Molecular tools to unravel the mechanism of action of M. tuberculosis phenotypic hits

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Moorey, Alice Ruth ORCID: https://orcid.org/0000-0003-3417-8643 (2022). Molecular tools to unravel the mechanism of action of M. tuberculosis phenotypic hits. University of Birmingham. Ph.D.

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Abstract

Until early 2020, tuberculosis (TB) was the deadliest disease caused by a single infectious agent. While Covid-19 has since gained the morbid accolade of the most lethal pathogen, TB remains a major threat to global health. The increasing rate of multi drug resistant (MDR) and extensively drug resistant (XDR) TB has led to the indisputable need for new anti-TB drugs. The primary aim of this research has been to investigate hits from drug screening programmes and, through the application of microbiological and molecular techniques, elucidate their targets. With a focus on re-examining existing therapeutics in the context of TB drug discovery, we tested three distinct groups of drugs to explore their activity against Mycobacterium tuberculosis (Mtb): BM212 series inhibitors of the transporter protein MmpL3; human diacylglycerol transferase (DGAT1) inhibitors and human kinase inhibitors. Inhibitors of MmpL3 are notoriously structurally diverse, and it is not uncommon for them to have alternative targets. Using chemoproteomic profiling the BM212 series of inhibitors were screened for alternative protein targets, revealing the binding of BM212 analogue compounds to the transcriptional regulator EthR2, which was confirmed using an in vitro tryptophan fluorescence assay. In the second chapter, we have explored the anti-TB activity of human DGAT1 inhibitors. Mtb is able to utilise host triacylglycerol as a source of energy and carbon and can store large amounts of TAG as intracellular lipid inclusion bodies (ILIs). Preliminary research showed that inhibiting host DGAT1 reduces TB infection in macrophages; we have demonstrated the activity of DGAT1 inhibitors against Mtb H37Rv and M. bovis BCG in vitro. Using a combination of whole cell assays, lipid profiling, and biochemical assays, we have observed the inhibition of bacterial triacylglycerol synthases by a DGAT1 inhibitor and have identified Tgs3 as the DGAT1 inhibitor protein target. Finally, we have selected phenotypic hits from a screen of human kinase inhibitors for their activity against Mtb and through the sequencing of spontaneous resistant mutants have identified a number of potential targets in Mtb.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):