Construction of Potent and Specific Inhibitors of M. Tuberculosis Redox Enzymes Using Fragment Screening Methods

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Mycobacterium tuberculosis (Mtb) poses a grave threat to global health. A third of the world's human population is infected by Mtb, and recent years have seen increasing failure of existing TB drugs (developed in the 1940s to 60s) due to Mtb drug resistance by mechanisms including target protein mutations that diminish or abolish binding of inhibitor drugs. We are in an era where Mtb variants resistant to multiple drugs have spread around the world - including strains resistant to at least the two leading drugs (rifampicin and isoniazid, MDR TB) through to those also resistant to second line drugs (extensively drug resistant, XDR TB) or to all major drugs (totally drug resistant, TDR TB). New antibiotics and new approaches to TB drug development are desperately needed to replace failing antibiotics. The approach proposed here involves developing inhibitors of Mtb cytochrome P450 protein systems, where importance of the selected enzymes to bacterial viability, survival in the host and virulence are established. Building on extensive preliminary work underpinning this proposal, we will use fragment screening methods combined with structural biology, enzymology and bacterial MIC determination in order to produce and validate new drug leads. These will be developed against (i) the essential CYP121A1 P450, where we will build on existing tight binding scaffolds to make derivatives with improved affinity and cell penetration; (ii) the cholesterol degrading CYPs 124, 125 and 142 - where we will exploit structural similarity of the enzymes to produce inhibitors that inhibit all these P450s to prevent host cholesterol catabolism; and (iii) CYP128A1 and its partner Stf3, which influence Mtb virulence by hydroxylation and sulfation of menaquinone. Collectively, we will generate, optimize and validate new TB drug leads that attack crucial redox systems in Mtb, and use these reagents in antimicrobial studies to probe the Mtb metabolome/transcriptome to define modes of action.

The proposal builds on substantial preliminary research done by the applicants to demonstrate that fragment screening technology can be used to produce potent inhibitors of M. tuberculosis (Mtb) cytochrome P450 (and partner) enzymes to provide new drug leads that are so desperately needed to treat infections with strains of Mtb that have become resistant to several antibiotics that once formed the basis of effective TB treatments. The emergence of drug resistant, multidrug resistant (MDR, resistant to at least rifampicin and isoniazid), extensively drug resistant (XDR, additionally resistant to a fluoroquinolone and to second line TB drugs) and totally drug resistant (TDR, resistant to all major TB drugs) strains of Mtb poses grave threats to human health worldwide. The appearance of these strains also brings focus on the lack of development of new anti-TB drugs in recent decades, with most of the "traditional" drugs effective against Mtb produced in the 1940s-60s. As resistance to these antibiotics emerged and spread, the lack of effective alternatives was exposed. While this has led to drug development in recent years (with two new drugs now available only for MDR TB treatment), there remains a dearth of new TB drugs at a time when these are most desperately needed. The research proposed in this programme is to progress a fragment screening approach to develop new types of anti-TB drugs that target P450 enzymes known to be crucial to bacterial viability, virulence and to ability to survive in the host through their ability to catabolise host cholesterol as a carbon source in the macrophage. Our previous research has proven the feasibility of this approach, particularly in developing potent inhibitors of the essential Mtb CYP121A1 enzyme. The current programme will extend this work and will also target the menaquinone hydroxylase CYP128A1 and its partner sulfotransferase Stf3, and the cholesterol hydroxylases (CYPs 124, 125 and 142) in order to produce novel compounds as effective new leads against a resilient human pathogen.

Beneficiaries from this research programme include researchers in the area of M. tuberculosis pathogenicity, compound screening and drug targeting. These include scientists in academia and industry who study the microbiology and biochemistry of Mtb with the aim of finding new reagents to treat drug-resistant forms of Mtb. In view of the current crisis in developing new TB drugs, this is an area of particular focus across the globe. Progress in this area has enormous potential to save lives and improve quality of life of millions of individuals. The success of a fragment-based strategy for TB drug development will also inspire researchers to adopt this relatively novel approach to produce drugs to address antibiotic resistance in other pathogenic microbes. This, in turn, should be transformative in the field and enhance the likelihood of identifying effective new antimicrobials through exploitation of new technological approaches. This should inspire further biomedical researchers to adopt the technology and biotechnologists/industrialists to make improvements to fragment screening strategies to streamline processes to enable faster development of potent drugs. Allied studies of the Mtb transcriptome and metabolome following treatment of Mtb with new compounds will also facilitate new insights into drug mechanisms and bacterial responses, and facilitate improvements in drug efficacy, highlighting to other researchers the value of these approaches in improving antibiotic potency. Researchers (PDRA and affiliated students) will be trained in areas including fragment screening, structural biology and transcriptomics, providing key skills for future employment in areas such as antimicrobial research and drug development in academic or industrial sectors. Collectively, this research study has potential to deliver important new drug molecules to provide a basis for new treatments for a deadly disease.