Structure and mechanism of the complex between cytochrome P4503A4 and ritonavir

Ritonavir is a HIV protease inhibitor routinely prescribed to HIV patients that also potently inactivates cytochrome P4503A4 (CYP3A4), the major human drug-metabolizing enzyme. By inhibiting CYP3A4, ritonavir increases plasma concentrations of other anti-HIV drugs oxidized by CYP3A4 thereby improving clinical efficacy. Despite the importance and wide use of ritonavir in anti-HIV therapy, the precise mechanism of CYP3A4 inhibition remains unclear. The available data are inconsistent and suggest that ritonavir acts as a mechanism-based, competitive or mixed competitive-noncompetitive CYP3A4 inactivator. To resolve this controversy and gain functional and structural insights into the mechanism of CYP3A4 inhibition, we investigated the ritonavir binding reaction by kinetic and equilibrium analysis, elucidated how the drug affects redox properties of the hemoprotein, and determined the 2.0 Å X-ray structure of the CYP3A4-ritonavir complex. Our results show that ritonavir is a type II ligand that perfectly fits into the CYP3A4 active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen, which decreases the redox potential of the protein and precludes its reduction with the redox partner, cytochrome P450 reductase.

[1]  J. Dawson,et al.  Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme. , 1982, The Journal of biological chemistry.

[2]  S. Vasavanonda,et al.  ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[4]  Ortiz de Montellano,et al.  Cytochrome P-450: Structure, Mechanism, and Biochemistry , 1986 .

[5]  F. Berthou,et al.  Inhibition of methadone and buprenorphine N-dealkylations by three HIV-1 protease inhibitors. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[6]  A. Munro,et al.  Determination of the redox properties of human NADPH-cytochrome P450 reductase. , 2001, Biochemistry.

[7]  W. Pryor.  Cytochrome P450: Structure, mechanism, and biochemistry , 1996 .

[8]  T. Shimada,et al.  Twenty years of biochemistry of human P450s: purification, expression, mechanism, and relevance to drugs. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[9]  C. Jefcoate,et al.  Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. , 1978, Methods in enzymology.

[10]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[11]  S. Sligar,et al.  Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: the effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium. , 2010, Biochimica et biophysica acta.

[12]  Y. Berger,et al.  Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. , 1996, The Journal of pharmacology and experimental therapeutics.

[13]  T. Sjögren,et al.  Structural basis for ligand promiscuity in cytochrome P450 3A4 , 2006, Proceedings of the National Academy of Sciences.

[14]  H. Yamazaki,et al.  Lack of Electron Transfer from Cytochrome b5 in Stimulation of Catalytic Activities of Cytochrome P450 3A4 , 1996, The Journal of Biological Chemistry.

[15]  S. Sligar,et al.  Spin state control of the hepatic cytochrome P450 redox potential. , 1979, Biochemical and biophysical research communications.

[16]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[17]  R. Silverman.  Mechanism-based enzyme inactivation : chemistry and enzymology , 1988 .

[18]  M Hendlich,et al.  LIGSITE: automatic and efficient detection of potential small molecule-binding sites in proteins. , 1997, Journal of molecular graphics & modelling.

[19]  F Peter Guengerich,et al.  Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4* , 2006, Journal of Biological Chemistry.

[20]  D. Greenblatt,et al.  In vitro metabolism of trazodone by CYP3A: inhibition by ketoconazole and human immunodeficiency viral protease inhibitors , 2000, Biological Psychiatry.

[21]  L. Pershing,et al.  Cytochrome P-450 metabolic-intermediate complex formation and induction by macrolide antibiotics; a new class of agents. , 1982, Xenobiotica; the fate of foreign compounds in biological systems.

[22]  M. Desai,et al.  Pharmacokinetic enhancers for HIV drugs. , 2009, Current opinion in investigational drugs.

[23]  A. Das,et al.  Redox potential control by drug binding to cytochrome P450 3A4. , 2007, Journal of the American Chemical Society.

[24]  F. Guengerich.  Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems. , 1983, Biochemistry.

[25]  Eric F. Johnson,et al.  The Structure of Human Microsomal Cytochrome P450 3A4 Determined by X-ray Crystallography to 2.05-Å Resolution* , 2004, Journal of Biological Chemistry.

[26]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. , 1964, The Journal of biological chemistry.

[27]  M. J. Coon,et al.  Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential. , 1975, The Journal of biological chemistry.

[28]  D. Greenblatt,et al.  Potent mechanism-based inhibition of human CYP3A in vitro by amprenavir and ritonavir: comparison with ketoconazole , 2000, European Journal of Clinical Pharmacology.

[29]  A. D. Rodrigues,et al.  Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes. , 1996, The Journal of pharmacology and experimental therapeutics.

[30]  H. Yamazaki,et al.  Roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome P450 3A4 and replacement of NADPH--cytochrome P450 reductase with other flavoproteins, ferredoxin, and oxygen surrogates. , 1995, Biochemistry.

[31]  Jose Cosme,et al.  Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone , 2004, Science.

[32]  Karthik Venkatakrishnan,et al.  Mechanism-Based Inactivation of Human Cytochrome P450 Enzymes and the Prediction of Drug-Drug Interactions , 2007, Drug Metabolism and Disposition.

[33]  C. Ernest,et al.  Mechanism-Based Inactivation of CYP3A by HIV Protease Inhibitors , 2005, Journal of Pharmacology and Experimental Therapeutics.

[34]  J. Bridges,et al.  Structure--activity relationship for 'safrole-type' cytochrome P-450 induction [proceedings]. , 1979, Biochemical Society Transactions.

[35]  P. Vouros,et al.  Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human intestinal microsomes and expressed cytochrome P4503A4/3A5: mechanism-based inactivation of cytochrome P4503A by ritonavir. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[36]  D. Back,et al.  Differential inhibition of cytochrome P450 isoforms by the protease inhibitors, ritonavir, saquinavir and indinavir. , 2003, British journal of clinical pharmacology.

[37]  F. Guengerich,et al.  Kinetics of ferric cytochrome P450 reduction by NADPH-cytochrome P450 reductase: rapid reduction in the absence of substrate and variations among cytochrome P450 systems. , 1997, Biochemistry.

[38]  J. Dawson,et al.  The diverse spectroscopic properties of ferrous cytochrome P-450-CAM ligand complexes. , 1983, The Journal of biological chemistry.

[39]  J. Lin,et al.  Role of cytochrome P450 3A4 in human metabolism of MK-639, a potent human immunodeficiency virus protease inhibitor. , 1996, Drug metabolism and disposition: the biological fate of chemicals.