0.5% MC) was orally administered at the indicated doses once a day from day 22 (A) or the indicated day (B) to day 36. Results are expressed as the mean ± S.E.M. (n = 6–8). ## p < 0.01 (vs. Normal group, Wilcoxon rank-sum test), * p < 0.05 (vs. Vehicle group, Steel test).

Thus, we examined whether the treatment start date affected the efficacy of JTE-151 in arthritis development. As we expected, the mean arthritis symptom score was the lowest in mice treated from day 19, followed by those treated from day 22 and those treated from day 25 (Fig. 6B).

DISCUSSION

In this report, we investigated the pharmacological properties of JTE-151, a novel orally available RORγ antagonist, through both in vitro and in vivo experiments. In the FRET assay, JTE-151 induced both the dissociation of co-activator peptide and the recruitment of co-repressor peptide in a concentration-related manner (Fig. 2A). In the luciferase reporter gene assay, JTE-151 inhibited the transcriptional activity of RORγ with an IC₅₀ value of about 30 nmol/L in humans, rats, and mice (Fig. 2B). JTE-151 had no effect on the transcriptional activity of other nuclear receptors including RORα and RORβ. These results suggested that JTE-151 inhibits the transcriptional activity of RORγ with high selectivity in human, mouse and rat. Furthermore, since RORγ was identified as a key regulator of Th17 cells, we investigated the effects of JTE-151 in the differentiation and activation of Th17 cells. As shown in Fig. 3, JTE-151 selectively inhibited the differentiation of naïve CD4⁺ T cells into Th17 cells without affecting the expression of RORγ itself. It should be noted that we have previously carried out some exploratory experiments to investigate the optimal conditions for the differentiation of naïve CD4⁺ T cells into Th17 cells and similar trends to those presented in Fig. 3B were observed under the same conditions as those shown in Fig. 3A in these experiments (data not shown). Furthermore, JTE-151 inhibited the production of IL-17 from activated helper T cells but not that of IFN-γ and IL-4 (Fig. 4). These results indicated that JTE-151 has inhibitory effects on Th17 cell functions but not other helper T cell subtype functions in vitro.

Our group reported that JTE-151 binds to the U-shaped ligand-binding pocket of RORγ and suggested that the binding of JTE-151 causes the shift of helix H11 in RORγ-LBD.^22,24) On the other hand, other RORγt inverse agonists with dissimilar chemical structures such as VTP-43742 or TAK-828F were reported to have different binding modes with RORγt-LBD.²⁰⁾ RORγt is an immune cell-specific isoform and has the same LBD as RORγ.²⁷⁾ For example, while VTP-43742 makes close contact with the “agonist lock” residues His479-Tyr502-Phe506 in RORγ,²⁰⁾ JTE-151 does not interact with agonist lock residues. Moreover, although TAK-828F forms hydrogen bonds with Phe377/Glu379 and makes water-mediated interactions with Arg364/Gln286,²⁸⁾ JTE-151 forms a hydrogen bond only with Phe377 and makes favorable van der Waals contacts with Leu324/Phe388/Leu391/Ile397.²²⁾ Taken together, these findings suggest that the conformational change of RORγ induced by JTE-151 might be different from that induced by other small molecules (VTP-43742 or TAK-828F). However, as indicated above, JTE-151 inhibits RORγ activities including the differentiation and activation of Th17 cells as well as other RORγ inhibitors, suggesting that JTE-151 is also a suitable small molecule to use for elucidating the role of RORγ in Th17 cells.

Next, we investigated the effects of JTE-151 in vivo. It was reported that RORγ-deficient mice and RAG2-deficient mice reconstituted with RORγ-deficient bone marrow showed decreased susceptibility to experimental autoimmune encephalomyelitis (EAE).¹⁹⁾ In other studies, the transfer of MOG-specific Th17 cells induced EAE symptoms in wild-type mice,²⁹⁾ and therapeutic neutralization of IL-17 or IL-23p19 ameliorated the disease course of EAE.^30,31) These reports suggested that RORγ, Th17 cells, and Th17-related cytokines play crucial roles in the pathogenesis of MOG peptide-induced inflammation and neurodegeneration. So, we assessed the effect of JTE-151 on IL-17 production using this EAE model. We showed that immunization with MOG_35-55 peptide increased plasma IL-17 concentrations in mice, and JTE-151 significantly suppressed it in a dose-related manner (Fig. 5). It is suggested that JTE-151 also inhibited the differentiation and activation of Th17 cells through the inhibition of RORγ activity in vivo as well as in vitro.

Th17 cells and Th17-related cytokines are also associated with the pathogenesis of arthritis in the murine CIA model, a widely used animal model of RA. Mice lacking IL-23 receptor (IL-23R) or IL-17RA are protected against the development of synovial inflammation and joint destruction in this model.³²⁾ Additionally, previous reports showed that the frequency of CD4⁺ IL-17⁺ T cells in lymph nodes was increased by the 1st immunization for CIA induction at day 10 and was further increased by the booster immunization at day 26, and that the treatment with anti-IL-6R antibody on the day of 1st immunization but not in the later phase suppressed the arthritis development in this model.³³⁾ Since IL-6 is a critical cytokine for Th17 differentiation and development from naïve CD4⁺ T cells,¹⁰⁾ it is suggested that the pathogenesis of arthritis in the mouse CIA model is mainly associated with the development of Th17 cells in the early phase, and with the maintenance and activation of Th17 cells in the later phase. In this study, we demonstrated that treatment with JTE-151 from day 22 ameliorated arthritis symptoms induced by booster collagen immunization (Fig. 6A), and that the suppressive efficacy of JTE-151 in this model was increased by starting the treatment earlier (Fig. 6B). Our results suggested that the treatment with JTE-151 in the later phase inhibited the maintenance and activation of Th17 cells, leading to the suppression of arthritis symptoms in this model. On the other hand, our group also reported that JTE-151 suppressed the differentiation of Th17 cells and ameliorated the severity of paralysis in the mouse EAE model.²²⁾ Taking these results into consideration, we expected that JTE-151 is expected to show its clinical effects on any symptoms of diseases whose pathogenesis is related to Th17 cells regardless of when treatment started.

There are several limitations that warrant discussion. Although we evaluated the effects of JTE-151 on the differentiation of naïve CD4⁺ T cells into Th17 cells and the activation of Th17 cells in vitro, we have not investigated their detail assessments in the mouse CIA model. The suppressive efficacy of JTE-151 on the arthritis score in the mouse CIA model varied depending on the start timing of administration (Fig. 6B), which implied that these differences were attributed to changes in the cell number and activation state of Th17 cells by the treatment with JTE-151. Therefore, further experiments are needed to fully understand the mechanisms of action of JTE-151 by examining the relationships between the improvement effects and plasma concentrations of JTE-151, the plasma IL-17 levels, as well as the number of Th17 cells in lymph nodes in these mice in the future.

In conclusion, we demonstrated that JTE-151 inhibits the transcriptional activity of RORγ leading to suppression of the differentiation and activation of Th17 cells, followed by mitigation of the Th17-related symptoms in mice in this study. Since there are various autoimmune diseases which is associated with Th17 cells such as psoriasis and rheumatoid arthritis, our findings indicate that JTE-151 deserves further study as a therapeutic compound for these indications.

Acknowledgments

We thank Morgan Fenn, Joseph Gatewood and Toshinobu Kato for their excellent technical assistance and analytical support.

Conflict of Interest

KA, SO, KK, SE, YO, YK, TY, MM are employees of Japan Tobacco Inc. HT, PC and ST are employees of Orphagen Pharmaceuticals, Inc.

Notes

Yoshihisa Okamoto (Deceased on 1st March, 2019.)

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