Molecular mechanics (MM) methods are computationally affordable tools for screening chemical libraries of novel compounds for sites of P450 metabolism. One challenge for MM methods has been the absence of a consistent and transferable set of parameters for the heme within the P450 active-site. Experimental data indicates that mammalian P450 enzymes vary greatly in the size, architecture, and plasticity of their active sites. Thus, obtaining x-ray based geometries for the development of accurate MM parameters for the major classes of hepatic P450 remains a daunting task. Our previous work with preliminary gas-phase quantum mechanics (QM) derived atomic partial charges, greatly improved the accuracy of docking studies of raloxifene to CYP3A4. We have therefore developed and tested a consistent set of transferable MM parameters based on gas-phase QM calculations of two model systems of the heme—a truncated (T-HM) and a full (F-HM) for four states of the P450 catalytic cycle. Our results indicate that the use of the atomic partial charges from the F-HM model further improves the accuracy of docked predictions for raloxifene to CYP3A4. Different patterns for substrate docking are also observed depending on the choice of heme model and state. Newly parameterized heme models are tested in implicit and explicitly solvated MD simulations in the absence and presence of enzyme structures, for CYP3A4, and appear to be stable on the nanosecond simulation timescale. The new force field for the various heme states may aid the community for simulations of P450 enzymes and other heme containing enzymes.

Activation of intracellular transient receptor potential vanilloid-1 (TRPV1) in human lung cells causes endoplasmic reticulum (ER) stress, increased expression of proapoptotic GADD153 (growth arrest-and DNA damage-inducible transcript 3), and cytotoxicity. However, in cells with low TRPV1 expression, cell death is not inhibited by TRPV1 antagonists, despite preventing GADD153 induction. In this study, chemical variants of the capsaicin analog nonivamide were synthesized and used to probe the relationship between TRPV1 receptor binding, ER calcium release, GADD153 expression, and cell death in TRPV1-overexpressing BEAS-2B, normal BEAS-2B, and primary normal human bronchial epithelial lung cells. Modification of the 3-methoxy-4-hydroxybenzylamide vanilloid ring pharmacophore of nonivamide reduced the potency of the analogs and rendered several analogs mildly inhibitory.Correlation analysis of analog-induced calcium flux, GADD153 induction, and cytotoxicity revealed a direct relationship for all three endpoints in all three lung cell types for nonivamide and N- (3,4-dihydroxybenzyl)nonanamide. However, the N-(3,4-dihydroxybenzyl)nonanamide analog also produced cytotoxicity through redox cycling/reactive oxygen species formation, shown by inhibition of cell death by N-acetylcysteine. Molecular modeling of binding interactions between the analogs and TRPV1 agreed with data for reduced potency of the analogs, and only nonivamide was predicted to form a "productive" ligand-receptor complex. This study provides vital information on the molecular interactions of capsaicinoids with TRPV1 and substantiates TRPV1-mediated ER stress as a conserved mechanism of lung cell death by prototypical TRPV1 agonists.

The use of molecular modeling in conjunction with site-directed mutagenesis has extensively been used to study substrate orientation within cytochrome P450 active sites, and to identify potential residues involved in positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies are highly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (PDB code:1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand-docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety increased accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation, and facilitating the identification of substrate/active site residue interactions. Based on the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme/substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.Cytochrome P450 3A4 (CYP3A4) is the most abundant human P450 found predominately in the liver and intestine, and is responsible for the metabolism of several endogenous compounds and a wide variety of xenobiotics compounds, including greater than 50% of drugs on the market (1). CYP3A4 catalyzes this metabolism by a variety of biochemical reactions, including hydroxylation, epoxidation, dealkylation, and dehydrogenation (desaturation) (2,3), with the potential to employ multiple reactions on the same substrate. Although the majority of CYP3A4-catalyzed reactions produce inactive and nontoxic * Correspondence should be addressed to this author. gyost@pharm.utah.edu Phone: (801) 585-3945. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 October 19. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript metabolites through the oxygenation of substrates, occasionally the less common dehydrogenation reaction generates highly reactive electrophiles that can form intracellular protein or DNA adducts, resulting in toxicities (4-7). Many of...

Background Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available x-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities. 4-hydroxy-tamoxifen (4OHT) is metabolized by CYP3A4 via competing hydroxylation and dehydrogenation reactions. Methods Ab initio gas-phase electronic structural characterization of 4OHT was used to develop a docking scoring scheme. Conformational sampling of CYP3A4 with molecular dynamics simulations along multiple trajectories were used to generate representative structures for docking studies using recently published heme parameters. A key predicted binding mode was tested experimentally using site-directed mutagenesis of CYP3A4 and liquid chromatography mass spectroscopy analysis. Results Docking with MD-refined CYP3A4 structures incorporating hexacoordinate heme parameters identifies a unique binding mode involving ARG212 and channel 4, unobserved in the starting PDB ID: 1TQN x-ray structure. The models supporting dehydrogenation are consistent with results from in vitro incubations. Conclusions and General significance Our models indicate that coupled structural contributions of the ingress, egress and solvent channels to the CYP3A4 active site geometries play key roles in the observed 4OHT binding modes. Thus adequate sampling of the conformational space of these drug-metabolizing promiscuous enzymes is important for substrates that may bind in malleable regions of the enzyme active-site.

Background Broad-spectrum drug screening is offered by many clinical laboratories to support investigation of possible drug exposures. The traditional broad-spectrum drug screen employed at our laboratory utilizes several different analytical platforms, thus requiring relatively high volumes of sample and a cumbersome workflow. Here we describe the development and validation of a consolidated broad-spectrum drug screen assay designed to qualitatively detect 127 compounds in urine (Ur) and serum/plasma (S/P) samples. Methods An LC-MS/MS method was developed using the Ultivo LC-MS/MS and designed to be qualitative with a 1-point calibration curve and 50% to 150% controls. Sample preparation included the addition of 122 internal standards (IS) followed by mixed-mode strong cation exchange solid-phase extraction and reverse-phase chromatographic separation on a biphenyl column. Results For the method described herein, ≥ 95% of analytes in urine and serum control samples had a CV of ≤20% for total imprecision. Accuracy testing included 46 external controls and demonstrated 99.9% accuracy. Method comparison studies to quantitative testing are discussed. The high level of coverage of the analytes with a stable isotope-labeled IS (SIL-IS) helped normalize for matrix effects when significant ion suppression (>25%) was present. Analyte stability in the matrix, the impact of potentially interfering compounds, and method ruggedness were demonstrated. Method limitations include limited detection of glucuronidated drugs and potential cross-contamination with samples at very high concentrations (>>100 × cutoff). Conclusions The broad-spectrum drug screen method developed here qualitatively detected 127 drugs and select metabolites. This method could be used to support investigations of possible drug exposures in a clinical setting.