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Allosteric effects on substrate dissociation from cytochrome P450 3A4 in nanodiscs observed by ensemble and single-molecule fluorescence spectroscopy.

ABSTRACT: Cytochrome P450 (CYP) 3A4 is a major human drug-metabolizing enzyme and displays pharmacologically relevant allosteric kinetics caused by multiple substrate and/or effector binding. Here, in the first single-molecule (SM) fluorescence studies of CYPs, we use total internal reflection fluorescence microscopy to measure residence times of the fluorescent dye Nile Red in CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, with and without the effector alpha-naphthoflavone. We find direct evidence that CYP3A4 effectors can decrease substrate off-rates, providing a possible mechanism for effector-mediated enhancement of substrate metabolism. These interesting results highlight the potential of SM methods in studies of CYP allosteric mechanisms.

SUBMITTER: Nath A

PROVIDER: S-EPMC2649694 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Allosteric effects on substrate dissociation from cytochrome P450 3A4 in nanodiscs observed by ensemble and single-molecule fluorescence spectroscopy.

Nath Abhinav A Koo Peter K PK Rhoades Elizabeth E Atkins William M WM

Journal of the American Chemical Society 20081101 47

Cytochrome P450 (CYP) 3A4 is a major human drug-metabolizing enzyme and displays pharmacologically relevant allosteric kinetics caused by multiple substrate and/or effector binding. Here, in the first single-molecule (SM) fluorescence studies of CYPs, we use total internal reflection fluorescence microscopy to measure residence times of the fluorescent dye Nile Red in CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, with and without the effector alpha-naphthoflavone. We find direct ev ...[more]

PMID: 18980315

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Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

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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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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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