The Experts below are selected from a list of 7611 Experts worldwide ranked by ideXlab platform
Thomas L Poulos - One of the best experts on this subject based on the ideXlab platform.
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Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions
Advances in Experimental Medicine and Biology, 2020Co-Authors: Irina F Sevrioukova, Thomas L PoulosAbstract:Cytochrome P450 3A4 (CYP3A4) is the major and most important drug-metabolizing enzyme in humans that oxidizes and clears over a half of all administered pharmaceuticals. This is possible because CYP3A4 is promiscuous with respect to substrate binding and has the ability to catalyze diverse oxidative chemistries in addition to traditional hydroxylation reactions. Furthermore, CYP3A4 binds and oxidizes a number of substrates in a cooperative manner and can be both induced and inactivated by drugs. In vivo, CYP3A4 inhibition could lead to undesired drug-drug interactions and drug toxicity, a major reason for late-stage clinical failures and withdrawal of marketed pharmaceuticals. Owing to its central role in drug metabolism, many aspects of CYP3A4 catalysis have been extensively studied by various techniques. Here, we give an overview of experimental and theoretical methods currently used for investigation and prediction of CYP3A4-ligand interactions, a defining factor in drug metabolism, with an emphasis on the problems addressed and conclusions derived from the studies.
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Ritonavir analogues as a probe for deciphering the Cytochrome P450 3A4 inhibitory mechanism
Current Topics in Medicinal Chemistry, 2020Co-Authors: Irina F Sevrioukova, Thomas L PoulosAbstract:Inactivation of human drug-metabolizing Cytochrome P450 3A4 (CYP3A4) could lead to serious adverse events such as drug-drug interactions and toxicity. However, when properly controlled, CYP3A4 inhibition may be beneficial as it can improve clinical efficacy of co-administered therapeutics that otherwise are quickly metabolized by CYP3A4. Currently, the CYP3A4 inhibitor ritonavir and its derivative cobicistat are prescribed to HIV patients as pharmacoenhancers. Both drugs were designed based on the chemical structure/activity relationships rather than the CYP3A4 crystal structure. To unravel the structural basis of CYP3A4 inhibition, we compared the binding modes of ritonavir and ten analogues using biochemical, mutagenesis and x-ray crystallography techniques. This review summarizes our findings on the relative contribution of the heme-ligating moiety, side chains and the terminal group of ritonavir-like molecules to the ligand binding process, and highlights strategies for a structure-guided design of CYP3A4 inactivators.
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conformational response of n terminally truncated Cytochrome P450 3A4 to ligand binding in solution
Biochemistry, 2019Co-Authors: Shihwei Chuo, Shuhao Liou, Leeping Wang, David R Britt, Thomas L Poulos, Irina F Sevrioukova, David B GoodinAbstract:Human Cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism ...
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structural basis for regiospecific midazolam oxidation by human Cytochrome P450 3A4
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Irina F Sevrioukova, Thomas L PoulosAbstract:Human Cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure–function relations in this important enzyme, improving the outcomes for drug metabolism predictions, and developing pharmaceuticals that have a decreased ability to undergo metabolism and cause detrimental drug–drug interactions. However, there is very limited structural information on CYP3A4–substrate interactions available to date. Despite the vast variety of drugs undergoing metabolism, only the sedative midazolam (MDZ) serves as a marker substrate for the in vivo activity assessment because it is preferentially and regioselectively oxidized by CYP3A4. We solved the 2.7 A crystal structure of the CYP3A4–MDZ complex, where the drug is well defined and oriented suitably for hydroxylation of the C1 atom, the major site of metabolism. This binding mode requires H-bonding to Ser119 and a dramatic conformational switch in the F–G fragment, which transmits to the adjacent D, E, H, and I helices, resulting in a collapse of the active site cavity and MDZ immobilization. In addition to providing insights on the substrate-triggered active site reshaping (an induced fit), the crystal structure explains the accumulated experimental results, identifies possible effector binding sites, and suggests why MDZ is predominantly metabolized by the CYP3A enzyme subfamily.
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dissecting Cytochrome P450 3A4 ligand interactions using ritonavir analogues
Biochemistry, 2013Co-Authors: Irina F Sevrioukova, Thomas L PoulosAbstract:Cytochrome P450 3A4 (CYP3A4) inhibitors ritonavir and cobicistat, currently administered to HIV patients as pharmacoenhancers, were designed on the basis of the chemical structure/activity relationships rather than the CYP3A4 crystal structure. To better understand the structural basis for CYP3A4 inhibition and the ligand binding process, we investigated five desoxyritonavir analogues to elucidate how substitution/elimination of the phenyl side groups (Phe-1 and Phe-2) and removal of the isopropyl-thiazole (IPT) moiety affect affinity, inhibitory potency, and the ligand binding mode. Our experimental and structural data indicate that the side group size reduction not only drastically lowers affinity and inhibitory potency for CYP3A4 but also leads to multiple binding modes. Regardless of the side group chemical nature and the number of molecules bound, the space adjacent to the 369–371 peptide and Arg105 (Phe-2 site) is always occupied and, hence, must be a critically important binding site. When possible...
Irina F Sevrioukova - One of the best experts on this subject based on the ideXlab platform.
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Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions
Advances in Experimental Medicine and Biology, 2020Co-Authors: Irina F Sevrioukova, Thomas L PoulosAbstract:Cytochrome P450 3A4 (CYP3A4) is the major and most important drug-metabolizing enzyme in humans that oxidizes and clears over a half of all administered pharmaceuticals. This is possible because CYP3A4 is promiscuous with respect to substrate binding and has the ability to catalyze diverse oxidative chemistries in addition to traditional hydroxylation reactions. Furthermore, CYP3A4 binds and oxidizes a number of substrates in a cooperative manner and can be both induced and inactivated by drugs. In vivo, CYP3A4 inhibition could lead to undesired drug-drug interactions and drug toxicity, a major reason for late-stage clinical failures and withdrawal of marketed pharmaceuticals. Owing to its central role in drug metabolism, many aspects of CYP3A4 catalysis have been extensively studied by various techniques. Here, we give an overview of experimental and theoretical methods currently used for investigation and prediction of CYP3A4-ligand interactions, a defining factor in drug metabolism, with an emphasis on the problems addressed and conclusions derived from the studies.
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Ritonavir analogues as a probe for deciphering the Cytochrome P450 3A4 inhibitory mechanism
Current Topics in Medicinal Chemistry, 2020Co-Authors: Irina F Sevrioukova, Thomas L PoulosAbstract:Inactivation of human drug-metabolizing Cytochrome P450 3A4 (CYP3A4) could lead to serious adverse events such as drug-drug interactions and toxicity. However, when properly controlled, CYP3A4 inhibition may be beneficial as it can improve clinical efficacy of co-administered therapeutics that otherwise are quickly metabolized by CYP3A4. Currently, the CYP3A4 inhibitor ritonavir and its derivative cobicistat are prescribed to HIV patients as pharmacoenhancers. Both drugs were designed based on the chemical structure/activity relationships rather than the CYP3A4 crystal structure. To unravel the structural basis of CYP3A4 inhibition, we compared the binding modes of ritonavir and ten analogues using biochemical, mutagenesis and x-ray crystallography techniques. This review summarizes our findings on the relative contribution of the heme-ligating moiety, side chains and the terminal group of ritonavir-like molecules to the ligand binding process, and highlights strategies for a structure-guided design of CYP3A4 inactivators.
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conformational response of n terminally truncated Cytochrome P450 3A4 to ligand binding in solution
Biochemistry, 2019Co-Authors: Shihwei Chuo, Shuhao Liou, Leeping Wang, David R Britt, Thomas L Poulos, Irina F Sevrioukova, David B GoodinAbstract:Human Cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism ...
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Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6',7'-Dihydroxybergamottin.
International Journal of Molecular Sciences, 2019Co-Authors: Irina F SevrioukovaAbstract:Human Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme. Some drugs and natural compounds can act as suicide (mechanism-based) inactivators of CYP3A4, leading to unanticipated drug-drug interactions, toxicity and therapeutic failures. Despite significant clinical and toxicological implications, the mechanism-based inactivation remains incompletely understood. This study provides the first direct insights into the interaction of CYP3A4 with three suicide substrates: mibefradil, an antihypertensive drug quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6′,7′-dihydroxybergamottin. Novel structural findings help better understand the suicide substrate binding and inhibitory mechanism, and can be used to improve the predictability of the binding ability, metabolic sites and inhibitory/inactivation potential of newly developed drugs and other chemicals relevant to public health.
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High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4.
Biochemistry, 2017Co-Authors: Irina F SevrioukovaAbstract:Human drug-metabolizing Cytochrome P450 3A4 (CYP3A4) is a dynamic enzyme with a large and highly malleable active site that can fit structurally diverse compounds. Despite extensive investigations, structure–function relationships and conformational dynamics in CYP3A4 are not fully understood. This study was undertaken to engineer a well-expressed and functionally active cysteine-depleted CYP3A4 that can be used in biochemical and biophysical studies. cDNA codon optimization and screening mutagenesis were utilized to boost the level of bacterial expression of CYP3A4 and identify the least harmful substitutions for all six non-heme-ligating cysteines. The C58A/C64M/C98A/C239T/C377A/C468S (Cys-less) mutant was found to be expressed as highly as the optimized wild-type (opt-WT) CYP3A4. The high-resolution X-ray structures of opt-WT and Cys-less CYP3A4 revealed that gene optimization leads to a different folding in the Phe108 and Phe189 regions and promotes binding of the active site glycerol that interlocks ...
Kyoung Tai No - One of the best experts on this subject based on the ideXlab platform.
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new combined model for the prediction of regioselectivity in Cytochrome P450 3A4 mediated metabolism
Journal of Chemical Information and Modeling, 2008Co-Authors: Won Seok Oh, Jihoon Jung, Kyoung Tai NoAbstract:Cytochrome P450 3A4 metabolizes nearly 50% of the drugs currently in clinical use with a broad range of substrate specificity. Early prediction of metabolites of xenobiotic compounds is crucial for cost efficient drug discovery and development. We developed a new combined model, MLite, for the prediction of regioselectivity in the Cytochrome P450 3A4 mediated metabolism. In the model, the ensemble catalyticphore- based docking method was implemented for the accessibility prediction, and the activation energy estimation method of Korzekwa et al. was used for the reactivity prediction. Four major metabolic reactions, aliphatic hydroxylation, N-dealkylation, O-dealkylation, and aromatic hydroxylation reaction, were included and the reaction data, metabolite information, were collected for 72 well-known substrates. The 47 drug molecules were used as the training set, and the 25 well-known substrates were used as the test set for the ensemble catalyticphore-based docking method. MLite predicted correctly about...
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New Combined Model for the Prediction of Regioselectivity in Cytochrome P450/3A4 Mediated Metabolism
Journal of Chemical Information and Modeling, 2008Co-Authors: Won Seok Oh, Jihoon Jung, Kyoung Tai NoAbstract:Cytochrome P450 3A4 metabolizes nearly 50% of the drugs currently in clinical use with a broad range of substrate specificity. Early prediction of metabolites of xenobiotic compounds is crucial for cost efficient drug discovery and development. We developed a new combined model, MLite, for the prediction of regioselectivity in the Cytochrome P450 3A4 mediated metabolism. In the model, the ensemble catalyticphore- based docking method was implemented for the accessibility prediction, and the activation energy estimation method of Korzekwa et al. was used for the reactivity prediction. Four major metabolic reactions, aliphatic hydroxylation, N-dealkylation, O-dealkylation, and aromatic hydroxylation reaction, were included and the reaction data, metabolite information, were collected for 72 well-known substrates. The 47 drug molecules were used as the training set, and the 25 well-known substrates were used as the test set for the ensemble catalyticphore-based docking method. MLite predicted correctly about...
James R Halpert - One of the best experts on this subject based on the ideXlab platform.
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the structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human Cytochrome P450 3A4
Biochemistry, 2011Co-Authors: Arthur G Roberts, James R Halpert, Sidney D. Nelson, Jing Yang, Kenneth T Thummel, William M. AtkinsAbstract:Human Cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1′-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1′-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T1 nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T1 NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ mo...
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Multiple substrate-binding sites are retained in Cytochrome P450 3A4 mutants with decreased cooperativity
Xenobiotica, 2010Co-Authors: Harshica Fernando, James R Halpert, Jessica A. O. Rumfeldt, Nadezhda Y. Davydova, Dmitri R. DavydovAbstract:The basis of decreased cooperativity in substrate binding in the Cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy.Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (nH) around 1.7 (S50 = 11.7 µM), the mutants showed no cooperativity (nH ≤ 1.1) with unchanged S50 values.Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high-affinity interaction is characterized by a dissociation constant (KD) ≤ 1.0 µM, whereas the KD of the second binding has the same magnitude as the S50.Theoretical analysis of a two-step binding model suggests that nH values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event.Alteration of cooperativity in the mutants is caused by a partial displacement of the “spin-shifting...
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Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4: The Stoichiometry of the Enzyme−Substrate Complexes Probed by FRET and Job's Titration†
Biochemistry, 2006Co-Authors: Harshica Fernando, James R Halpert, Dmitri R. DavydovAbstract:To explore the mechanism of homotropic cooperativity in human Cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and brom...
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Kinetics of dithionite-dependent reduction of Cytochrome P450 3A4: heterogeneity of the enzyme caused by its oligomerization.
Biochemistry, 2005Co-Authors: Dmitri R. Davydov, Stephen G. Sligar, Bradley J. Baas, Harshica Fernando, James R HalpertAbstract:To explore the basis of apparent conformational heterogeneity of Cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decre...
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structures of Cytochrome P450 3A4
Trends in Biochemical Sciences, 2005Co-Authors: Emily E Scott, James R HalpertAbstract:Cytochrome P450 3A4 (CYP3A4) catalyzes the initial step in the clearance of many pharmaceuticals and foreign chemicals. The structurally diverse nature of CYP3A4 substrates complicates rational prediction of their metabolism and identification of potential drug interactions. The first molecular structures of human CYP3A4 were recently determined, revealing an active site of sufficient size and topography to accommodate either large ligands or multiple smaller ligands, as suggested by the heterotropic and homotropic cooperativity of the enzyme.
William M. Atkins - One of the best experts on this subject based on the ideXlab platform.
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the structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human Cytochrome P450 3A4
Biochemistry, 2011Co-Authors: Arthur G Roberts, James R Halpert, Sidney D. Nelson, Jing Yang, Kenneth T Thummel, William M. AtkinsAbstract:Human Cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1′-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1′-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T1 nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T1 NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ mo...
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allosteric activation of Cytochrome P450 3A4 by α naphthoflavone branch point regulation revealed by isotope dilution analysis
Biochemistry, 2011Co-Authors: Caleb M Woods, Cristina Fernandez, Kent L. Kunze, William M. AtkinsAbstract:Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic metabolizing CYP. Despite great interest in CYP enzymology, two in vitro aspects of CYP3A4 catalysis are still not well understood, namely, sequential metabolism and allosteric activation. We have therefore investigated such a system in which both phenomena are present. Here we report that the sequential metabolism of Nile Red (NR) is accelerated by the heterotropic allosteric effector α-naphthoflavone (ANF). ANF increases the rates of formation for NR metabolites M1 and M2 and also perturbs the metabolite ratio in favor of M2. Thus, ANF has as an allosteric effect on a kinetic branch point. Co-incubating deuterium-labeled NR and unlabeled M1, we show that ANF increases kcat/koff ∼1.8-fold in favor of the kcat of M2 production. Steady-state metabolic experiments are analyzed using a kinetic model in which the enzyme and substrates are not in rapid equilibrium, and this distinction allows for the estimation of rates of catalysis for the formation of b...
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Nile Red is a fluorescent allosteric substrate of Cytochrome P450 3A4.
Biochemistry, 2007Co-Authors: Jed N. Lampe, Cristina Fernandez, And Abhinav Nath, William M. AtkinsAbstract:Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 μM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized ...
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Time-resolved fluorescence studies of heterotropic ligand binding to Cytochrome P450 3A4
Biochemistry, 2006Co-Authors: Jed N. Lampe, William M. AtkinsAbstract:Cytochrome P450 3A4 (CYP3A4) is a major enzymatic determinant of drug and xenobiotic metabolism that demonstrates remarkable substrate diversity and complex kinetic properties. The complex kinetics may result, in some cases, from multiple binding of ligands within the large active site or from an effector molecule acting at a distal allosteric site. Here, the fluorescent probe TNS (2-p-toluidinylnaphthalene-6-sulfonic acid) was characterized as an active site fluorescent ligand. UV−vis difference spectroscopy revealed a TNS-induced low-spin heme absorbance spectrum with an apparent Kd of 25.4 ± 2 μM. Catalytic turnover using 7-benzyloxyquinoline (7-BQ) as a substrate demonstrated TNS-dependent inhibition with an IC50 of 9.9 ± 0.1 μM. These results suggest that TNS binds in the CYP3A4 active site. The steady-state fluorescence of TNS increased upon binding to CYP3A4, and fluorescence titrations yielded a Kd of 22.8 ± 1 μM. Time-resolved frequency-domain measurement of TNS fluorescence lifetimes indicates a...
