Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity

Huh, Jun R.; Leung, Monica; Huang, Peiying; Ryan, David L.; Krout, Michael R.; Malapaka, Raghu R.V.; Chow, Jonathan; Manel, Nicolas; Ciofani, Maria; Kim, Sangwon V.; Cuesta, Adolfo; Santori, Fabio R.; Lafaille, Juan J.; Xu, H. Eric; Gin, David Y.; Rastinejad, Fraydoon; Littman, Dan R.

doi:10.1038/nature09978

Cited by 490 publications

(495 citation statements)

References 30 publications

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“…Several RORgt small-molecule inhibitors have been discovered and shown to block Th17 cell differentiation and IL-17A production, as well as demonstrate efficacy in EAE studies (22)(23)(24). In this article, we report the discovery of RORgt inverse agonist TMP778 and its functionally inactive diastereomer TMP776.…”

Section: Discussionmentioning

confidence: 99%

“…Digoxin, SR2211, SR1001, and ursolic acid are a variety of small-molecule RORgt inhibitors that have been recently discovered (22)(23)(24)42). These compounds inhibit Th17 cell differentiation in vitro and reduce severity of EAE, a mouse model of multiple sclerosis.…”

Section: Discussionmentioning

confidence: 99%

“…The critical role of RORgt in the generation of IL-17-producing T cells led to the identification of small molecules targeting RORgt; these compounds not only show inhibitory activity against Th17 cell differentiation and IL-17 production, but have also demonstrated variable levels of efficacy in EAE studies (22)(23)(24). In this study, we describe a novel class of selective and potent RORgt inverse agonists, exemplified by a lead compound TMP778 and its structurally identical but functionally inactive diastereomer TMP776.…”

Section: D4mentioning

confidence: 99%

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Pharmacologic Inhibition of RORγt Regulates Th17 Signature Gene Expression and Suppresses Cutaneous Inflammation In Vivo

Skepner¹,

Ramesh²,

Trocha³

et al. 2014

The Journal of Immunology

128

120

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IL-17–producing CD4+Th17 cells, CD8+Tc17 cells, and γδ T cells play critical roles in the pathogenesis of autoimmune psoriasis. RORγt is required for the differentiation of Th17 cells and expression of IL-17. In this article, we describe a novel, potent, and selective RORγt inverse agonist (TMP778), and its inactive diastereomer (TMP776). This chemistry, for the first time to our knowledge, provides a unique and powerful set of tools to probe RORγt-dependent functions. TMP778, but not TMP776, blocked human Th17 and Tc17 cell differentiation and also acutely modulated IL-17A production and inflammatory Th17-signature gene expression (Il17a, Il17f, Il22, Il26, Ccr6, and Il23) in mature human Th17 effector/memory T cells. In addition, TMP778, but not TMP776, inhibited IL-17A production in both human and mouse γδ T cells. IL-23–induced IL-17A production was also blocked by TMP778 treatment. In vivo targeting of RORγt in mice via TMP778 administration reduced imiquimod-induced psoriasis-like cutaneous inflammation. Further, TMP778 selectively regulated Th17-signature gene expression in mononuclear cells isolated from both the blood and affected skin of psoriasis patients. In summary, to our knowledge, we are the first to demonstrate that RORγt inverse agonists: 1) inhibit Tc17 cell differentiation, as well as IL-17 production by γδ T cells and CD8+ Tc17 cells; 2) block imiquimod-induced cutaneous inflammation; 3) inhibit Th17 signature gene expression by cells isolated from psoriatic patient samples; and 4) block IL-23–induced IL-17A expression. Thus, RORγt is a tractable drug target for the treatment of cutaneous inflammatory disorders, which may afford additional therapeutic benefit over existing modalities that target only IL-17A.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: D4mentioning

confidence: 99%

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Pharmacologic Inhibition of RORγt Regulates Th17 Signature Gene Expression and Suppresses Cutaneous Inflammation In Vivo

Skepner¹,

Ramesh²,

Trocha³

et al. 2014

The Journal of Immunology

128

120

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“…Due to its important pharmacological activities, UA has been used for treatment of liver diseases and skin cancer (15). These clinical practices and its relatively low toxicity provide UA a great advantage over other ROR inhibitors recently reported (22,23) in developing therapeutics against Th17-mediated autoimmune diseases. Considering the broad function of Th17 cells in inflammatory diseases and cancer, it is of interest in assessing whether the therapeutic effects of UA are via inhibition of ROR␥t or Th17 cell function.…”

Section: Ua Inhibits Il-17 Expression In Mature Th17mentioning

confidence: 99%

Ursolic Acid Suppresses Interleukin-17 (IL-17) Production by Selectively Antagonizing the Function of RORγt Protein

Wang

Zhong

et al. 2011

Journal of Biological Chemistry

194

149

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Th17 cells have recently emerged as a major player in inflammatory and autoimmune diseases via the production of pro-inflammatory cytokines IL-17, IL-17F, and IL-22. The differentiation of Th17 cells and the associated cytokine production is directly controlled by ROR␥t. Here we show that ursolic acid (UA), a small molecule present in herbal medicine, selectively and effectively inhibits the function of ROR␥t, resulting in greatly decreased IL-17 expression in both developing and differentiated Th17 cells. In addition, treatment with UA ameliorated experimental autoimmune encephalomyelitis. The results thus suggest UA as a valuable drug candidate or leading compound for developing treatments of Th17-mediated inflammatory diseases and cancer. Th17 cells have been recently discovered as the third effector CD4ϩ T helper subset (1, 2). Th17 cells produce 4). Although Th17 cells play important roles in host defense against bacterial and fungal infections, they have been also linked to many immune-related diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, periodontal diseases, and asthma/airway inflammatory diseases (4, 5). Anti-IL-17 was recently shown to have good efficacy in treatment of multiple human diseases (6).In Th17 cells, the transcription of IL-17 and IL-17F is mediated by Th17-specific transcriptional regulators ROR␥t 6 and ROR␣, although the latter plays a less significant role in mice (7,8). Mice deficient in ROR␥ and those deficient in both ROR␥t and ROR␣ are defective in production of IL-17 and IL-17F and are resistant to experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (7,8). Therefore, developing ROR inhibitors represents a promising therapeutic strategy in treatment of Th17-mediated diseases.In the current study, we screened a small chemical library and identified ursolic acid (UA), a natural carboxylic acid ubiquitously present in plants, as a strong and selective inhibitor for ROR␥t function. UA inhibited IL-17 production not only in developing Th17 cells but also in mature Th17 cells. Mice receiving UA were resistant to EAE, indicating that UA can be used for developing treatment of Th17-mediated diseases. EXPERIMENTAL PROCEDUREST Cell Analysis-Human and mouse T cell differentiation and retroviral transduction were performed and analyzed by intracellular staining or by quantitative real-time RT-PCR assays as described (8 -10). UA (dissolved in DMSO) or DMSO was added into the culture medium for inhibition assays.Luciferase Reporter Assays-The CNS2-Il17a and RORE reporter constructs were used for Dual-Luciferase reporter assays in EL4 and 293T cells, respectively, as reported (8, 11). The reporter activity was normalized against Renilla luciferase activity.Co-activator Binding Assays-The effect of UA on the interaction of coactivator peptides with ROR␥ was determined by terbium-mediated time-resolved fluorescence energy transfer assays using the LanthaScreen TR-FRET from Invitrogen. The experiments were conduc...

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“…For example, bortezomib, the positive control compound used in the present study, directly binds the proteasome β5 subunit and inhibits CT-L activity and other activities of the proteasome [30] . As cardiac steroids have been shown to bind to intracellular targets [31] , the possibility that BF211 directly binds proteasome subunits cannot be excluded. However, BF211 exhibited no binding affinity to the β2 or β5 subunits and only weakly bound to the β1 subunit.…”

Section: Discussionmentioning

confidence: 99%

Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly

et al. 2016

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Aim: Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells. Methods: A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis. Results: The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome β1 subunit and no binding affinity to the β2 and β5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome β1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the β1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity. Conclusion: BF211 inhibits proteasome activity in A549 cells by decreasing β1 subunit expression and disrupting proteasome assembly.Keywords: bufalin; ChanSu; cardiac steroids; proteasome β1 subunit; proteasome assembly; A549 human lung cancer cells; In the present study, to identify the possible signaling network activated by BF211 in cancer cells, we conducted a SILAC-based proteomic analysis and compared the protein expression profiles of A549 human lung cancer cells treated with either BF211 or a solvent control. Our findings suggested that BF211 influences proteasome function, and we further evaluated the effects and potential mechanisms mediating this phenomenon.

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Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity

Cited by 490 publications

References 30 publications

Pharmacologic Inhibition of RORγt Regulates Th17 Signature Gene Expression and Suppresses Cutaneous Inflammation In Vivo

Pharmacologic Inhibition of RORγt Regulates Th17 Signature Gene Expression and Suppresses Cutaneous Inflammation In Vivo

Ursolic Acid Suppresses Interleukin-17 (IL-17) Production by Selectively Antagonizing the Function of RORγt Protein

Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly

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