WITHDRAWN: Fibulin 2, a tyrosine o-sulfated protein, is up-regulated following retinal detachment.

The manuscript has been withdrawn by the author.

A systematic analysis of predicted phosphorylation sites within the human pregnane X receptor protein.

The pregnane X receptor (PXR, NR1I2) regulates the expression of genes that encode drug-metabolizing enzymes and drug transporter proteins in liver and intestine. Understanding the molecular mechanisms that modulate PXR activity is therefore critical for the development of effective therapeutic strategies. Several recent studies have implicated the activation of kinase signaling pathways in the regulation of PXR biological activity, although direct evidence and molecular mechanisms are currently lacking. We therefore sought to characterize potential phosphorylation sites within the PXR protein by use of a rational, comprehensive, and systematic site-directed mutagenesis approach to generate phosphomimetic mutations (Ser/Thr --> Asp) and phospho-deficient mutations (Ser/Thr --> Ala) at 18 predicted consensus kinase recognition sequences in the human PXR protein. Here, we identify amino acid residues Ser8, Thr57, Ser208, Ser305, Ser350, and Thr408 as being critical for biological activity of the PXR protein. Mutations at positions 57 and 408 abolish ligand-inducible PXR activity. Mutations in the extreme N terminus and in the PXR ligand-binding domain at positions Ser8, Ser305, Ser350, and Thr408 decrease the ability of PXR to form heterodimers with retinoid X receptor alpha. Mutations at positions Ser208, Ser305, Ser350, and Thr408 alter PXR-protein cofactor interactions. Finally, the subcellular localization of the PXR protein is profoundly affected by mutations at position Thr408. These data suggest that PXR activity can potentially be regulated by phosphorylation at specific amino acid residues within several predicted consensus kinase recognition sequences to differentially affect PXR biological activity.

An in vitro evaluation of the victim and perpetrator potential of the anticancer agent laromustine (VNP40101M), based on reaction phenotyping and inhibition and induction of cytochrome P450 enzymes.

Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. Laromustine generates two types of reactive intermediates: 90CE and methylisocyanate. When incubated with rat, dog, monkey, and human liver microsomes, [(14)C]laromustine was converted to 90CE (C-8) and seven other radioactive components (C-1-C-7). There was little difference in the metabolite profile among the species examined, in part because the formation of most components (C-1-C-6 and 90CE) did not require NADPH but involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Laromustine caused direct inhibition of CYP2B6 and CYP3A4/5 (the two enzymes involved in C-7 formation) as well as of CYP2C19. K(i) values were 125 microM for CYP2B6, 297 muM for CYP3A4/5, and 349 microM for CYP2C19 and were greater than the average clinical plasma C(max) of laromustine (25 microM). There was evidence of time-dependent inhibition of CYP1A2, CYP2B6, and CYP3A4/5. Treatment of primary cultures of human hepatocytes with up to 100 microM laromustine did not induce CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5, but the highest concentration of laromustine decreased the activity and levels of immunoreactive CYP3A4. The results of this study suggest the laromustine has 1) negligible victim potential with respect to metabolism by cytochrome P450 enzymes, 2) negligible enzyme-inducing potential, and 3) the potential in some cases to cause inhibition of CYP2B6, CYP3A4, and possibly CYP2C19 during and shortly after the duration of intravenous administration of this anticancer drug, but the clinical effects of such interactions are likely to be insignificant.

Guggulsterone activates multiple nuclear receptors and induces CYP3A gene expression through the pregnane X receptor.

Gugulipid is an extract of the guggul tree, Commiphora mukul, that is used to treat hyperlipidemia in humans. The lipid-lowering activity is found in the stereoisomers and plant sterols Z-guggulsterone and E-guggulsterone. The molecular basis for the lipid-lowering action of guggulsterone has been suggested to be antagonism of the farnesoid X receptor, a member of the nuclear receptor superfamily of ligand-activated transcription factors. To determine whether guggulsterone has the ability to function as an agonist of other nuclear receptor family members, we screened a panel of these proteins for their ability to transactivate reporter genes. Here, we show that guggulsterones activate the estrogen receptor alpha isoform, progesterone receptor, and pregnane X receptor. Concentration-response analysis using reporter gene assays indicate that guggulsterones activate these three receptors with EC(50) values in the low micromolar range. Furthermore, we show that guggulsterone-mediated activation of the pregnane X receptor induces the expression of CYP3A genes in both rodent and human hepatocytes. Protein interaction assays indicate that guggulsterones interact directly with pregnane X receptor, thereby modulating interaction with protein cofactors. We introduce a novel method to screen herbal remedies for their ability to activate pregnane X receptor. Pregnane X receptor activation is known to cause herb-drug interactions, and our data suggest that gugulipid therapy should be used cautiously in patients taking prescription medications that are metabolized by CYP3A family members. Moreover, our data suggest the need for additional studies of guggulsterones agonist activity against estrogen receptor alpha isoform and the progesterone receptor.