Development of mode of action tools using lysyl-tRNA synthetase to support Cryptosporidium drug discovery

Abstract

Cryptosporidium is a major cause of diarrhoeal disease particularly in young children, where it accounted for nearly 50,000 deaths in 2016. There are currently no effective treatments for cryptosporidiosis in the patient groups where the disease burden is felt most harshly. This unmet need has led to the development of several promising new therapeutics, including those which target lysyl-tRNA synthetase (KRS). Alongside the development of these novel therapeutics, it is vital that tools are available to identify and characterise the targets of these drugs. This ensures that the Cryptosporidium drug discovery pipeline is populated by compounds acting on known targets that are essential for parasite survival. To achieve this, I aimed to leverage the known target of KRS and its inhibitor, Compound 5, to establish mode of action tools that support Cryptosporidium drug discovery.

By probing the basic biology of this target, I found it to be expressed in all life cycle stages but particularly highly expressed in asexual stages. Conditional knockdown of KRS prevented parasite shedding in a mouse model of infection and inhibition of KRS with Compound 5 caused asexual parasite death. I then used this KRS-Compound 5 drug-target pair to optimise and establish thermal proteome profiling (TPP) for Cryptosporidium. This establishes TPP as the first target blind method of drug target identification in Cryptosporidium.

I then used genetic approaches to generate independent strains which overexpress KRS or express KRS with a single amino acid substitution. In each case, these modifications generated resistance to Compound 5. I then combined these mechanisms of resistance to generate a novel selection marker, KRSR. This selection marker can be used in conjunction with the established NeoR to generate and select for Cryptosporidium genetic crosses, greatly expanding Cryptosporidium genetic engineering.

Date of Award	2023
Original language	English
Awarding Institution	University of Dundee
Sponsors	Medical Research Council, Wellcome Trust, Royal Society & Carnegie Trust for the Universities of Scotland
Supervisor	Mattie Christine Pawlowic (Supervisor) & Susan Wyllie (Supervisor)