(BBSRC DTP) Development of novel inhibitors of cholesterol oxidases in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb, the causative agent of TB) represent a huge threat to human life worldwide, with one third of the world’s population thought to be infected by the bacterium. Numerous strains of Mtb have developed resistance to some or all of the frontline antibiotics, and these drug-, multidrug- and even totally drug-resistant strains pose major challenges for the successful treatment of TB. New strategies and new target enzymes in the bacterium are desperately required to provide new antibiotics that can provide effective treatments against Mtb. In this project, the aim is to develop novel inhibitors against key Mtb cytochrome P450 enzymes that are crucial for the bacterium to survive while engulfed in the human macrophage. The P450s CYP125A1, CYP142A1 and (to a lesser extent) CYP124A1 catalyse the primary step in the catabolism of host cholesterol, which is a major energy source for Mtb in the macrophage. Inhibition of these enzymes provides a new route to killing Mtb bacteria engulfed in macrophages, where they otherwise might survive in a dormant state for several years. The strategy used would be fragment based screening – a relatively new method in which target proteins are screened for binding to small compounds (“fragments”), using high throughput NMR and/or calorimetry methods to identify “hit” molecules. The structures of the target P450 enzymes in complex with these various molecules are then solved using X-ray crystallography, which then guides modelling work to guide compound development through e.g. chemically linking/merging molecules together to form larger and tighter-binding inhibitors, or by chemically elaborating the original hits to extend their structures in order to fill adjacent parts of the enzyme active site to produce more effective and specific inhibitors. This strategy has already proved successful in our ongoing work on other Mtb P450s, including CYP121A1, which is essential for Mtb viability1,2, as well as in studies of other Mtb P450s. The targeted cholesterol hydroxylases have been successfully expressed and purified in large yield in our group3-5, and we have determined the crystal structures of these enzymes, which will facilitate fragment screening for these P450s to allow development of novel inhibitors, and enable testing these compounds for their effectiveness against Mtb bacteria. The project will involve training in areas including molecular biology, protein expression, structural biology, enzymology, spectroscopic techniques and inhibitor development. The project also opportunities to work in collaboration with Chris Abell’s group at Cambridge to learn fragment screening technology.

Qualifications
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Contact for further Information
For more details contact Professor Andrew Munro ([Email Address Removed])

https://www.research.manchester.ac.uk/portal/Andrew.Munro.html
https://www.research.manchester.ac.uk/portal/David.Leys.html