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Investigating orphan cytochromes P450 from Mycobacterium tuberculosis: The search for potential drug targets
[Thesis]. Manchester, UK: The University of Manchester; 2011.
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Abstract
Tuberculosis (TB) is a disease that the World Health Organisation (WHO)regards as a global pandemic. There is a great need for new drugs to combat thisthreat. Drug resistant strains of the causative agent, Mycobacterium tuberculosis(Mtb), have increased the urgency of this quest for novel anti-mycobacterialmedicines. Publication of the Mtb genome sequence revealed a large number ofcytochrome P450 (CYP) enzymes [Cole, S. T. et al. 1998]. These mono-oxygenaseenzymes have been studied for many years and are responsible for metabolicfunctions in every kingdom of life. Research on the Mtb P450s to date has highlightedseveral of them as having critcal roles within the organism. CYP121 and CYP128 havebeen implicated as essential through gene knockout studies. It has been demonstratedthat CYP125 is not essential for viability. However, it is part of a gene cluster highlyimportant for Mtb infectivity and virulence. Due to the prospective importance ofP450s to Mtb, this group of enzymes is under investigation as a source of novel drugtargets. CYP142 was discovered as a potential drug target after it was located to a genecluster involved in cholesterol catabolism during Mtb dormancy. As part of this PhDproject, it was demonstrated that CYP142 performs an almost identical role to thatreported for CYP125. These enzymes both perform C27 hydroxylation andcarboxylation of the cholesterol side chain. However, variations in the level ofoxidation have been identified, dependent upon the redox system with which theseP450s are associated. A crystal structure of CYP142 showing high similarity in activesite architecture to CYP125 supports the physiological role of CYP142 in cholesterolcatabolism. Combining this with in vitro data which demonstrates that CYP142possesses high affinity for a range of azole anti-fungal agents [Ahmad, Z. et al. 2005,2006] supports the suggestion that it is a candidate target for the next generation ofanti-mycobacterial drugs. CYP144 was highlighted as being important during the latentphase of Mtb growth, a phase that is not targeted by any of the current antimycobacterials.Work performed as part of this PhD has shown that manycharacteristics of CYP144 are highly comparable to those reported for other MtbP450s. CYP144 shows high affinity and specificity towards many azole molecules.Econazole, clotrimazole and miconazole have repeatedly been shown to bind to MtbP450s, including CYP144 and CYP142, with high affinity and are excellent potentialcandidates as novel anti-mycobacterial agents. An N-terminally truncated form ofCYP144, CYP144-T, has been investigated in the pursuit of a CYP144 crystal structure. Itis hoped that this will enable the elucidation of a physiological role for CYP144.Both CYP142 and CYP144 have demonstrated biochemical and biophysicalcharacteristics that contribute to our knowledge of P450 enzymes. This PhD hasestablished that CYP142 exhibits an equilibrium between P450 and P420 species in itsCO-bound, ferrous form. A conversion from P420, and stabilisation of P450, uponsubstrate binding was also demonstrated. CYP144 displays unusual azole coordinationcharacteristics when examined by EPR and removal of the CYP144 gene from Mtbincreased sensitivity of the strain to clotrimazole. Studies of these enzymes hasadvanced knowledge of P450 and Mtb redox chemistry, established roles for the MtbP450 cohort and identified the potential of anti-mycobacterial drugs and associatedtargets.