Creating effective drug discovery methods in Mycobacterium abscessus

With the inevitability of resistance emergence, the only way to ensure future antibiotic efficacy is through continued development of new drugs capable of evading resistance mechanisms. However, over the past 30 years the antibiotic drug development space has been characterised by the withdrawal of recourses, large pharmaceutical companies, and funding. This is a result of a mismatch between development costs and potential revenue, where high development costs are largely due to the attrition of compounds at different stages of development. There is an urgent need for better antibiotic discovery methods, both to repopulate the current pipeline, and to reliably discover novel compounds suitable for development. In this thesis I have focussed on developing tools which overcome the limitations of phenotypic drug discovery, namely that there is limited information on how a compound works available when critical decisions of resource commitment are made. I identify hit identification and hit-to-lead progression as two key components of early discovery and have created tools which can be used to improve decision making during these stages. These tools include a phenotypic screening selection funnel, a CRISPRi arrayed library, and transcriptomic predictions of drug action. Increasing the focus of antibiotic discovery has been on narrow spectrum, species specific compounds, and I have focussed on developing tools in a single bacterium, Mycobacterium abscessus. M. abscessus is an emergent human pathogen which causes lung infections in those with underlying lung conditions, most significantly patients with cystic fibrosis. M. abscessus is also highly drug resistant, and currently has very limited therapeutic options, and even with triple- antibiotic-therapy culture negativity is rarely achieved. Initially I performed a phenotypic screen using a diversity orientated synthesis library, demonstrating that functional diversity of compound libraries results in a chemically diverse selection of hits. Further I have developed a hit triage approach which aims to specifically select for compounds which have features good for clinical infection, selecting 4 compounds from a library of 1562. I describe the generation of a structure activity relationship for two of these compounds, TD178 and 14F8. Identifying target identification as a limiting factor in hit to lead progression I have also developed a method for the creation and characterisation of a CRISPR-interference arrayed library in M. abscessus. This CRISPRi library could be used for target identification and validation. Finally, to further allow the identification of novel mechanism of action I have used RNAseq to characterise the transcriptional impact of different antibiotics and examine whether there is mechanism of action specific signatures.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Morris, Bethan

Advisor dc:contributor.advisor

• Floto, Andres

• Antibiotics
• Drug discovery

Licence

• https://www.repository.cam.ac.uk/bitstreams/51021309-72d7-4d1d-b6cc-15e1d8e9a2e1/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.122748

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/391731