Mohamed Boudjelal

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.

Pharmacological modulation of circadian rhythms by synthetic activators of the deacetylase SIRT1.

Circadian rhythms govern a wide variety of physiological and metabolic functions in many organisms, from prokaryotes to humans. We previously reported that silent information regulator 1 (SIRT1), a NAD+-dependent deacetylase, contributes to circadian control. In addition, SIRT1 activity is regulated in a cyclic manner in virtue of the circadian oscillation of the coenzyme NAD+. Here we used specific SIRT1 activator compounds both in vitro and in vivo. We tested a variety of compounds to show that the activation of SIRT1 alters CLOCK:BMAL1-driven transcription in different systems. Activation of SIRT1 induces repression of circadian gene expression and decreases H3 K9/K14 acetylation at corresponding promoters in a time-specific manner. Specific activation of SIRT1 was demonstrated in vivo using liver-specific SIRT1-deficient mice, where the effect of SIRT1 activator compounds was shown to be dependent on SIRT1. Our findings demonstrate that SIRT1 can fine-tune circadian rhythm and pave the way to the development of pharmacological strategies to address a broad range of therapeutic indications.

Raised hepatic bile acid concentrations during pregnancy in mice are associated with reduced farnesoid X receptor function.

Pregnancy alters bile acid homeostasis and can unmask cholestatic disease in genetically predisposed but otherwise asymptomatic individuals. In this report, we show that normal pregnant mice have raised hepatic bile acid levels in the presence of procholestatic gene expression. The nuclear receptor farnesoid X receptor (FXR) regulates the transcription of the majority of these genes, and we show that both ablation and activation of Fxr prevent the accumulation of hepatic bile acids during pregnancy. These observations suggest that the function of Fxr may be perturbed during gestation. In subsequent in vitro experiments, serum from pregnant mice and humans was found to repress expression of the Fxr target gene, small heterodimer partner (Shp), in liver‐derived Fao cells. Estradiol or estradiol metabolites may contribute to this effect because coincubation with the estrogen receptor (ER) antagonist fulvestrant (ICI 182780) abolished the repressive effects on Shp expression. Finally, we report that ERα interacts with FXR in an estradiol‐dependent manner and represses its function in vitro. Conclusion: Ligand‐activated ERα may inhibit FXR function during pregnancy and result in procholestatic gene expression and raised hepatic bile acid levels. We propose that this could cause intrahepatic cholestasis of pregnancy in genetically predisposed individuals. HEPATOLOGY 2010

The normal mechanisms of pregnancy-induced liver growth are not maintained in mice lacking the bile acid sensor Fxr

Rodents undergo gestational hepatomegaly to meet the increased metabolic demands on the maternal liver during pregnancy. This is an important physiological process, but the mechanisms and signals driving pregnancy-induced liver growth are not known. Here, we show that liver growth during pregnancy precedes maternal body weight gain, is proportional to fetal number, and is a result of hepatocyte hypertrophy associated with cell-cycle progression, polyploidy, and altered expression of cell-cycle regulators p53, Cyclin-D1, and p27. Because circulating reproductive hormones and bile acids are raised in normal pregnant women and can cause liver growth in rodents, these compounds are candidates for the signal driving gestational liver enlargement in rodents. Administration of pregnancy levels of reproductive hormones was not sufficient to cause liver growth, but mouse pregnancy was associated with increased serum bile acid levels. It is known that the bile acid sensor Fxr is required for normal recovery from partial hepatectomy, and we demonstrate that Fxr(-/-) mice undergo gestational liver growth by adaptive hepatocyte hyperplasia. This is the first identification of any component that is required to maintain the normal mechanisms of gestational hepatomegaly and also implicates Fxr in a physiologically normal process that involves control of the hepatocyte cell cycle. Understanding pregnancy-induced hepatocyte hypertrophy in mice could suggest mechanisms for safely increasing functional liver capacity in women during increased metabolic demand.

Optimized chemical probes for REV-ERBα

REV-ERBα has emerged as an important target for regulation of circadian rhythm and its associated physiology. Herein, we report on the optimization of a series of REV-ERBα agonists based on GSK4112 (1) for potency, selectivity, and bioavailability. (1) Potent REV-ERBα agonists 4, 10, 16, and 23 are detailed for their ability to suppress BMAL and IL-6 expression from human cells while also demonstrating excellent selectivity over LXRα. Amine 4 demonstrated in vivo bioavailability after either iv or oral dosing.

Intestinal detoxification limits the activation of hepatic pregnane X receptor by lithocholic acid.

The intestinal-derived secondary bile acid (BA) lithocholic acid (LCA) is hepatotoxic and is implicated in the pathogenesis of cholestatic diseases. LCA is an endogenous ligand of the xenobiotic nuclear receptor pregnane X receptor (PXR), but there is currently no consensus on the respective roles of hepatic and intestinal PXR in mediating protection against LCA in vivo. Under the conditions reported here, we show that mice lacking Pxr are resistant to LCA-mediated hepatotoxicity. This unexpected phenotype is found in association with enhanced urinary BA excretion and elevated basal expression of drug metabolism enzymes and the hepatic sulfate donor synthesis enzyme Papss2 in Pxr(−/−) mice. By subsequently comparing molecular responses to dietary and intraperitoneal administration of LCA, we made two other significant observations: 1) LCA feeding induces intestinal, but not hepatic, drug-metabolizing enzymes in a largely Pxr-independent manner; and 2) in contrast to LCA feeding, bypassing first-pass gut transit by intraperitoneal administration of LCA did induce hepatic detoxification machinery and in a Pxr-dependent manner. These data reconcile important discrepancies in the reported molecular responses to this BA and suggest that Pxr plays only a limited role in mediating responses to gut-derived LCA. Furthermore, the route of administration must be considered in the future planning and interpretation of experiments designed to assess hepatic responses to BAs, orally administered pharmaceuticals, and dietary toxins.

The application of BacMam technology in nuclear receptor drug discovery

The nuclear receptor (NR) superfamily represents a major class of drug targets for the pharmaceutical industry. Strategies for the development of novel, more selective and safer compounds aimed at these receptors are now emerging. Reporter assays have been used routinely for the identification and characterisation of NR ligands. As the NR drug development process evolves, the increase in screening demand in terms of both capacity and complexity has necessitated the development of novel assay formats with increased throughput and flexibility. BacMam technology, a modified baculovirus system for over-expressing genes of interest in mammalian cells has helped answer this requirement. BacMam has many advantages over traditional gene delivery systems including high transduction efficiencies, broad cell host range, speed, cost and ease of generation and use. As outlined in this review, the technology has shown itself to be robust and efficient in various NR assay formats including transactivation (ER alpha/beta, MR, PR and PXR) and transrepression (GR-NFkappaB). In addition, the flexibility of this system will allow greater multiplexing of receptor, reporter, and cell host combinations as NR assays become more complex in order to relate better to relevant cellular and biological systems.

Sequencing and functional assessment of hPXR (NR1I2) variants in intrahepatic cholestasis of pregnancy.

1. The purpose of this study was to evaluate the role of coding variation in hPXR (NR1I2) in intrahepatic cholestasis of pregnancy (ICP) and to functionally asses the response of PXR variants to ligands of interest in ICP. 2. The coding region of hPXR was sequenced in a cohort of 121 Caucasian ICP patients and exon 2 was sequenced in an additional 226 cases. Reporter assays were used to evaluate the function of all known hPXR variants in response to the secondary bile acid lithocholic acid and therapeutic agents rifampicin, ursodeoxycholic acid and dexamethasone. 3. Two coding single nucleotide polymorphisms (C79T and G106A) were detected in the ICP cohort at frequencies consistent with healthy populations. These do not alter hPXR function in response to ligands of interest to ICP. Analysis of all known coding hPXR variants demonstrates that while subtle changes in experimental design mask or may unveil the functional effects of genetic variation, these are not maintained in a standard functional assay. 4. Coding genetic variation in hPXR does not contribute to the aetiology of ICP in Caucasian populations.

Interlink between Nuclear Receptors, Posttranslational Modifications and the Biological Clock in Health and Diseases

The day and night cycle influences the body function in health and disease through the modulation of the biological clock in via the suprachiasmatic nucleus (SCN) which in turn acts as the master clock in aligning the downstream clocks in the peripheral organs. The peripheral clocks can also be entrained independently by different factors such as external nutritional, hormonal and chemical cues that in many cases come from ligand modulation of nuclear receptors and kinase activators. Disruption of the molecular clock is associated with many diseases including cancer, immune diseases and aging. In addition, administration of toxic drugs such as anti-cancer can disturb the clock. These examples lead to changes in the sleep pattern of the patient and consequently can accelerate the manifestation of disease. This review details the function of the molecular clock in health and disease and how nuclear receptors and posttranslational modifications interact with the clock to regulate its function. In addition this review describes how chronotherapy can be applied in the clinical setting to treat cancer patients