Anthony Y. H. Lu

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug–drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (–)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

Metabolic bioactivation and drug-related adverse effects: current status and future directions from a pharmaceutical research perspective

Retrospective studies indicate that many drugs that cause clinical adverse reactions, such as hepatotoxicity, undergo metabolic bioactivation, resulting in the formation of electrophilic intermediates capable of covalently modifying biological macromolecules. A logical extension of these findings is a working hypothesis that compounds with reduced levels of bioactivation should be inherently safer drug molecules and thus have a greater likelihood of success in drug development. Whereas some research-based pharmaceutical companies have adopted a strategy of addressing metabolic bioactivation early in drug discovery, much skepticism remains on whether such an approach would enable the industry to reach the desired objectives. The debate is centered on the question of whether there is a quantitative correlation between bioactivation and the severity of drug-treatment–related toxicity, and whether covalent protein modification represents only one of several possible mechanisms underlying observed tissue injury. This communication is intended to briefly review the current understanding of drug-induced hepatotoxicity and to discuss the controversy and future directions with respect to the effort of minimizing the probability of clinical adverse reactions.

Critical Amino Acid Residues for Nicotine 5′-Hydroxylation in Human CYP2A Enzymes

Abstract Objective We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-(3-pyridyl)-1-butanone(NNK) and aflatoxin B1(AFB1) carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods A series of reciprocally substituted mutants of CYP2A6Ile 300 → Phe, CYP2A6Gly 301 Ala, CYP2A6Ser 369 → Gly, CYP2A13Phe 300 → Ile, CYP2A13Ala 301 → Gly and CYP2A13Gly 369 → Ser were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5′hydroxylatin by wild type and mutant CYP2A proteins was performed. Results All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile 300 → Phe and CYP2A6Gly 301 →Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe 300 →Ile and CYP2A13Ala 301 →Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by V max variations rather than K m changes. Substitution of #369 residue significantly affected both K m and V max values. CYP2A6Ser 369 → Gly increase the catalytic efficiency via a significant K m decrease versus V max enhancement, while the opposite effects were seen with CYP2A13Gly 369 → Ser. Conclusion #300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5′-oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Ser 369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu 370 which is crucial for many hydroxylations.

Covalent binding of chemical residues: health impact.

Risk assessment of covalent binding of reactive intermediates to proteins in humans is dependent on whether the exposure of these chemicals is direct or indirect. It is well established that direct exposure of drugs and environmental chemicals capable of covalent binding to proteins in laboratory and food-producing animals and humans could result in potential risk. The degree of toxicological concern is dependent on the dose, chemical nature of the reactive intermediates, protein targets and other factors. Humans could also be exposed to food containing covalently bound chemical residues, mostly meat from drug-treated food-producing animals, and agriculture products from pesticide-treated crops. Health impact from this type of indirect exposure (i.e. protein bound residues rather than the free parent compound) is more difficult to evaluate in humans.