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Identification of Tetraazacyclic Compounds as Novel Potent Inhibitors Antagonizing ROR?t Activity and Suppressing Th17 Cell Differentiation.

ABSTRACT: CD4+ T-helper cells that produce interleukin-17 (Th17 cells) are characterized as pathological T-helper cells in autoimmune diseases. Differentiation of human and mouse Th17 cells requires a key transcription regulator, retinoic acid receptor-related orphan receptor ?t (ROR?t), which is a potential therapeutic target for autoimmune diseases. To develop a therapeutic agent for Th17-mediated autoimmune diseases, we have established a high-throughput screening (HTS) assay for candidate screening, in which the luciferase activity in ROR?t-LBD positive and negative Jurkat cells were analyzed to evaluate induction of ROR?t activity by compounds. This technique was applied to screen a commercially-available drug-like chemical compound library (Enamine) which contains 20155 compounds. The screening identified 17 compounds that can inhibit ROR?t function in the HTS screen system. Of these, three tetraazacyclic compounds can potently inhibit ROR?t activity, and suppress Th17 differentiation and IL-17 production. These three candidate compounds could significantly attenuate the expression of the Il17a by 65%- 90%, and inhibit IL-17A secretion by 47%, 63%, and 74%, respectively. These compounds also exhibited a potent anti-ROR?t activity, with EC50 values of 0.25 ?M, 0.67 ?M and 2.6 ?M, respectively. Our data demonstrated the feasibility of targeting the ROR?t to inhibit Th17 cell differentiation and function with these tetraazacyclic compounds, and the potential to improve the structure of these compounds for autoimmune diseases therapeutics.

SUBMITTER: Ding Q

PROVIDER: S-EPMC4569406 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of Tetraazacyclic Compounds as Novel Potent Inhibitors Antagonizing RORγt Activity and Suppressing Th17 Cell Differentiation.

Ding Qingfeng Q Zhao Mei M Yu Bolan B Bai Chuan C Huang Zhaofeng Z

PloS one 20150914 9

CD4+ T-helper cells that produce interleukin-17 (Th17 cells) are characterized as pathological T-helper cells in autoimmune diseases. Differentiation of human and mouse Th17 cells requires a key transcription regulator, retinoic acid receptor-related orphan receptor γt (RORγt), which is a potential therapeutic target for autoimmune diseases. To develop a therapeutic agent for Th17-mediated autoimmune diseases, we have established a high-throughput screening (HTS) assay for candidate screening, i ...[more]

PMID: 26368822

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Project description:CD4+ T helper lymphocytes that express interleukin-17 (Th17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in Th17 cell differentiation and function with susceptibility to Crohn’s disease, rheumatoid arthritis, and psoriasis1-3. Thus, the pathway towards differentiation of Th17 cells and, perhaps, of related innate lymphoid cells with similar effector functions4, 5, is an attractive target for therapeutic applications. Mouse and human Th17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of Th17-dependent autoimmune disease in mice6. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine Th17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives, 20,22-dihydrodigoxin-21,23-diol (Dig(dhd)) and digoxin-21-salicylidene (Dig(sal)), specifically inhibited induction of IL-17 in human CD4+ T cells. Using these small molecule compounds, we demonstrated that RORγt is imporant for the maintenance of IL-17 expression in mouse and human effector T cells. These data suggest that derivatives of digoxin can be used as chemical probes for development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease. We performed gene expression profiling with total RNA samples isolated from DMSO- or digoxin-treated wildtype or RORγt-deficient cells cultured in Th17 conditions. Total RNA was extracted with TRIzol. RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX11.5.

Dataset Information

Identification of Tetraazacyclic Compounds as Novel Potent Inhibitors Antagonizing ROR?t Activity and Suppressing Th17 Cell Differentiation.

Publications

Identification of Tetraazacyclic Compounds as Novel Potent Inhibitors Antagonizing RORγt Activity and Suppressing Th17 Cell Differentiation.

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