Differential activation of human constitutive androstane receptor and its SV23 and SV24 splice variants by rilpivirine and etravirine

Abstract

BACKGROUND AND PURPOSERilpivirine and etravirine are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) indicated for the treatment of HIV/AIDS. The constitutive androstane receptor (CAR) regulates the expression of genes involved in various biological processes, including the transport and biotransformation of drugs. We investigated the effect of rilpivirine and etravirine on the activity of the wild-type human CAR (hCAR-WT) and its hCAR-SV23 and hCAR-SV24 splice variants, and compared it with first-generation NNRTIs (efavirenz, nevirapine, and delavirdine). EXPERIMENTAL APPROACHReceptor activation, ligand-binding domain (LBD) transactivation, and co-activator recruitment were investigated in transiently transfected, NNRTI-treated HepG2 cells. Nuclear translocation of green fluorescent protein-tagged hCAR-WT and CYP2B6 gene expression were assessed in NNRTI-treated human hepatocytes. KEY RESULTSRilpivirine and etravirine activated hCAR-WT, but not hCAR-SV23 or hCAR-SV24, and without transactivating the LBD or recruiting steroid receptor coactivators SRC-1, SRC-2, or SRC-3. Among the first-generation NNRTIs investigated, only efavirenz activated hCAR-WT, hCAR-SV23, and hCAR-SV24, but none of them transactivated the LBD of these receptors or substantively recruited SRC-1, SRC-2, or SRC-3. Rilpivirine, etravirine, and efavirenz triggered nuclear translocation of hCAR-WT and increased hCAR target gene (CYP2B6) expression. CONCLUSION AND IMPLICATIONSNNRTIs activate hCAR-WT, hCAR-SV23, and hCAR-SV24 in a drug-specific and isoform-selective manner. The activation occurs by a mechanism that does not appear to involve binding to the LBD or recruitment of SRC-1, SRC-2, or SRC-3. AbbreviationsCAR, constitutive androstane receptor; CAS, Chemical Abstracts Service; CITCO, 6-(4-chlorophenyl)imidazo [2,1-b] [1,3]

Rilpivirine attenuates liver fibrosis through selective STAT1-mediated apoptosis in hepatic stellate cells

“…Importantly, RPV decreased severe liver inflammation and fibrosis progression when administered in combination with the toxic insult, and it reduced hepatic fibrosis and inflammation when administered after consolidation of fibrosis, thus highlighting the potential of this compound to reverse fibrosis. Mechanistic studies revealed an interesting and selective pro-apoptotic effect of RPV in activated HSC through STAT1 signalling, which was not observed in hepatocytes, in accordance with previous reports of a lack of cytotoxic effects in parenchymal cells 18 19. Furthermore, we describe an interesting interplay between HSC and hepatocytes via JAK-STAT signalling, which leads to liver regeneration: RPV treatment also restored the number of proliferative hepatocytes through STAT3 activation, thereby re-establishing parenchymal homeostasis.…”

Small-molecule modulators of the constitutive androstane receptor

“…Many alternatively spliced variants of CAR have been identified [15, 47, 50, 51], with varying tissue expression profiles and different ligand specificities [5, 48, 52–55]. Although there is some preliminary data about the expression levels of the variants of CAR in liver [47, 56], further investigation of organ-specific induction of CAR target genes is needed to understand the tissue-specific variation in drug metabolism and drug–drug interactions.…”

Negative Regulation of Human Pregnane X Receptor by MicroRNA-18a-5p: Evidence for Suppression of MicroRNA-18a-5p Expression by Rifampin and Rilpivirine

“…Total RNA was reverse transcribed and total cDNA concentrations were quantified (Sharma et al, 2013). hPXR cDNA, CYP3A4 cDNA, hypoxanthine phosphoribosyltransferase 1 (HPRT) cDNA, and cyclophilin cDNA were amplified using gene-specific primers (Lau et al, 2011;Sharma et al, 2015) in a StepOnePlus Real-Time PCR System. Each 20 ml of a PCR reaction contained 10 ml of EvaGreen 2Â qPCR MasterMix-ROX, 5 ml of gene-specific primers (0.3 mM final concentration), and 5 ml of the RT product (i.e., 10 ng total cDNA for the amplification of CYP3A4 cDNA and hPXR cDNA, 5 ng total cDNA for the amplification of HPRT cDNA, and 0.5 ng total cDNA for the amplification of cyclophilin cDNA).…”

“…The PCR cycling condition was 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, 55°C for 10 seconds, and 60°C for 50 seconds. The levels of hPXR and CYP3A4 mRNA were quantified and normalized to HPRT mRNA level and cyclophilin mRNA level (Lau et al, 2010;Sharma et al, 2015).…”