Periodic Reporting for period 1 - Mtb CoaBC (CoaBC from the Coenzyme A pathway of Mycobacterium tuberculosis as an antimicrobial drug target) Reporting period: 2018-04-19 to 2020-04-18 Summary of the context and overall objectives of the project Tuberculosis (TB) is one of the top ten causes of death worldwide, accounting for 1.8 million fatalities per year. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb), the pathogen causing TB, is of particular concern. To overcome this substantial threat to public health, it will be crucial to have access to new anti-tubercular drugs. The biosynthesis pathway of coenzyme A (CoA) is essential in bacteria, and inhibiting the CoA pathway is considered a viable antibiotic strategy that has not yet been thoroughly explored. In Mtb, an important step in the biosynthesis of CoA is the formation of 4’-phosphopantetheine from 4’-phosphopantothenate and L-cysteine. This two-step process is catalysed by the bifunctional enzyme CoaBC. Given its significant different to the human orthologue, CoaBC is considered a highly promising target for the development of novel selective anti-tubercular drugs. While there is genetic evidence indicating that CoaBC is a vulnerable target in Mtb, at the start of this project, there was no existing inhibitors of CoaBC that also showed activity on whole-cell Mtb. Our aim was to develop first CoaBC inhibitors that would also be active on Mtb. This would confirm that targeting CoaBC is a promising new antitubercular strategy with potential against MDR and XDR strains, underlining the viability CoaBC as a new TB drug target. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the course of this this project, we have deployed a fragment-based drug discovery approach to develop novel inhibitors of MtbCoaBC. Starting from initial fragment hits identified by screening a small fragment library, we relied on X-ray crystallography for the structure-guided design of potent MtbCoaBC inhibitors. We used an iterative fragment-growing approach to ultimately obtain a lead compound series of drug-like small-molecules inhibiting the MtbCoaBC enzyme at nanomolar concentrations. We further demonstrated that this series of compounds possesses remarkable activity on whole cell Mtb. These results are a proof-of-concept for the druggability of MtbCoaBC and open avenues to develop a new class of antitubercular drugs that could help addressing emerging resistant mutants. Manuscripts with further details of the findings are in preparation and will be submitted for publication in due course.I have also been involved in the development and testing of other series of MtbCoaBC inhibitors. Some are thought to bind to a cryptic allosteric site in the CoaB subunit of MtbCoaBC in the course of a study that was recently published in Nature Communications (https://doi.org/10.1038/s41467-020-20224-x). Other compounds were found to have modest activity on whole-cell Mtb, providing a first indication for the putative druggability of CoaBC in Mtb. This study was recently published in ACS Infectious Diseases (https://doi.org/10.1021/acsinfecdis.0c00904). More broadly, we have developed inhibitors of E. coli CoaBC and demonstrated that native mass spectrometry can be used as a screening tool to identify novel inhibitors of this enzyme. This work was recently published in the Biochemical Journal (https://doi.org/10.1042/BCJ20190318). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We have rationally developed first-in-class series of non-substrate like small-molecule MtbCoaBC inhibitors that were selective and non-toxic. With these compounds, we demonstrated the vulnerability of the CoA pathway towards chemical inhibition via a new inhibitory mechanism. We also established a proof-of-concept of the druggability of MtbCoaBC by identifying candidate CoaBC inhibitors that also possessed remarkable activity on whole-cell Mtb. These compounds will be useful in the future for the study of the CoA pathway in Mtb, to establish CoA salvage mechanisms, and – with some compounds found to have residual activity on other CoaBC proteins – to study inter-organism differences of the CoaBC enzyme.Moreover, the developed compounds represent an interesting lead series for further development towards antitubercular drugs with a new mechanism of action, thus possessing the potential to target emerging resistant Mtb strains. Antimicrobial resistance is a major public health challenge, and with the rise of drug-resistant mycobacteria, innovative solutions that could lead to the potential development of a pre-clinical candidate active against resistant Mtb are extremely precious. Clearly, knowledge generated during the course of this project has the potential to broadly benefit society.