Inhibition of Cytochrome P450 Enzymes

7 Inhibition of Cytochrome P450 Enzymes Maria Almira Correia and Paul R. Ortiz de Monteflano 1. Introduction of P450 inhibitors are available in various reviews"^^^. This chapter focuses on the mecha Three steps in the catalytic cycle of nisms of inactivation; thus, most of the chapter is cytochrome P450 (P450, CYP; see Chapters 5 and devoted to the discussion of agents that require 6) are particularly vulnerable to inhibition: (a) the P450 catalysis to fiilfill their inhibitory potential. binding of substrates, (b) the binding of molecular The mechanisms of reversible competitive and oxygen subsequent to the first electron transfer, noncompetitive inhibitors, despite their practical and (c) the catalytic step in which the substrate is importance, are relatively straightforward and are actually oxidized. Only inhibitors that act at one of discussed more briefly. these three steps will be considered in this chapter. Inhibitors that act at other steps in the catalytic cycle, such as agents that interfere with the 2. Reversible Inhibitors electron supply to the hemoprotein by accepting electrons directly from P450 reductase^"^, are not Reversible inhibitors compete with substrates discussed here. for occupancy of the active site and include agents P450 inhibitors can be divided into three that (a) bind to hydrophobic regions of the active mechanistically distinct classes: Agents that site, (b) coordinate to the heme iron atom, or (a) bind reversibly, (b) form quasi-irreversible (c) enter into specific hydrogen bonding or ionic complexes with the heme iron atom, and (c) bind interactions with active-site residues"*"^^. The first irreversibly to the protein or the heme moiety, or mechanism, in which the inhibitor simply competes accelerate the degradation and/or oxidative frag for binding to lipophilic domains of the active site, mentation of the prosthetic heme. Agents that is often responsible for the inhibition observed interfere in the catalytic cycle prior to the actual when two substrates compete for oxidation by a sin oxidative event are largely reversible competitive gle P450 isoform. A clear example of such an inter or noncompetitive inhibitors. Those that act dur action is provided by the mutual in vitro and in vivo ing or subsequent to the oxygen transfer step are inhibition of benzene and toluene metabolism^^. generally irreversible or quasi-irreversible inhibitors This form of inhibition, which is optimal when the and often fall into the category of mechanism- inhibitory compound is bound tightly but is a poor based (or suicide) inactivators. Extensive lists substrate, is usually not highly effective but can Maria Almira Correia • Department of Cellular and Molecular Pharmacology, Department of Pharmaceutical Chemistry, Department of Biopharmaceutical Sciences and the Liver Center, University of California, San Francisco, CA. Paul R. Ortiz de Montellano • Department of Pharmaceutical Chemistry, and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3e, edited by Paul R. Ortiz de Montellano Kluwer Academic / Plenum Publishers, New York, 2005. 247 248 M.A. Correia and P.R. Ortiz de Montellano cause physiologically relevant metabolic changes 2.2. Coordination to and clinically significant interactions^"*. Ferrous Heme 2.1. Coordination to In its simplest form, inhibition through coordi nation to the heme iron is exemplified by carbon Ferric Heme monoxide, a neutral ligand that binds exclusively The binding of a strong sixth ligand to the pen- to the ferrous (reduced) form of P450. The tacoordinated heme iron atom of a P450 enzyme, binding of carbon monoxide involves donation of or displacement of a weak ligand in a P450 hexa- electrons from the carbon to the iron through a coordinated state by a strong ligand, causes a shift a-bond as well as back-donation of electrons from of the iron from the high- to the low-spin state. the occupied ferrous iron d-orbitals to the empty This shift is characterized by a "type H" binding antibonding ir-orbitals of the ligand^^. P450 spectrum with a Soret maximum at 425-435 nm enzymes (P450, CYP) are so named because their and a trough at 390-405 nm^^~^^. The change in carbon monoxide complexes have spectroscopic the redox potential of the enzyme associated with absorption maxima at approximately 450 nm^^. this spin state change makes its reduction by P450 Early studies with model ferroporphyrins, which reductase more difficult (see Chapter 5)^^' ^^. This indicated that only those with a thiolate ligand change in reduction potential, as much as physical trans to the carbon monoxide yielded the 450-nm occupation of the sixth coordination site, is absorption, provided key evidence for the pres responsible for the inhibition associated with the ence of a thiolate fifth ligand in P450.^' Inhibition binding of strong iron ligands. by carbon monoxide is a signature of P450- Cyanide and other ionic ligands bind preferen catalyzed processes, although the sensitivities of tially, albeit weakly, to the ferric state of a P450 different P450 isoforms to carbon monoxide dif- enzyme^^' ^^. The three positive charges of the iron fer^^ and a few P450-catalyzed reactions are are matched in the ferric hemoprotein by the three resistant to inhibition by carbon monoxide^^"^^. In negative charges of its permanent ligands (i.e., the particular, the sensitivity of aromatase^^' ^^ and two porphyrin nitrogens and the thiolate ion), but P450g(.^^^^ to inhibition by carbon monoxide in the reduced state there is a charge imbalance of decreases drastically as the enzymes traverse the three ligand negative charges but only two ferrous conformational and ligand states inherent in their iron positive charges. The cyanide ion, a nega multistep catalytic sequences. Among the human tively charged species, binds more readily to the liver P450 isoforms, the susceptibility of different neutral (ferric) than the negative (ferrous) families to carbon monoxide inhibition appears to enzyme. Indeed, cyanide binds more weakly to decrease in the order 2D > 2C> 3A^^. ferric P450 than to ferric myoglobin because the P450 thiolate ligand places a higher electron den sity on the iron than does the imidazole ligand of 2.3. Heme Coordination and myoglobin^^. The chelation of ionic ligands is dis Lipophilic Binding favored, in addition, by the lipophilic nature of the P450 active site^^. Agents that simultaneously bind to lipophilic P450 enzymes are inhibited by nitric oxide regions of the active site and to the heme iron atom (NO), a molecule of great interest because of its (Figure 7.1) are inherently more effective P450 role in diverse physiological and pathological inhibitors than agents that only exploit one of these processes. Inhibition initially involves reversible binding interactions. The effectiveness of these coordination of the nitrogen to the iron but a sub agents as P450 inhibitors is governed both by their sequent time-dependent, irreversible inactivation hydrophobic character and the strength of the bond of the enzyme by an undefined mechanism has between their heteroatomic lone pair and the heme been reported^"^^^. Inhibition by endogenous NO iron. Agents such as alcohols, ethers, ketones, lac of P450 enzymes involved in endogenous sub tones, and other structures in which an oxygen strate metabolism, including eicosanoid formation atom of the ligand coordinates to the iron, or which and sterol metabolism, may have physiological act by stabilizing the coordination of the distal consequences^^' •^^. water ligand, are relatively poorly bound and are Inhibition of Cytochrome P450 Enzymes 249 MeCON O^ O N-X^ N.^ r,-^^^^CI , CI Ketoconazole Metyrapone Cimetidine Figure 7.1. Schematic illustration of the binding of an inhibitor to a P450 active site by both coordinating to the heme-iron atom and interacting with the surrounding protein residues. The structures of three agents that inhibit P450 by binding in this manner are shown. weak inhibitors^'^' ^^^^. The Soret band of such affinity of the ligand nitrogen electron pair for the complexes is found at approximately 415 nm^^' ^^. heme iron, (b) the degree to which the intrinsic In contrast, agents that interact strongly with both affinity of the ligand for the iron is moderated by the protein and the heme iron atom are often highly steric interactions with substituents on the effective reversible inhibitors'*"^^. As already noted, inhibitor^^' ^^, (c) the lipophilicity of the nonligat- the binding of such inhibitors yields a "type IF' ing portion of the inhibitor^^' ^^, and, naturally, difference spectnmi with a Soret maximum at (d) the congruence between the geometry of 430 nm^^' ^^' ^^^ ^^. The structures of these powerful the inhibitor and that of the active site. The inhibitors usually incorporate nitrogen-containing synergism that results from binding simultane aliphatic or aromatic functions. ously through lipophilic interactions and coordi P)nridine, imidazole, and triazole derivatives nation with the heme iron is illustrated by the have proven particularly useful in P450 inhibitors^. fact that imidazole and benzene individually Metyrapone (Figure 7.1), one of the first P450 are weak inhibitors, but when coupled together as inhibitors to be widely employed, first gained in phenylimidazole they produce a powerful prominence as an inhibitor of 11 p-hydroxylase, inhibitor^^. Optimization of these structural fea the enzyme that catalyzes the final step

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