Genetic and microbiological tools to unravel the mechanism of action of novel anti-tuberculosis new chemical entities

Burke, Christopher E. ORCID: 0000-0003-3933-3403 (2021). Genetic and microbiological tools to unravel the mechanism of action of novel anti-tuberculosis new chemical entities. University of Birmingham. Ph.D.

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Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, is the leading cause of mortality from a single infectious agent. The treatment of this disease is a long and difficult process requiring full patient compliance, otherwise risking a recurrent infection and/or antibiotic resistance. The treatment time of drug susceptible tuberculosis is six months and uses drugs that are tolerable for the patient. Rifampicin resistant tuberculosis and multi-drug resistant tuberculosis treatments rely on drugs that are either poorly tolerated by the patient or have an unknown mechanism of action. Identifying new drugs that could shorten the treatment time of tuberculosis or have reduced side effects is imperative. This thesis focuses on drug discovery with an emphasis on using a target specific high-throughput phenotypic screening to identify potential hits and leads. The resulting hits are then re-examined, using microbiological, genetic, and biochemical techniques to confirm the target protein/complex of the hits. The first project uses a media supplementation assay to identify hits that inhibit amnio acid biosynthesis. These pathways are well-validated using known inhibitors of branched chain amino acid biosynthesis. The second project focuses on a large phenotypic screen designed to identify inhibitors of the protein synthesis pipeline. Finally, the third project uses numerous biochemical assays to assess the interactions of hits selected from an overexpression screen of the Antigen 85 protein, fbpA. The hits were then evaluated using a combination of biochemical assays, microbiological techniques, and whole genome sequencing to confirm the targets of the hit compounds. While the success rate of identifying hits from compound libraries is low in these projects, these methods have all been validated and would be suitable for a more industrialised method of high-throughput screening of compound libraries.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):