Addressing antimicrobial resistance in Mycobacterium tuberculosis - a fragment screening-led approach

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Resistance to antibiotics poses a grave threat to human health worldwide, but is an inevitable consequence of antibiotic overuse. A major threat is posed by strains of Mycobacterium tuberculosis (Mtb) that have developed resistance to leading drugs (e.g. rifampicin, streptomycin and isoniazid) that have been the backbone of successful TB treatments for decades1. Mtb strains resistant to the leading drugs (multidrug resistant, MDR) or to these plus other TB antibiotics (extensively drug resistant, XDR; and even totally drug resistant, TDR strains) are widespread. This prompted the World Health Organization to declare a "global emergency" in a drive to promote research into development of new types of antibiotics that have new modes of action and that attack different Mtb targets. In preliminary work, our group and collaborators have addressed this challenge through studies of cytochrome P450 (P450) enzymes from the virulent strain Mtb H37Rv2. The Mtb H37Rv genome encodes 20 different P450s, and preliminary work revealed that several of these enzymes are crucial to the viability of the bacterium and/or its ability to infect and to sustain infection in the human host3. In collaboration with researchers at the University of Cambridge, we have exploited the relatively new approach of fragment based screening (FBS) in order to identify small ligands that bind to important Mtb P450 enzymes (using calorimetric and NMR-based methods), and to determine how they bind to their P450s using X-ray crystallography. Through chemical "elaboration" of fragments using synthetic chemistry and guided by structural information, several new molecules have been developed that bind tightly to Mtb P450s, including the genetically essential CYP121A1 - a cyclic dipeptide oxidase, and the host cholesterol oxidizing CYP125A1 and CYP124A1 P450s that are crucial for Mtb to sustain infection when engulfed by the human macrophage3. We have reached a point where we have FBS-derived inhibitors of several key MtbP450s4,5, and now require to optimize their structures for both effective target inhibition and for chemical properties conducive to good Mtb cell penetration. The student will progress this project through development of HPLC-/GC-MS based assays to identify products formed by key Mtb P450s and through demonstrating and quantifying P450 inhibition by FBS-derived compounds. Research will also include expression/purification of novel Mtb P450s, identification of new ligands/inhibitors of these P450 by fragment screening, and structural characterization of key Mtb P450s in complex with new inhibitors en route to production of novel classes of Mtb antibiotics.