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Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations

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Departments of Medicinal Chemistry and Pharmaceutics, Box 357610, University of Washington, Seattle, Washington 98195-7610, and Icos Corporation, Bothell, Washington 98021
Cite this: Biochemistry 2006, 45, 20, 6341–6353
Publication Date (Web):April 28, 2006
https://doi.org/10.1021/bi0600042
Copyright © 2006 American Chemical Society

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    The heme-containing cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug−drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low-spin heme iron complexes via formation of a nitrogen−ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is ∼25 Å from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed, and one of the two binding components resolved by curve fitting exhibits “equilibrium overshoot”. Preloading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV−vis difference absorbance titrations yield low-spin spectra and KD values that are consistent with the high-affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly dissociating inhibitory mode. Most importantly, they provide the first example of a SPR-based method for the kinetic characterization of binding of a drug to any human CYP, including mechanistic insight not available from other methods.

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     This work was supported by NIH Grants GM32165 (W.M.A., N.I., and K.L.K.) and GM07750 (J.T.P.).

     Department of Medicinal Chemistry, University of Washington.

    §

     Icos Corp.

     Department of Pharmaceutics, University of Washington.

    *

     To whom correspondence should be addressed. Telephone:  (206) 685-0379. Fax:  (206) 685-3252. E-mail:  [email protected].

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    Preparation and loading of chips, as well as demonstration of their stability, and algebraic formalism for the equivalence of the parallel binding trajectory model and the analyte heterogeneity model (or parallel binding trajectory model) found in the BIAevaluation software. This material is available free of charge via the Internet at http://pubs.acs.org.

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