Kiumars Shahrokh

University of Utah, Salt Lake City, UT, United States

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Publications (7)15.53 Total impact

  • Kiumars Shahrokh, Thomas E Cheatham, Garold S Yost
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    ABSTRACT: 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. 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. Docking with MD-refined CYP3A4 structures incorporating hexa-coordinate 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. 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.
    Biochimica et Biophysica Acta 06/2012; 1820(10):1605-17. · 4.66 Impact Factor
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    ABSTRACT: 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 indicate 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 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.
    Journal of Computational Chemistry 01/2012; 33(2):119-33. · 3.60 Impact Factor
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    ABSTRACT: 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.
    Journal of Pharmacology and Experimental Therapeutics 02/2011; 337(2):400-10. · 3.89 Impact Factor
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    ABSTRACT: The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 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 improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate-active site residue interactions. On the basis of 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.
    Biochemistry 10/2010; 49(41):9011-9. · 3.38 Impact Factor
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    ABSTRACT: Objectives: We have used P450-specific metabolism of the Selective Estrogen Receptor Modulators (SERM): tamoxifen & raloxifene and of several inhaled glucocorticoids (GC) as molecular probes of competing metabolic pathways by the three CYP3A enzymes to refine an integrated computational approach for predicting drug metabolism and bioactivation. The unstable products of these bioactivation reactions are electrophilic intermediates that are linked to biomolecular adduct formation which results in toxicities that range from altered drug metabolism (mechanism-based inactivation) to carcinogenesis. Methods: To remove structural artifacts potentially introduced by the crystallography conditions, molecular dynamics (MD) simulations were performed with the x-ray structure of CYP3A4 (PDB code: 1TQN) and homology models of CYP3A5 & CYP3A7along multiple trajectories. Each MD trajectory was then subjected to cluster analysis, and one representative structure was generated from the largest cluster of each trajectory. These structures were then modified with quantum mechanics (QM)-based parameters for the heme during different stages of the P450 catalytic cycle and were used as docking templates for multiple SERMs and GCs. Results: An integrated approach of docking using QM optimized substrate structures and MD-refined protein structures modified with QM-based heme parameters produced as the most highly populated conformations those that correctly predicted observed metabolism of SERMs and GCs by the three CYP3A enzymes (CYP3A4/5/7). The accuracy of these models was validated by site-directed mutagenesis and in vitro incubations. Specifically, for CYP3A4,-arginine 212 and for CYP3A5-lysine212 within the active site play important roles in determination of competing reaction mechanisms catalyzed by the CYP3As. Conclusions: An integrated computational approach for the structure-based prediction of metabolism and bioactivation chemically distinct drugs by the three CYP3A enzymes has been developed. This computational approach of greatly improved predictions over x-ray structure-based modelling, and confirmed the importance of accurately modelling the electronic and thermodynamic contributions to both substrate and enzyme structures to predict P450-mediated metabolism of xenobiotics. Supported by NIH grants # GM074249 from the National Institute of General Medical Sciences, NICHD Grant # HD060559 and # NCRR 1 S10 RR17214-01 from the National Center for Research Resources, NSF grant #MCA017S027 and the University of Utah Center for High Performance Computing.
    9th International International society for the study of xenobiotics Meeting;
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    ABSTRACT: Recent x-ray crystallography structures of mammalian P450 enzymes with and without substrates demonstrate highly variable levels of conformational plasticity during P450-substrate binding. In an effort to understand the contribution of substrate binding and crystallographic packing forces to the observed x-ray structures, we have performed a series of 100 ns-scale molecular dynamics (MD) simulations on three different crystal structures of the CYP2B4 isozyme. CYP2B4 displays some of the largest-scale rearrangements with different x-ray crystallography conditions. Our simulations of a single ligand-free monomeric unit of 2B4 in the "open" structure from the crystallographic dimer (PDB code: 1PO5), bound to a small ligand: 4-(4-chlorophenyl)imidazole (PDB code: 1SUO), and bound with a larger ligand: bifonazole (PDB code: 2BDM) suggest that the 1PO5 structure adopts a stable conformation different from the crystal conformation The 2BDM structure is unstable and adopts a conformation with a collapsed active-site, while the 1SUO conformation is stable with and without substrate. Our MD simulations of these structures with and without substrates also provide important insights into the role of classically defined substrate recognition sequences and newly identified plastic regions. Supported by NIH grant # GM074249, NIH grant # NCRR 1 S10 RR17214-01 and the Arches Metacluster at the University of Utah Center for High Performance Computing.
    15th North American Regional International society for the study of xenobiotics Meeting;