Fig 1.
Cpd 1 is a potent RORγt inhibitor and binds to the ligand binding pocket of RORγt.
(A) Chemical structure of low-molecular-weight cpd 1. (B) TR-FRET assay measuring RIP140 co-factor displacement from the human RORγt-LBD by cpd 1. Representative concentration-dependent curve from ten independent experiments with duplicate readings are shown. Inhibitory concentration to achieve 50% of the signal (IC50) is indicated in the graph. (C) Results of thermal stability studies by DSLS. Solid lines show the scattered light intensity as a function of temperature. The dotted line is the negative of the 1st derivative of this function, with a minimum at the inflection point, indicating the value of Tagg(inf), which characterizes the midpoint of the transition. A comparison with apo shows that cpd 1 binds with high affinity to the RORγt-LBD leading to ΔTagg(inf) of 13.7 oC. All experiments were performed independently for at least three times with similar results.
Fig 2.
Cpd 1 is a potent and selective RORγt inhibitor in in vitro cellular assays.
(A) Overexpression of RORγt results in IL-17A production. HUT78 T-cells, stably transduced with human full-length RORγt or control vector, were stimulated with anti-CD3 antibody plus PMA for 48 hrs and IL-17A production was quantified by ELISA. (B) HUT78 T-cells expressing RORγt were incubated with serial dilutions of cpd 1 at the beginning of the stimulation with PMA and anti-CD3 antibody and after 48 hrs IL-17A concentrations were measured by ELISA. Representative example of a concentration-response curve from five independent experiments with triplicate readings is shown. Activity of cpd 1 against the nuclear hormone receptors RORα (C) or RORβ (D). Jurkat T-cells stably expressing GAL4 upstream activator sequences that control the transcription of the luciferase reporter gene were transfected with a GAL4-DNA binding domain fusion construct containing the RORα or RORβ-LBD. Cells were incubated for 24 hrs with cpd 1, followed by cell lysis and measurement of luciferase activity. Graphs are representative of three independent experiments performed in duplicates.
Fig 3.
Cpd 1 specifically impairs human Th17 cell polarization and Th17-signature cytokine production.
(A) Total CD4+ T-cells were incubated with the RORγt inhibitor and were activated under Th17 cell favoring conditions for 72 hrs followed by quantification of IL-17A production. Data are representative of five experiments with triplicate readings. (B) Th17 cells were treated with PMA/ionomycin and Brefeldin A for 4 hrs and frequencies of IL-17A producing cells in gated CD4+ T-cells were determined by intracellular staining. Data represent duplicate measurements of two independent experiments. (C and D) Supernatants from polarized Th17 cells were taken to determine IL-17F and IL-22 cytokine concentrations. Naïve (E) and memory (F) human CD4+ T-cells were purified from PBMCs and were cultured under Th17 skewing conditions for 7 days in the presence of cpd 1. IL-17A production was quantified by ELISA. Representative examples of concentration-response curves from two experiments with duplicate readings are shown. (G) Human CD4+ T-cells were stimulated with anti-CD3 plus anti-CD28 antibodies only (Th0) or incubated with IL-12 and anti-IL-4 antibody (Th1) or with IL-4 and anti-IFN-γ antibody (Th2). After 48 hrs, Th subset signature cytokines including IL-2, IFN-γ and IL-13 were analyzed by ELISAs. Representative examples from two experiments with triplicate readings are shown.
Fig 4.
Cpd 1 blocks IL-17A production by differentiated Th17 cells and by Tc17 and γδT-cells.
(A) Human CD4+ T-cells were stimulated with anti-CD3 plus anti-CD28 antibodies in the presence of Th17 cell promoting conditions for 7 days without compound, followed by extensive washing of cells to remove IL-17A and re-stimulation with anti-CD3 plus anti-CD28 antibodies in the presence of cpd 1. After 3 days, supernatants were collected and IL-17A concentrations were determined. (B) Purified CD8+ T-cells were stimulated with anti-CD3 and anti-CD28 antibodies under Th17-polarizing cytokines for 72 hrs, and IL-17A production was quantified. (C) Human γδ T-cells were incubated with cpd 1 and stimulated with PMA/ionomycin for 24 hrs, followed by quantification of IL-17A concentration by ELISA. Representative examples from three independent experiments with triplicate readings are shown.
Fig 5.
Downregulated RORγt-regulated target gene expression by cpd 1.
(A-F) Purified human CD4+ T-cells were polarized towards Th17 cells and were treated at the beginning of the cell culture with various concentrations of cpd 1 or with DMSO control (Co). After 72 hrs, mRNA was extracted and transcript levels were quantified by RT-PCR. Gene expression was normalized to β-glucoronidase levels and expressed as arbitrary units. All graphs are representative of three independent experiments containing three technical replicates.
Fig 6.
Cpd 1 blocks IL17A/IL23R gene expression by inhibiting permissive chromatin remodeling at their promoter regions.
(A) RORγt or empty vector transduced HUT78 T-cells were stimulated with anti-CD3 antibody and PMA. IL17A and IL23R gene expression was analyzed by RT-PCR, which was performed on mRNA isolated after 24 hrs of stimulation. (B) RORγt transduced HUT78 T-cells were stimulated as described above in the presence of cpd 1 (10 μM) or DMSO and mRNA was prepared followed by analysis of IL17A, IL23R and RORC gene expression via RT-PCR. Gene expression levels are shown relative to DMSO treated cells (100%). (C and D) Stimulated cell lysates from cpd 1(10 μM) or DMSO-treated RORγt transduced HUT78 T-cells were cross-linked with 1% formaldehyde, sonicated and chromatin preparations were immunoprecipitated with H3K9/14 acetyl or H3K4me3-specific or isotype-specific control antibodies. The precipitated DNA was quantified by qPCR with primers specific for IL17A and IL23R promoter regions. The results were normalized to an input control. (E) RORγt transduced HUT78 T-cells were treated with cpd 1(10 μM) or DMSO, stimulated with anti-CD3 antibody/PMA or left unstimulated (No stim) for 2 hrs and nuclear extracts were prepared. Nuclear extracts were equally divided into two and each half was subjected to pull-down experiments using mutated or wild-type biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. After extensive washing pull-down complexes (left panel) or nuclear extracts (right panel) were subjected to SDS PAGE and RORγ Western blot analysis. Graphs and Western blots are representative of two independent experiments.
Fig 7.
Cpd 1 inhibits IL-17A production by whole blood cells and blocks rat Th17 differentiation.
(A and B) Diluted human whole blood cells were stimulated with ConA and IL-23 in the presence of cpd 1. After 72 hrs, IL-17A (A) or IL-2 (B) production was measured by ELSA. Data are representative from five (A) or two (B) independent experiments with triplicate measurements. (C) Purified CD4+ Tcells originating from splenocytes from Lewis rats were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of Th17-skewing conditions. After 72 hrs, IL-17A concentrations in supernatants were determined by ELISA. Representative example of concentration-response curve from two experiments with triplicate readings is shown.
Fig 8.
Cpd 1 ameliorates antigen-induced arthritis (AiA) responses in Lewis rats.
(A) Rats were immunized twice with methylated BSA (mBSA). Three weeks later, the right knees of the animals were challenged with mBSA in 5% glucose, while the left knees were injected with vehicle (5% glucose). Starting just prior to antigen challenge, cpd 1 was administered twice daily via oral gavage at the indicated doses for 7 days and knee swelling was monitored. The mean ± SEM of the swelling ratios between antigen-challenged and vehicle injected knees are shown (n = 5). Results are representative of two experiments with 5 rats per treatment group. (B) Draining lymph node cells were prepared 7 days after mBSA challenge and stimulated ex vivo with mBSA (200 μg/ml). Frequencies of IL-17A secreting antigen-specific cells were quantified by ELISPOT after 6 h. (C) Diluted sublingual whole blood cells originating from cpd 1- or vehicle-treated animals were re-stimulated with mBSA (50 μg/ml) and after 96 hrs, IL17A concentrations were determined by ELISA. Each point represents mean ± SEM from an individual rat (n = 4–5 readings/animal). *, P < 0.05; **, P < 0.01; ***, P < 0.001 Dunnett´s test.