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Kinetics of cytochrome P450 3A4 inhibition by heterocyclic drugs defines a general sequential multistep binding process.

ABSTRACT: Cytochrome P450 (P450) 3A4 is the enzyme most involved in the metabolism of drugs and can also oxidize numerous steroids. This enzyme is also involved in one-half of pharmacokinetic drug-drug interactions, but details of the exact mechanisms of P450 3A4 inhibition are still unclear in many cases. Ketoconazole, clotrimazole, ritonavir, indinavir, and itraconazole are strong inhibitors; analysis of the kinetics of reversal of inhibition with the model substrate 7-benzoyl quinoline showed lag phases in several cases, consistent with multiple structures of P450 3A4 inhibitor complexes. Lags in the onset of inhibition were observed when inhibitors were added to P450 3A4 in 7-benzoyl quinoline O-debenzylation reactions, and similar patterns were observed for inhibition of testosterone 6β-hydroxylation by ritonavir and indinavir. Upon mixing with inhibitors, P450 3A4 showed rapid binding as judged by a spectral shift with at least partial high-spin iron character, followed by a slower conversion to a low-spin iron-nitrogen complex. The changes were best described by two intermediate complexes, one being a partial high-spin form and the second another intermediate, with half-lives of seconds. The kinetics could be modeled in a system involving initial loose binding of inhibitor, followed by a slow step leading to a tighter complex on a multisecond time scale. Although some more complex possibilities cannot be dismissed, these results describe a system in which conformationally distinct forms of P450 3A4 bind inhibitors rapidly and two distinct P450-inhibitor complexes exist en route to the final enzyme-inhibitor complex with full inhibitory activity.

SUBMITTER: Guengerich FP

PROVIDER: S-EPMC7948456 | biostudies-literature |

REPOSITORIES: biostudies-literature

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N-Heterocyclic Carbene Capture by Cytochrome P450 3A4.

Project description:Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH <7.4, the spectra are definitively type I, whereas at pH ?7.4 the spectra have split Soret bands, including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development.

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Comparative study of the effects of antituberculosis drugs and antiretroviral drugs on cytochrome P450 3A4 and P-glycoprotein.

Project description:Predicting drug-drug interactions (DDIs) related to cytochrome P450 (CYP), such as CYP3A4 and one of the major drug transporters, P-glycoprotein (P-gp), is crucial in the development of future chemotherapeutic regimens to treat tuberculosis (TB) and TB/AIDS coinfection cases. We evaluated the effects of 30 anti-TB drugs, novel candidates, macrolides, and representative antiretroviral drugs on human CYP3A4 activity using a commercially available screening kit for CYP3A4 inhibitors and a human hepatocyte, HepaRG. Moreover, in order to estimate the interactions of these drugs with human P-gp, screening for substrates was performed. For some substrates, P-gp inhibition tests were carried out using P-gp-expressing MDCK cells. As a result, almost all the compounds showed the expected effects on human CYP3A4 both in the in vitro screening and in HepaRG cells. Importantly, the unproven mechanisms of DDIs caused by WHO group 5 drugs, thioamides, and p-aminosalicylic acid were elucidated. Intriguingly, clofazimine (CFZ) exhibited weak inductive effects on CYP3A4 at >0.25 ?M in HepaRG cells, while an inhibitory effect was observed at 1.69 ?M in the in vitro screening, suggesting that CFZ autoinduces CYP3A4 in the human liver. Our method, based on one of the pharmacokinetics parameters in humans, provides more practical information associated with not only DDIs but also with drug metabolism.

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Project description:Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic-metabolizing enzyme in the liver and intestine and is involved in the disposition of more than 50% of drugs. Because of its ability to bind multiple substrates, its reaction kinetics are complex, and its association with the microsomal membrane confounds our understanding of how this enzyme recognizes and recruits diverse substrates. Testosterone (TST) hydroxylation is the prototypical CYP3A4 reaction, displaying positive homotropic cooperativity with three binding sites. Here, exploiting the capability of accelerated molecular dynamics (aMD) to sample events in the millisecond regime, I performed >25-?s aMD simulations in the presence of three TST molecules. These simulations identified high-occupancy surface-binding sites as well as a pathway for TST ingress into the CYP3A4 active site originating in the membrane. Adaptive biasing force analysis of the latter pathway revealed a metastable intermediate that could constitute a third binding site at high TST concentrations. Prompted by the observation that interactions between TST and the G'-helix mobilize the ligand into the active site, a free-energy analysis of TST distribution in the membrane was conducted and revealed that the depth of the G'-helix is optimal for extracting TST. In summary, these simulations confirm separate, but adjacent substrate-binding sites within the enzyme and the existence of an auxiliary TST-binding site. The broader impact of these simulations is that they support a mechanism in which cytochromes P450 directly recruit membrane-solubilized substrates.

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| S-EPMC3099659 | biostudies-literature

Polymorphism in Cytochrome P450 3A4 Is Ethnicity Related.

Project description:Can mutations in Cytochrome P450 3A4 (CYP3A4), the major food- and drug-metabolizing enzyme, serve as biomarkers for personalized precise medicine? Classical genetic studies provide only limited data regarding the frequencies of CYP3A4 mutations and their role in food-drug interactions. Here, in an analysis of one large database of 141,456 individuals, we found 856 SNPs (single nucleotide polymorphism), of which 312 are missense mutations, far more than the previously reported dozens. Analyzing the data further, it is demonstrated that the frequency of mutations differs among ethnic groups. Hierarchical clustering divided the mutations to seven groups, each corresponding to a specific ethnicity. To the best of our knowledge this is the first comprehensive analysis of CYP3A4 allele frequencies in distinct ethnic groups. We suggest ethnicity based classification of CYP3A4 SNPs as the first step toward precise diet and medicine. Understanding which and when polymorphism might have clinical significance is a tremendously complex task. Using modeling approach, we could predict changes in the binding poses of ligands in the active site of single variants. These changes might imply clinical effects of the overlooked protein-altering CYP3A4 mutations, by modifying drug metabolism and FDI. It may be concluded that dietary habits, and hence FDI, are matters of ethnicity. Consequently, ethnic-related polymorphism in CYP3A4 and diet may be one underlying mechanism of response to medical regimes. The approaches presented here have the power to highlight mutations of clinical relevance in any gene of interest, thus to complement the arsenal of classic genetic screening tools.

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Structural Dynamics of Cytochrome P450 3A4 in the Presence of Substrates and Cytochrome P450 Reductase.

Project description:Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen-deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein-protein interactions (helix C) and substrate binding and specificity (helices B' and E, and loop K/β1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes.

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Project description:Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of approximately 50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4-ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal structures of human CYP3A4 in complex with two well characterized drugs: ketoconazole and erythromycin. In contrast to previous reports, the protein undergoes dramatic conformational changes upon ligand binding with an increase in the active site volume by >80%. The structures represent two distinct open conformations of CYP3A4 because ketoconazole and erythromycin induce different types of coordinate shifts. The binding of two molecules of ketoconazole to the CYP3A4 active site and the clear indication of multiple binding modes for erythromycin has implications for the interpretation of the atypical kinetic data often displayed by CYP3A4. The extreme flexibility revealed by the present structures also challenges any attempt to apply computational design tools without the support of relevant experimental data.

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A Combined Molecular Docking/Dynamics Approach to Probe the Binding Mode of Cancer Drugs with Cytochrome P450 3A4.

Project description:Cytarabine, daunorubicin, doxorubicin and vincristine are clinically used for combinatorial therapies of cancers in different combinations. However, the knowledge about the interaction of these drugs with the metabolizing enzyme cytochrome P450 is limited. Therefore, we utilized computational methods to predict and assess the drug-binding modes. In this study, we performed docking, MD simulations and free energy landscape analysis to understand the drug-enzyme interactions, protein domain motions and the most populated free energy minimum conformations of the docked protein-drug complexes, respectively. The outcome of docking and MD simulations predicted the productive, as well as the non-productive binding modes of the selected drugs. Based on these interaction studies, we observed that S119, R212 and R372 are the major drug-binding residues in CYP3A4. The molecular mechanics Poisson-Boltzmann surface area analysis revealed the dominance of hydrophobic forces in the CYP3A4-drug association. Further analyses predicted the residues that may contain favorable drug-specific interactions. The probable binding modes of the cancer drugs from this study may extend the knowledge of the protein-drug interaction and pave the way to design analogs with reduced toxicity. In addition, they also provide valuable insights into the metabolism of the cancer drugs.

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