William Cairns

Cloning and functional expression of a human orthologue of rat vanilloid receptor-1

&NA; Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor‐1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor‐1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor‐1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

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

Characterization of the human HCN1 channel and its inhibition by capsazepine

The human hyperpolarization‐activated cyclic nucleotide‐gated 1 (hHCN1) subunit was heterologously expressed in mammalian cell lines (CV‐1 and CHO) and its properties investigated using whole‐cell patch‐clamp recordings. Activation of this recombinant channel, by membrane hyperpolarization, generated a slowly activating, noninactivating inward current. The pharmacological properties of hHCN1‐mediated currents resembled those of native hyperpolarization‐activated currents (Ih), that is, blockade by Cs+ (99% at 5 mM), ZD 7288 (98% at 100 μM) and zatebradine (92% at 10 μM). Inhibition of the hHCN1‐mediated current by ZD 7288 was apparently independent of prior channel activation (i.e. non‐use‐dependent), whereas that induced by zatebradine was use‐dependent. The VR1 receptor antagonist capsazepine inhibited hHCN1‐mediated currents in a concentration‐dependent (IC50=8 μM), reversible and apparently non‐use‐dependent manner. This inhibitory effect of capsazepine was voltage‐independent and associated with a leftward shift in the hHCN1 activation curve as well as a dramatic slowing of the kinetics of current activation. Elevation of intracellular cAMP or extracellular K+ significantly enhanced aspects of hHCN1 currents. However, these manipulations did not significantly affect the capsazepine‐induced inhibition of hHCN1. The development of structural analogues of capsazepine may yield compounds that could selectively inhibit HCN channels and prove useful for the treatment of neurological disorders where a role for HCN channels has been described.

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.

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.

Evaluation of Cell-Based Assays for Steroid Nuclear Receptors Delivered by Recombinant Baculoviruses

The authors describe the use of modified baculoviruses containing mammalian expression cassettes (BacMam technology) in steroid nuclear receptor reporter assays designed for screening and profiling agonist and antagonist compounds. Baculo-viruses were constructed that express full-length human genes for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), progesterone receptor A (PR-A), and progesterone receptor B (PR-B) from the cytomegalovirus immediate early promoter. A virus carrying the mouse mammary tumor virus–firefly luciferase (MMTV-Luc) cassette was generated to provide a suitable reporter construct. Feasibility studies with BacMam-MR in single-dose tests of 1000 compounds showed high correlation to the standard transfection-based assay results. Likewise, in dose-response experiments, BacMam-based assays for GR and PR-B produced potency and efficacy values similar to transfection assay results. At various receptor/reporter ratios, the BacMam assays showed good flexibility, demonstrating consistent signal-to-background (S/B) ratios and compound potencies. Increasing transduction time from 24 to 48 h provided no benefit, actually reducing overall assay performance as measured by S/B and Z′ values. The BacMam technology was applied in studies of isoforms PR-A and PR-B, which showed similar responses to a series of agonists. Taken together, the results demonstrate the utility of steroid nuclear receptor BacMam constructs for compound screening procedures with high reproducibility, reduced turnaround time, and lower cost.

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.

The identification a novel, selective, non-steroidal, functional glucocorticoid receptor antagonist.

The identification of novel, potent, non-steroidal/small molecule functional GR antagonist GSK1564023A selective over PR is described. Associated structure-activity relationships and the process of optimisation of an initial HTS hit are also described.