Mycobacterium tuberculosis (Mtb), a deadly pathogen, has scourged mankind for many centuries and has remained a major threat to global world health. Tuberculosis, the disease caused by this bacterium, is a major cause of death in developing nations and there is potential for its re-emergence in developed countries. An alarming rise in cases of multidrug-resistant and extremely-drug resistant tuberculosis (MDR-TB and XDR-TB) that do not respond to the customary first-line antibiotics necessitates the urgent need for development of new anti-TB drugs. Mtb becomes engulfed in human macrophages post infection of the host, but persists in the harsh environment of the human lungs by utilization of host cholesterol as a carbon source. The P450s CYP125A1, CYP142A1 and CYP124A1 are responsible for catalysing the side-chain degradation of cholesterol, which is critical for cholesterol to be used in the Mtb β-oxidation pathway for energy production. This PhD thesis focuses on understanding the structure/mechanism of the Mtb cholesterol 27-oxidases with the aim of facilitating the development of novel inhibitors of these P450s, which are crucial for Mtb to infect the host and to sustain infection. CYP142A1 and CYP124A1 were purified through three chromatographic steps with contaminating proteins successfully removed to give highly pure forms of these enzymes following the final purification step. Spectrophotometric titrations indicate that CYP142A1 and CYP124A1 bind tightly to cholesterol and cholestenone (and also to branched-chain methyl lipids for CYP124A1), highlighting their physiological roles in sterol and fatty acid metabolism, respectively. Binding analyses with a range of azole antibiotics revealed tight binding to bifonazole, clotrimazole, miconazole and econazole, and weak binding to fluconazole. Studies with compounds from a fragment screening library revealed weak binding to fragment hits for the cholesterol oxidases, but much tighter binding to these enzymes was found for ‘elaborated’ hits from a previous fragment screen on the Mtb cyclodipeptide oxidase CYP121A1, indicative of improved ligand potency achieved via ‘fragment merging’ strategies, and of structural similarities between these diverse Mtb P450s. Light scattering data indicate that CYP142A1 exists in dimeric form in solution, but becomes monomeric when treated with DTT; while CYP124A1 is completely monomeric. Crystal structures of CYP142A1 and CYP124A1 in complex with cholestenone, econazole and fragment library hits were determined. CYP142A1 crystal structures with econazole and fragment hits revealed heme coordination via the heterocyclic nitrogen in an azole group, and provide important data towards design of superior inhibitor drugs. The binding of cholestenone within the active site channels of CYP124A1 and CYP142A1 revealed an alignment favourable for C27 hydroxylation of the cholestenone side chain, which supports the physiological roles of CYP142A1 and CYP124A1 (as well as CYP125A1) in host cholesterol catabolism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:694265 |
| Date | January 2016 |
| Creators | Amadi, Cecilia Nwadiuto |
| Contributors | Munro, Andrew |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/biochemical-and-drug-targeting-studies-of-mycobacterium-tuberculosis-cholesterol-oxidase-p450-enzymes(16cbca7a-b8b2-4ec4-bbd7-977785ed65b9).html |
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