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Molecular Genetics and Immunobiology of Mycobacteria |
Department of Biochemistry, University of Leicester, The Adrian Building, University Road, Leicester LE1 7RH, UK1
Department of Biosciences, University of Strathclyde, The Royal College, 204 George Street, Glasgow G1 1XL, UK2
Department of Genetics, John Innes Centre, Norwich NR4 7UH, UK3
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK4
Author for correspondence: Andrew W. Munro. Tel: +44 116 252 3464. Fax: +44 116 252 3630. e-mail: awm9{at}le.ac.uk
The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14
-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of <0·2 and 0·3 µM, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1·2 and 36·5 µM, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with Kd values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor 
CYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.
Keywords: tuberculosis, azole drugs, CYP121, P450 inhibitors
Abbreviations: g.t., Generation time; Kd, binding constant; Mtb, Mycobacterium tuberculosis; s.g.r., specific growth rate; P450, cytochrome P450 mono-oxygenase
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