Development of novel inhibitors of cholesterol oxidases in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis) represents a huge threat to human life worldwide. A third of the world's population is 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 tuberculosis (TB). New strategies and new target enzymes in the bacterium are desperately needed to provide novel antibiotics that can deliver effective TB treatments. In this project, the aim is to develop new inhibitors of key Mtb cytochrome P450 enzymes (P450s) 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 in the macrophage, where they otherwise might survive in a dormant state for several years. The strategy used will 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 P450s in complex with these various molecules are then solved using X-ray crystallography, which then informs 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 in order to produce more effective and specific inhibitors. This strategy has proved successful in our ongoing work on other Mtb P450s, including CYP121A1, which is essential for Mtb viability, as well as in studies of other Mtb P450s. The targeted cholesterol hydroxylases have been successfully expressed and purified in large yield in our group, and we have determined the crystal structures of these enzymes. This will facilitate fragment screening for these P450s to allow development of novel inhibitors and testing of these compounds for their effectiveness against Mtb bacteria. The project will involve training of the student in areas including molecular biology, protein expression, structural biology, enzymology/biocatalysis, spectroscopic techniques and inhibitor development. The project also provides an opportunity to work with our research collaborators at Cambridge to gain further skills in fragment screening technology. The project aligns with BBSRC remit in priority areas such as "Technology Development for the Biosciences" (through the use of fragment screening and inhibitor development methods, together with structural biology to produce new compounds effective against key Mtb enzymes) and "Combatting Antimicrobial Resistance" (through producing new lead compounds derived from fragment screening approaches, and demonstrating their activity against Mtb P450 enzymes crucial for bacterial survival in the host). The project also aligns with the DTP theme of "World Class Underpinning Biosciences" (through application of novel fragment screening techniques in order to provide new structure-led strategies for the development of potent Mtb enzyme inhibitors, and their validation through collaboration with collaborators at the Crick Institute.