Pharmacologic inhibition of RORγt regulates Th17 signature gene expression and suppresses cutaneous inflammation in vivo.

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.

In vivo regulation of gene expression and T helper type 17 differentiation by RORγt inverse agonists.

The orphan nuclear receptor, retinoic acid receptor-related orphan nuclear receptor γt (RORγt), is required for the development and pathogenic function of interleukin-17A-secreting CD4(+) T helper type 17 (Th17) cells. Whereas small molecule RORγt antagonists impair Th17 cell development and attenuate autoimmune inflammation in vivo, the broader effects of these inhibitors on RORγt-dependent gene expression in vivo has yet to be characterized. We show that the RORγt inverse agonist TMP778 acts potently and selectively to block mouse Th17 cell differentiation in vitro and to impair Th17 cell development in vivo upon immunization with the myelin antigen MOG35-55 plus complete Freund's adjuvant. Importantly, we show that TMP778 acts in vivo to repress the expression of more than 150 genes, most of which fall outside the canonical Th17 transcriptional signature and are linked to a variety of inflammatory pathologies in humans. Interestingly, more than 30 genes are related with SMAD3, a transcription factor involved in the Th17 cell differentiation. These results reveal novel disease-associated genes regulated by RORγt during inflammation in vivo, and provide an early read on potential disease indications and safety concerns associated with pharmacological targeting of RORγt.

Chronic treatment with epoxyeicosatrienoic acids modulates insulin signaling and prevents insulin resistance in hepatocytes.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites produced by cytochrome P450 epoxygenases which are highly expressed in hepatocytes. The functions of EETs in hepatocytes are not well understood. In this study, we investigated the effects of 14,15-EETs treatment on the insulin signal transduction pathway in hepatocytes. We report that chronic treatment, not acute treatment, with 30 µM 14,15-EETs prevents palmitate induced insulin resistance and potentiates insulin action in cultured HepG2 hepatocytes. 14,15-EETs increase Akt phosphorylation at S473, activating Akt, in an insulin dependent manner in HepG2 cells. Under insulin resistant conditions induced by palmitate, 14,15-EETs restore the insulin response by increasing S473-phosphorylated Akt. 8,9-EETs and 11,12-EETs demonstrated similar effects to 14,15-EETs. Furthermore, 14,15-EETs potentiate insulin-suppression of gluconeogenesis in cultured H4IIE hepatocytes. To elucidate the mechanism of EETs function, we analyzed the insulin signaling factors upstream of Akt. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 attenuated the 14,15-EETs-induced activating phosphorylation of Akt. 14,15-EETs reduced palmitate-stimulated phosphorylation of IRS-1 on S312 and phosphorylation of c-Jun N-terminal kinase (JNK) at threonine 183 and tyrosine 185 residues. The regulation of insulin sensitivity in cultured hepatocytes by chronic 14,15-EETs treatment appears to involve the JNK-IRS-PI3K pathway. The requirement of chronic treatment with EETs suggests that the effects of EETs on insulin response may be indirect.

Discovery and characterization of QPT-1, the progenitor of a new class of bacterial topoisomerase inhibitors.

QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (-)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization. Biochemical and genetic characterization showed that the QPT-1 targets the beta subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

Modulation of EZH2 expression in T cells improves efficacy of anti-CTLA-4 therapy.

Enhancer of zeste homolog 2-mediated (EZH2-mediated) epigenetic regulation of T cell differentiation and Treg function has been described previously; however, the role of EZH2 in T cell-mediated antitumor immunity, especially in the context of immune checkpoint therapy, is not understood. Here, we showed that genetic depletion of EZH2 in Tregs (FoxP3creEZH2fl/fl mice) leads to robust antitumor immunity. In addition, pharmacological inhibition of EZH2 in human T cells using CPI-1205 elicited phenotypic and functional alterations of the Tregs and enhanced cytotoxic activity of Teffs. We observed that ipilimumab (anti-CTLA-4) increased EZH2 expression in peripheral T cells from treated patients. We hypothesized that inhibition of EZH2 expression in T cells would increase the effectiveness of anti-CTLA-4 therapy, which we tested in murine models. Collectively, our data demonstrated that modulating EZH2 expression in T cells can improve antitumor responses elicited by anti-CTLA-4 therapy, which provides a strong rationale for a combination trial of CPI-1205 plus ipilimumab.

Targeting Th17 Effector Cytokines for the Treatment of Autoimmune Diseases.

Interleukin (IL)-17-producing T cells, especially T helper (Th)17 cells, play a critical role in the pathogenesis of a variety of autoimmune inflammatory diseases. The pathogenic function of Th17 cells results from their production of Th17 effector cytokines, namely IL-17 (or IL-17A), IL-17F, IL-22 and IL-26. The importance of IL-17 has been demonstrated by antibody neutralization studies in both animal models of autoimmune diseases as well as in human clinical trials. This review highlights the current knowledge of the clinical aspects of the Th17 cytokines as well as therapeutic antibodies against IL-17, IL-17F, IL-17 receptor, IL-22, IL-26 and granulocyte macrophage colony-stimulating factor for the future treatment of autoimmune inflammatory diseases.

Targeting Th17 cells in autoimmune diseases.

T helper 17 (Th17) cells have been implicated in the pathogenesis of most common autoimmune diseases, including psoriasis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and multiple sclerosis (MS). Although anti-interleukin-17 (IL-17) antibodies show marked clinical efficacy in psoriasis, targeting IL-17 alone is not sufficient to improve clinical end points in other autoimmune conditions, namely RA and Crohn's disease. Given that Th17 cells express IL-17 together with many other proinflammatory cytokines [IL-17F, IL-22, IL-26, and granulocyte-macrophage colony-stimulating factor (GM-CSF)], targeting the Th17 cell lineage may be superior to blocking a single effector cytokine. Here, we discuss the rationale for targeting two checkpoints in the development and inflammatory function of Th17 cells, retinoid-related orphan receptor-γt (RORγt) and IL-23, and we review recent progress in the development of both RORγt small molecule inhibitors and IL-23 neutralizing antibodies.

Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.