Alfin D. N. Vaz

Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis: implications for treatment of tuberculosis

Although antibiotics against Mycobacterium tuberculosis have decreased the incidence of tuberculosis infections significantly, the emergence of drug-resistant strains of this deadly pathogen renders current treatments ineffective. Therefore, it is imperative to identify biochemical pathways in M. tuberculosis that can serve as targets for new anti-mycobacterial drugs. We recently cloned, expressed, and purified MT CYP51, a soluble protein from M. tuberculosis that is similar in sequence to CYP51 (lanosterol-14alpha-demethylase) isozymes, pharmacological targets for several anti-mycotic compounds. Its striking amino acid sequence similarity to that of mammalian and fungal CYP51s led to the hypothesis that MT CYP51 plays an important role in mycobacterial biology that can be targeted for drug action. In this manuscript, we established through spectral analysis that several azole antifungals bind MT CYP51 with high affinity. The effects of several azole compounds on the growth of M. bovis and M. smegmatis, two mycobacterial species that closely resemble M. tuberculosis were examined. We established a correlation between the affinity of azole compounds to MT CYP51 and their ability to impair the growth of M. bovis and M. smegmatis. These results suggest that the metabolic functions of MT CYP51 may be comparable to those of CYP51 in yeast and fungi and may lead to the development of a new generation of anti-mycobacterial agents.

Cytochrome P450 2: peroxidative reactions of diversozymes.

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoprote‐ ins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive β‐scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides undergo reductive β‐cleavage to give hydrocarbons and aldehydic acids. One of these products, trans‐4‐hydroxynonenal, inactivates P450, particularly alcohol‐inducible 2E1, in what may be a negative regulatory process. Al though a P450 iron‐oxene species is believed to be the oxygen donor in most hydroxylation reactions, an iron‐peroxy species is apparently involved in the deformylation of many aldehydes with desaturation of the remaining structure, as in aromatization reactions.—Coon, M. J., Vaz, A. D. N., Bestervelt, L. L. Peroxidative reaction of diversozymes. FASEB J. 10, 428‐434 (1996)

Metabolic Activation of trans-4-Hydroxy-2-nonenal, a Toxic Product of Membrane Lipid Peroxidation and Inhibitor of P450 Cytochromes

Lipid peroxidation in biological membranes is known to yield reactive aldehydes, of whichtrans-4-hydroxy-2-nonenal (HNE) is particularly cytotoxic. This laboratory previously reported that purified liver microsomal P450 cytochromes are directly inactivated to varying extents by HNE. We have now found a mechanism-based reaction in which P450s are inactivated by HNE in the presence of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase. The sensitivity of the various isozymes in the two pathways is different as follows: P450 2B4 and the orthologous 2B1 are inactivated to the greatest extent and 2C3, 1A2, 2E1, and 1A1 to a somewhat lesser extent by the pathway in which HNE undergoes metabolic activation. In contrast, 2B4 and 2B1 are insensitive to direct inactivation, and the reductase is unaffected by HNE by either route. Recent studies on the catalytic activities of the T302A mutant of P450 2B4 have shown that the rate of oxidation of a variety of xenobiotic aldehydes to carboxylic acids is decreased, but the rates of aldehyde deformylation and mechanism-based inactivation of the cytochrome are stimulated over those of the wild-type enzyme (Raner, G. M., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry36, 4895–4902). Inactivation by those aldehydes apparently occurs by homolytic cleavage of a peroxyhemiacetal intermediate to yield formate and an alkyl radical that reacts with the heme. In sharp contrast, the rate of mechanism-based inactivation by HNE is decreased with the T302A mutant relative to that of the wild-type P450 2B4, and mass spectral analysis of the heme adduct formed shows that deformylation does not occur. We therefore propose that the metabolic activation of HNE involves formation of an acyl carbon radical that leads to the carboxylic acid or alternatively reacts with the heme.

Adrenal proteins bound by a reactive intermediate of mitotane

Abstract Purpose: Mitotane (o,p′-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources. Methods: With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450SCC and adrenodoxin. Results: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450SCC, but the band at 11.5 kDa did not correspond to adrenodoxin. Conclusions: The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.

[28] Reductive cleavage of hydroperoxides by cytochrome P-450

Publisher Summary This chapter describes the reductive cleavage of hydroperoxides by cytochrome P- 450. Various forms of liver microsomal cytochrome P-450 (cytochrome P- 450 LM ) are known to catalyze numerous chemical reactions, including the hydroxylation of fatty acids, steroids, and a variety of foreign compounds, such as drugs and carcinogens. These monooxygenation reactions require aerobic conditions and nicotine adenine dinucleotide phosphate hydrogenase (NADPH) as the electron donor in microsomal suspensions or in the reconstituted system containing purified cytochrome P-450 LM , purified NADPH-cytochrome-P-450 reductase, and phosphatidylcholine. On the other hand, molecular oxygen, NADPH, and the reductase are not required when organic hydroperoxides or related compounds serve as the oxygen donor in cytochrome P-450 LM -catalyzed substrate hydroxylations. In such reactions, the hydroperoxides are converted to the corresponding alcohols so that cumyl hydroperoxide, for example, is converted to cumyl alcohol. Other model hydroperoxides in addition to cumyl serve as substrates in the reductive cleavage reaction. α-Methylbenzyl hydroperoxide yields acetophenone as well as benzaldehyde because either a hydrogen atom or a methyl group can be lost, benzyl hydroperoxide yields benzaldehyde, and tert -butyl hydroperoxide yields acetone and methane. With limiting amounts of cytochrome P-450 form present, the turnover numbers, expressed as nanomoles of NADPH oxidized per minute per nanomole of the cytochrome, are 60, 30, 24, and 6 for tert -butyl, cumyl, α-methylbenzyl, and benzyl hydroperoxides, respectively. Pentane has been identified as a cleavage product of the 15-hydroperoxide derived from arachidonic acid as well as from the 13-hydroperoxide derived from linoleic acid.

RADICAL INTERMEDIATES IN THE CATALYTIC CYCLES OF CYTOCHROME P-450

Schemes are presented summarizing current knowledge of the mechanism of action of cytochrome P-450 when it functions either as a monooxygenase with molecular oxygen as the oxygen donor or as a peroxygenase with peroxy compounds as the oxygen donor. In the process, a large variety of physiologically occurring and foreign compounds undergo hydroxylation and oxy and peroxy radicals are generated. In addition, cytochrome P-450 catalyzes reductive reactions, including a recently discovered reaction in which organic hydroperoxides are cleaved to yield hydrocarbons and aldehydes or ketones. The reaction is believed to involve homolysis of the oxygen-oxygen bond and generation of an alkoxy radical, with beta-scission of the latter followed by reduction of the secondary radical to the hydrocarbon. Evidence has been obtained that lipid hydroperoxides are physiological substrates for this reductive cleavage reaction catalyzed by cytochrome P-450.

Radical intermediates in peroxide-dependent reactions catalyzed by cytochrome P-450

Highly purified liver microsomal cytochrome P-450 acts as a peroxygenase in catalyzing the reaction, RH+ XOOH→ROH+XOH, Where RH represents any of a large variety of foreign or physiological substrates and ROH the corresponding product, and XOOH represents any of a series of peroxy compounds such as hydroperoxides or peracids serving as the oxygen donor and XOH the resulting alcohol or acid. Several experimental approaches in this and other laboratories have yielded results compatible with a homolytic mechanism of oxygen-oxygen bond cleavage but not with the heterolytic formation of a common iron-oxo intermediate from the various peroxides.Recently, we have found a new reaction, catalyzed by the reconstituted system containing the phenobarbital-inducible form of P-450, which catalyzes the reductive cleavage of hydroperoxides: XRR’C-OOH+ NADPH+H+→ XR’CO + R’H+H2O + NADP+ Thus, cumyl hydroperoxide yields acetophenone and methane, and 13-hydroperoxyoctadeca-9, 11-dienoic acid yields pentane and an as yet unidentified additional product. Since hydroperoxide reduction does not produce the corresponding alcohol, it is concluded that homolytic cleavage of the oxygen-oxygen bond occurs with rearrangement of the resulting alkoxy radical. Studies are in progress to determine how broad a role the new hydroperoxide cleavage reaction plays in the biological peroxidation of lipids.