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Multiple Sequential Steps Involved in the Binding of Inhibitors to Cytochrome P450 3A4

2007; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês

10.1074/jbc.m610346200

ISSN

1083-351X

Autores

Emre M. Isin, F. Peter Guengerich,

Tópico(s)

Drug Transport and Resistance Mechanisms

Resumo

Cytochrome P450 (P450) 3A4 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved. Cytochrome P450 (P450) 3A4 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved. Cytochrome P450 (P450) 2The abbreviations used are: P450, cytochrome P450 (also termed "heme thiolate P450" (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar)); ITC, isothermal titration calorimetry; SVD, singular value decomposition.2The abbreviations used are: P450, cytochrome P450 (also termed "heme thiolate P450" (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar)); ITC, isothermal titration calorimetry; SVD, singular value decomposition. 3A4 is one of the 57 human P450 enzymes (2Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Plenum Publishers, New York2005: 377-530Crossref Scopus (274) Google Scholar) that catalyze mixed-function oxidation reactions (3Ortiz de Montellano P.R. DeVoss J.J. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Plenum Publishers, New York2005: 183-245Crossref Scopus (203) Google Scholar). P450 3A4 is expressed mainly in the intestine (4Zhang Q.-Y. Dunbar D. Ostrowska A. Zeisloft S. Yang J. Kaminsky L. Drug Metab. Dispos. 1999; 27: 804-809PubMed Google Scholar) and liver (5Shimada T. Yamazaki H. Mimura M. Inui Y. Guengerich F.P. J. Pharmacol. Exp. Ther. 1994; 270: 414-423PubMed Google Scholar), where it is the major P450 and is involved in the metabolism of >50% of marketed drugs (6Evans W.E. Relling M.V. Science. 1999; 286: 487-491Crossref PubMed Scopus (2095) Google Scholar, 7Guengerich F.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 1-17Crossref PubMed Scopus (1034) Google Scholar). The capability of P450 3A4 to accommodate and oxidize a large number of structurally diverse drug molecules as well as endogenous substrates has made it an extensively studied enzyme (2Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Plenum Publishers, New York2005: 377-530Crossref Scopus (274) Google Scholar, 8Poulos T.L. Johnson E.F. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Plenum Publishers, New York2005: 87-114Crossref Scopus (110) Google Scholar, 9Williams P.A. Cosme J. Vinkovic D.M. Ward A. Angove H.C. Day P.J. Vonrhein C. Tickle I.J. Jhoti H. Science. 2004; 305: 683-686Crossref PubMed Scopus (709) Google Scholar, 10Yano J.K. Wester M.R. Schoch G.A. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 38091-38094Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 11Ekroos M. Sjögren T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13682-13687Crossref PubMed Scopus (635) Google Scholar). Most of the studies on P450 3A4 aimed at understanding its mechanism are based on steady-state kinetic methods (2Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Plenum Publishers, New York2005: 377-530Crossref Scopus (274) Google Scholar, 12Ekins S. Stresser D.M. Williams J.A. Trends Pharmacol. Sci. 2003; 24: 161-166Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). These studies indicate that P450 3A4 displays homotropic and heterotropic cooperative behavior in a number of substrate oxidations (13Shimada T. Guengerich F.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 462-465Crossref PubMed Scopus (344) Google Scholar, 14Shou M. Grogan J. Mancewicz J.A. Krausz K.W. Gonzalez F.J. Gelboin H.V. Korzekwa K.R. Biochemistry. 1994; 33: 6450-6455Crossref PubMed Scopus (362) Google Scholar, 15Ueng Y.-F. Kuwabara T. Chun Y.-J. Guengerich F.P. Biochemistry. 1997; 36: s381Crossref Scopus (383) Google Scholar, 16Kenworthy K.E. Clarke S.E. Andrews J. Houston J.B. Drug Metab. Dispos. 2001; 29: 1644-1651PubMed Google Scholar), providing a basis for some earlier observations made in microsomal systems (17Kapitulnik J. Poppers P.J. Buening M.K. Fortner J.G. Conney A.H. Clin. Pharmacol. Ther. 1977; 22: 475-485Crossref PubMed Scopus (55) Google Scholar, 18Buening M.K. Fortner J.G. Kappas A. Conney A.H. Biochem. Biophys. Res. Commun. 1978; 82: 348-355Crossref PubMed Scopus (58) Google Scholar). The unusual kinetic behavior is not unique to P450 3A4; P450 2C9 (19Hutzler J.M. Hauer M.J. Tracy T.S. Drug Metab. Dispos. 2001; 29: 1029-1034PubMed Google Scholar, 20Hummel M.A. Dickmann L.J. Rettie A.E. Haining R.L. Tracy T.S. Biochem. Pharmacol. 2004; 67: 1831-1841Crossref PubMed Scopus (26) Google Scholar, 21Locuson C.W. Gannett P.M. Tracy T.S. Arch. Biochem. Biophys. 2006; 449: 115-129Crossref PubMed Scopus (31) Google Scholar), P450 1A2 (22Venkatakrishnan K. Greenblatt D.J. von Moltke L.L. Schmider J. Harmatz J.S. Shader R.I. J. Clin. Pharmacol. 1998; 38: 112-121Crossref PubMed Scopus (140) Google Scholar, 23Miller G.P. Guengerich F.P. Biochemistry. 2001; 40: 7262-7272Crossref PubMed Scopus (61) Google Scholar), and P450 2B6 (24Ekins S. VandenBranden M. Ring B.J. Wrighton S.A. Pharmacogenetics. 1997; 7: 165-179Crossref PubMed Scopus (104) Google Scholar) also show such phenomena. Several models have been proposed to explain the cooperative behavior (12Ekins S. Stresser D.M. Williams J.A. Trends Pharmacol. Sci. 2003; 24: 161-166Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 25Atkins W.M. Expert Opin. Drug Metab. Toxicol. 2006; 2: 573-579Crossref PubMed Scopus (41) Google Scholar, 26Atkins W.M. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 291-310Crossref PubMed Scopus (160) Google Scholar). Occupancy of the active site with multiple ligands has been discussed frequently (14Shou M. Grogan J. Mancewicz J.A. Krausz K.W. Gonzalez F.J. Gelboin H.V. Korzekwa K.R. Biochemistry. 1994; 33: 6450-6455Crossref PubMed Scopus (362) Google Scholar, 27Shou M.G. Methods Enzymol. 2002; 357: 261-276Crossref PubMed Scopus (21) Google Scholar, 28Shou M. Dai R. Cui D. Korzekwa K.R. Baillie T.A. Rushmore T.H. J. Biol. Chem. 2001; 276: 2256-2262Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 29Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar), although direct evidence is very limited (30Dabrowski M.J. Schrag M.L. Wienkers L.C. Atkins W.M. J. Am. Chem. Soc. 2002; 124: 11866-11867Crossref PubMed Scopus (84) Google Scholar). Another proposal is the presence of an effector site distinct from the active site (16Kenworthy K.E. Clarke S.E. Andrews J. Houston J.B. Drug Metab. Dispos. 2001; 29: 1644-1651PubMed Google Scholar), which may have some support from two recent crystal structures (9Williams P.A. Cosme J. Vinkovic D.M. Ward A. Angove H.C. Day P.J. Vonrhein C. Tickle I.J. Jhoti H. Science. 2004; 305: 683-686Crossref PubMed Scopus (709) Google Scholar, 31He Y.-A. Gajiwala K.S. Wu M. Parge H. Burke B. Lee C.A. Wester M.R. Abstracts of the 16th International Symposium on Microsome Drug Oxidations (MDO September 3-7, 2006, Budapest, Hungary). Diamond Congress, Ltd., Budapest, Hungary2006: 114Google Scholar). Although the models may provide insight into mechanisms of P450-catalyzed oxidations, these are based mainly on steady-state kinetic experiments and do not directly provide information on the individual steps of the catalytic cycle. Our own focus has been on substrate binding by P450 3A4, the first step in the catalytic cycle (29Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar), and several other laboratories have also examined certain aspects of substrate binding (32Baas B.J. Denisov I.G. Sligar S.G. Arch. Biochem. Biophys. 2004; 430: 218-228Crossref PubMed Scopus (151) Google Scholar, 33Roberts A.G. Campbell A.P. Atkins W.M. Biochemistry. 2005; 44: 1353-1366Crossref PubMed Scopus (66) Google Scholar, 34Cameron M.D. Wen B. Allen K.E. Roberts A.G. Schuman J.T. Campbell A.P. Kunze K.L. Nelson S.D. Biochemistry. 2005; 44: 14143-14151Crossref PubMed Scopus (50) Google Scholar, 35Lampe J.N. Atkins W.M. Biochemistry. 2006; 45: 12204-12215Crossref PubMed Scopus (31) Google Scholar, 36Fernando H. Halpert J.R. Davydov D.R. Biochemistry. 2006; 45: 4199-4209Crossref PubMed Scopus (48) Google Scholar). The recent availability of x-ray crystal structures provides further information on the binding of ligands by P450 3A4 (9Williams P.A. Cosme J. Vinkovic D.M. Ward A. Angove H.C. Day P.J. Vonrhein C. Tickle I.J. Jhoti H. Science. 2004; 305: 683-686Crossref PubMed Scopus (709) Google Scholar, 10Yano J.K. Wester M.R. Schoch G.A. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 38091-38094Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 11Ekroos M. Sjögren T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13682-13687Crossref PubMed Scopus (635) Google Scholar, 31He Y.-A. Gajiwala K.S. Wu M. Parge H. Burke B. Lee C.A. Wester M.R. Abstracts of the 16th International Symposium on Microsome Drug Oxidations (MDO September 3-7, 2006, Budapest, Hungary). Diamond Congress, Ltd., Budapest, Hungary2006: 114Google Scholar), although the multiplicity of observed sites raises additional questions (37Scott E.E. Halpert J.R. Trends Biochem. Sci. 2005; 30: 5-7Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 38Guengerich F.P. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13565-13566Crossref PubMed Scopus (53) Google Scholar). Recently we utilized transient-state kinetic methods as well as equilibrium binding studies to study the steps involved in substrate binding by P450 3A4 and demonstrated that binding is a complex process involving multiple-sequential steps for five substrates, testosterone, midazolam, bromocriptine, flavone, and α-naphthoflavone (39Isin E.M. Guengerich F.P. J. Biol. Chem. 2006; 281: 9127-9136Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This complexity is in contrast with the simple single-step enzyme-ligand binding interactions commonly observed for other P450s (40Griffin B.W. Peterson J.A. Biochemistry. 1972; 11: 4740-4746Crossref PubMed Scopus (112) Google Scholar, 41Yun C.-H. Kim K.-H. Calcutt M.W. Guengerich F.P. J. Biol. Chem. 2005; 280: 12279-12291Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In the present work we extended our studies to the binding of inhibitors to P450 3A4 using the structurally diverse drug molecules ketoconazole, itraconazole, clotrimazole, and indinavir plus the synthetic peptide morphiceptin (YPFP-NH2) (Fig. 1) (29Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar). Our transient kinetic experiments show that inhibitor binding by P450 3A4 is even more complex than substrate binding. Kinetic modeling yielded a minimal binding model consistent with our previous three-step model for substrate binding (39Isin E.M. Guengerich F.P. J. Biol. Chem. 2006; 281: 9127-9136Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Stopped-flow fluorescence studies using morphiceptin yielded evidence for this "silent" step, providing further confirmation for our previous observations with bromocriptine and α-naphthoflavone. Sequential-mixing stopped-flow experiments were used to eliminate an alternate multiple enzyme populations model (42Koley A.P. Robinson R.C. Friedman F.K. Biochimie (Paris). 1996; 78: 706-713Crossref PubMed Scopus (48) Google Scholar, 43Koley A.P. Buters J.T.M. Robinson R.C. Markowitz A. Friedman F.K. J. Biol. Chem. 1997; 272: 3149-3152Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 44Koley A.P. Robinson R.C. Markowitz A. Friedman F.K. Biochem. Pharmacol. 1997; 53: 455-460Crossref PubMed Scopus (53) Google Scholar, 45Davydov D.R. Fernando H. Baas B.J. Sligar S.G. Halpert J.R. Biochemistry. 2005; 44: 13902-13913Crossref PubMed Scopus (82) Google Scholar) in favor of sequential-step binding, and thus, we demonstrate the general applicability of our model (39Isin E.M. Guengerich F.P. J. Biol. Chem. 2006; 281: 9127-9136Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) for binding of ligands by P450 3A4. Chemicals—Indinavir was kindly provided by J. H. Lin (Merck). Itraconazole, clotrimazole, ketoconazole, and morphiceptin (the peptide YPFP-NH2) were purchased from Sigma. Itraconazole and ketoconazole were used as racemates. Testosterone was obtained from Steraloids (Newport, RI). All other reagents and solvents were obtained from general commercial suppliers. All chemicals were used without further purification. Enzymes—Recombinant P450 3A4 with a C-terminal His5 tag (46Gillam E.M.J. Baba T. Kim B.-R. Ohmori S. Guengerich F.P. Arch. Biochem. Biophys. 1993; 305: 123-131Crossref PubMed Scopus (380) Google Scholar) was expressed in Escherichia coli and purified as described previously (29Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar). Spectroscopy—An Aminco DW2a/OLIS or a Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA) was used to collect steady-state absorbance spectra. Most stopped-flow experiments were done with an OLIS RSM-1000 instrument using a 4 × 20-mm cell for absorbance measurements and a 4 × 4-mm cell for fluorescence measurements. Sequential mixing stopped-flow experiments were conducted using an SX-18MV stopped-flow instrument (Applied Photophysics, Leatherhead, UK) equipped with a 20-μl observation cell (10-mm path length). For all sequential mixing experiments, a non-return valve was fitted before the inlet port on the stop valve, and the pressure-held option was selected during the data collection to minimize pressure artifacts. Spectral Binding Titrations—Spectrophotometric equilibrium binding experiments were carried out by titrating P450 3A4 (1 μm) with the ligand (at 23 °C) in a total volume of 1.0 ml of 100 mm potassium phosphate buffer (pH 7.4). Final CH3OH concentrations were ≤2% (v/v). The reference cuvette, containing an equal concentration of enzyme in buffer, was titrated with an equal volume of the vehicle solvent. UV-visible spectra (350-500 nm) were recorded after each addition, and the absorbance differences (at the wavelength maximum and minimum) were plotted against the added ligand concentrations. Spectral dissociation constants (Ks, indicates a spectrally estimated dissociation constant) were estimated as described previously (39Isin E.M. Guengerich F.P. J. Biol. Chem. 2006; 281: 9127-9136Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) using GraphPad Prism software (GraphPad software, San Diego, CA) or DynaFit (47Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1329) Google Scholar) simulation software (Biokin, Pullman, WA). Because of the high affinities of the ligands (Ks within 5-fold of the P450 concentration), a nonlinear regression analysis using a quadratic equation was applied to determine the Ks for all the ligands reported in this study: ΔA = A0 + (Bmax/2[E]){(Ks + [E] + [L]) - {(Ks + [E] + [L])2 - 4[E][L]}1/2}, where A is the absorbance difference, Bmax is the maximum absorbance difference extrapolated to infinite ligand concentration, L is the ligand concentration, and E is the total enzyme concentration (A0 is a coefficient in each analysis and not relevant here). In all cases reported in this study, the quadratic equation proved to result in more satisfactory fits compared with the hyperbolic equation (ΔA = Bmax[L]/(Ks + [L])). With ketoconazole, the best fit was obtained with the Hill equation ΔA = Bmax[L]n/(Kns + [L]n), where n is a measure of cooperativity. Single-mixing Stopped-flow Experiments—Binding kinetics of ligands to P450 3A4 was monitored at 23 °C using the OLIS RSM-1000 stopped-flow instrument by observing the changes in heme Soret spectra as a function of time (in the absorbance mode) or the fluorescence quenching of ligands (in the fluorescence mode). One of the drive syringes contained purified P450 3A4, diluted to 2-6 μm in 100 mm potassium phosphate buffer (pH 7.4). The second drive syringe contained the ligand solution (indinavir, dissolved in H2O, was diluted in buffer to the desired concentration, and the stock solutions of all other ligands, dissolved in CH3OH, were diluted in buffer to ≤2% (v/v) final CH3OH concentration). Immediately after mixing equal amounts (150 μl) of reagent from both syringes, UV-visible spectra (350-500 nm) were collected in the rapid-scanning mode with a 16 × 1-mm scan disk, which is a component of the instrument and is used to acquire spectra rapidly. Depending on the data collection time, between 10 and 1000 scans s-1 were acquired. Time-resolved spectra were collected with at least five ligand concentrations for each ligand. Kinetic traces were extracted from the acquired spectra utilizing either ΔA390 or ΔA390-A418 (or ΔA390-A420) measurements and were analyzed using the manufacturer's software (OLIS), GraphPad Prism, or DynaFit. In the fluorescence experiments, after mixing the contents of the drive syringes, emission spectra were collected as a function of time with a mid-plane photomultiplier accessory and using the 16 × 1-mm scan disk. The excitation wavelength was 280 nm for morphiceptin. In general, an excitation monochromator slit of 1.24 mm was used, corresponding to an 8-nm bandpass. A 0.6-mm slit was used before the chamber housing the scan disk and mid-plane photomultiplier for the time-resolved scans. Time-resolved scans were analyzed and deconvoluted using the OLIS SVD global fitting software (48Golub G.H. Reinsch C. Numerische Mathematik 1. 1970; 4: 403-420Crossref Scopus (1913) Google Scholar, 49Chan T.F. ACM Transactions on Mathematical Software. 1982; 8: 72-83Crossref Scopus (152) Google Scholar, 50Matheson I.B.C. Anal. Instrum. 1987; 16: 345-373Crossref Scopus (7) Google Scholar, 51Matheson I.B.C. Comput. Chem. 1989; 13: 299-304Crossref Scopus (13) Google Scholar, 52Matheson I.B.C. Comput. Chem. 1990; 14: 49-57Crossref Scopus (48) Google Scholar, 53Maeder M. Zuberbühler A.D. Anal. Chem. 1990; 62: 2220-2224Crossref Scopus (412) Google Scholar, 54Knutson J.R. Beechem J.M. Brand L. Chem. Phys. Lett. 1983; 102: 501-507Crossref Scopus (540) Google Scholar). Alternatively, data were collected using a >285-nm long-pass filter in the single-wavelength mode. Generally, averages of the results of four to eight individual experiments were used in the subsequent data analyses, and S.E. indicate the goodness of the fit to the average of the multiple experiments. Sequential-mixing Stopped-flow Experiments—In the sequential mixing experiments (done in the Applied Photophysics SX-18MV instrument), P450 3A4 (8 μm, 110 μl) was mixed with an equal volume of the first ligand, and the mixture was "aged" in the delay loop. After a pre-determined amount of time, the contents of the delay loop were pushed into the mixing chamber using 90 μl of 100 mm potassium phosphate buffer (pH 7.4) and combined with the second ligand (90 μl). The changes in the Soret spectrum were monitored as ΔA390 in the single-wavelength mode. ITC—ITC titrations were carried out at 25 °C using a VP-ITC instrument (MicroCal, Northampton, MA) as described previously (39Isin E.M. Guengerich F.P. J. Biol. Chem. 2006; 281: 9127-9136Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In a typical experiment, before the titration, P450 3A4 was dialyzed twice against 100 volumes of 100 mm potassium phosphate buffer (pH 7.4) at 4 °C for 4 h. The clotrimazole solution (5 μm in dialysis buffer) in the ITC cell was titrated with P450 3A4 (32 μm) loaded into the ITC syringe. The first injection (2 μl, omitted from analysis) was followed by 4 injections of 5 μl and 23 injections of 10 μl with 8-min intervals between injections. The cell contents were stirred at 450 rpm to provide immediate mixing. The thermal power (heat per unit time) required to keep the cell temperature constant was monitored with time. The peaks observed in the power versus time plots (thermograms, not shown) were integrated using the ORIGIN software (MicroCal). The heats of dilution were obtained from the saturating part of the thermograms and subtracted from each integrated peak. The total heat change was plotted versus the concentration of P450 added to give a titration curve (binding isotherm), expressed in units of kJ mol-1. Kinetic Modeling of Data—Kinetic binding data were fit to various proposed models using DynaFit software (47Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1329) Google Scholar). Rate constants were estimated by globally fitting the raw kinetic data obtained at five different ligand concentrations to the proposed models. The initial ligand concentrations were allowed to float within 10% of the starting value in the system, and in most cases the concentration adjustment was <5%. Sample scripts and some of the pertinent results are included in the supplemental. Spectral Equilibrium Binding Titrations—Spectrophotometric titrations were first carried out to estimate the binding affinities of various ligands to P450 3A4, and spectral dissociation constants (Ks) were obtained from the titration curves as described under "Experimental Procedures." The P450 3A4 inhibitors indinavir, clotrimazole, itraconazole, ketoconazole, and morphiceptin (the peptide YPFP-NH2) all showed a "type II" shift in the heme Soret spectra (Fig. 2A), typical of a ligand (nitrogen atom) coordinating to the heme iron (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar, 3Ortiz de Montellano P.R. DeVoss J.J. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 3rd Ed. Plenum Publishers, New York2005: 183-245Crossref Scopus (203) Google Scholar, 55Schenkman J.B. Remmer H. Estabrook R.W. Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar, 56Imai Y. Horie S. Yamano T. Iizuka T. Sato R. Omura T. Cytochrome P-450. Academic Press, Inc., New York1978: 37-135Google Scholar). Estimated Ks values are summarized in Table 1. The amplitudes of the changes varied due to the specific interactions of the various inhibitors with the heme iron (Fig. 2).TABLE 1Binding rates and affinities of ligands to P450 3A4LigandConcentration used for initial analysisSoret shiftKskaaFrom initial analysis. ΔA = Aae−kat + Abe−kbtkbaFrom initial analysis. ΔA = Aae−kat + Abe−kbtAaaFrom initial analysis. ΔA = Aae−kat + Abe−kbtAbaFrom initial analysis. ΔA = Aae−kat + Abe−kbtμMTypebFrom Ref. 55.mms−1s−1Clotrimazole10II0.03 ± 0.010.72 ± 0.010.05 ± 0.010.050 ± 0.0010.083 ± 0.001Indinavir10II0.3cFrom Ref. 29.0.54 ± 0.010.04 ± 0.010.021 ± 0.0010.035 ± 0.001Itraconazole4II0.20 ± 0.012.5 ± 0.10.2 ± 0.10.008 ± 0.0010.011 ± 0.001Morphiceptin100II7.8 ± 2.4cFrom Ref. 29.0.5 ± 0.10.05 ± 0.010.023 ± 0.0010.049 ± 0.001Ketoconazole5II0.5 ± 0.1 (n = 1.4 ± 0.1)dFit to the Hill equation ΔA = Bmax[L]n/(Ksn + [L]n) with n values given in parentheses.7.7 ± 0.10.75 ± 0.010.043 ± 0.0010.043 ± 0.001a From initial analysis. ΔA = Aae−kat + Abe−kbtb From Ref. 55Schenkman J.B. Remmer H. Estabrook R.W. Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar.c From Ref. 29Hosea N.A. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 5929-5939Crossref PubMed Scopus (225) Google Scholar.d Fit to the Hill equation ΔA = Bmax[L]n/(Ksn + [L]n) with n values given in parentheses. Open table in a new tab All of the inhibitors except morphiceptin showed tight binding to P450 3A4 (Table 1), clotrimazole being the highest affinity ligand with an estimated Ks of 30 nm. The results obtained for the azole inhibitors are consistent with the previously obtained inhibition potencies for these compounds (57Wanchana S. Yamashita F. Hashida M. Pharmacol. Res. 2003; 20: 1401-1408Crossref Scopus (54) Google Scholar, 58Stresser D.M. Blanchard A.P. Turner S.D. Erve J.C.L. Dandeneau A.A. Miller V.P. Crespi C.L. Drug Metab. Dispos. 2000; 28: 1440-1448PubMed Google Scholar). The titration plot obtained for ketoconazole binding displayed some sigmoidicity, and a fit to the Hill equation was more satisfactory compared with a hyperbolic (results not shown) or quadratic equation (which did not consider cooperative behavior) (Fig. 2B). Binding Kinetic Experiments—The kinetics of binding of type II ligands to P450 3A4 were studied using stopped-flow absorbance by monitoring the changes in the heme Soret spectra over time. Spectra collected over time showed a decrease in absorbance at ∼390 nm and an increase in absorbance at ∼420 nm for all the ligands, consistent with coordination to the heme iron (Fig. 3A). Kinetic traces extracted at these wavelengths were used to describe the kinetics of ligand binding (Fig. 3B). In all cases, a single-exponential fit (results not shown) applied to the kinetic data (ΔA390-A420) was not adequate to describe the binding kinetics, suggesting a complex multi-step binding event. Therefore, bi-exponential fitting was used in an attempt to describe the binding kinetics of these ligands with P450 3A4 (Fig. 4, A, C, E, and G). Although reasonable fits were obtained, visual inspection of the residuals plots (Fig. 4, A, C, E, and G, top panels) suggests that the binding events are probably even more complex than can be defined by a classical bi-exponential binding fit. This deviation from a bi-exponential fit became more apparent at increasing ligand concentrations. The data were also fit to a tri-exponential equation (Fig. 4, B, D, F, and H), resulting in more evenly distributed residuals plots (Fig. 4, B, D, F, and H, top panels).FIGURE 4Binding kinetics of ligands to P450 3A4. A and B, the kinetic trace (ΔA428-A390) obtained for clotrimazole (10 μm) binding to P450 3A4 was fit to a bi-exponential plot with rates of 0.72 and 0.05 s-1 (A) and to a tri-exponential fit with rates of 2.3, 0.22, and 0.03 s-1 (B). C and D, the kinetic trace (ΔA433-A405) obtained for ketoconazole (5 μm) binding to P450 3A4 was fit to a bi-exponential plot with rates of 7.7 and 0.75 s-1 (C) and to a tri-exponential fit with rates of 14.3, 3.0, and 0.46 s-1 (D). E and F, the kinetic trace (ΔA428-A390) obtained for indinavir (10 μm) binding to P450 3A4 was fit to a bi-exponential plot with rates of 0.54 and 0.04 s-1 (E) and to a tri-exponential fit with rates of 2.4, 0.26, and 0.03 s-1 (F). G and H, the kinetic trace (ΔA428-A390) obtained for morphiceptin (100 μm) binding to P450 3A4 was fit to a bi-exponential plot with rates of 0.49 and 0.05 s-1 (G) and to a tri-exponential fit with rates of 1.4, 0.19, and 0.03 s-1 (H). Residuals analyses for the fits are shown in the upper panels of all parts.View Large Image Figure ViewerDownload Hi-res image

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