Methionine Synthases as Targets for Antifungal Drug Development

Abstract

Fungal infections pose an urgent threat to human health and are responsible for approximately 1.6 million deaths annually. Current therapeutic options for life-threatening mycoses, such as those caused by Aspergillus fumigatus, Candida albicans or Cryptococcus neoformans, are severely limited and even under antifungal treatment these infections have mortality rates of ~50%. To improve patient outcomes there is a critical need for development of novel antimycotic drugs. This urgency is further exacerbated by the pharmacological weaknesses of current chemotherapeutics, surges in susceptible patient groups and rising antifungal resistance.   Fungal pathogenic potential is strongly dependent on metabolic versatility. Pathways required for cellular metabolism and nutrient homeostasis in host tissues are fundamental for infection and represent exciting targets for drug discovery. The fungal cobalamin-independent methionine synthase (metH), which belongs to a different enzyme class to the cobalamin-dependent version found in humans, forms a junction between two critical primary metabolic pathways: the transsulfuration and one-carbon cycles. The enzyme has been shown to be essential for the viability and/ or virulence of A. fumigatus and other clinically relevant fungal species indicating it has potential as a fungal specific target with broad applications.   However, the reason why MetH is indispensable for fungi beyond methionine auxotrophy, has so far not been identified. To improve the likelihood of success in the drug discovery process and fully exploit the enzyme’s potential as an antifungal target this thesis investigated the mechanistic basis of MetH’s essentiality in A. fumigatus. Phenotypic analyses using point-mutated versions of MetH revealed that enzymatic activity converting homocysteine to methionine is indispensable for A. fumigatus viability. Overexpression of genes that prevent accumulation of the potentially toxic substrate homocysteine did not rescue growth, implying that its accumulation is not responsible for the enzyme’s essentiality. Supplementation of the growth media with several metabolites which are potentially depleted in the absence of MetH’s enzymatic activity restored fungal viability with very limited growth. This suggested that methionine synthases’ essentiality is explained by the depletion of critical molecules. Metabolomics analyses indicated that suppressing metH reduces cell energetics and phenotypic experiments confirmed that this was the additional cause of MetH essentiality in A. fumigatus, as supplementation with compounds selected to enhance energetics was able to fully rescue growth of the fungus in the absence of methionine synthase enzymatic activity. Taken together, our results suggest that cellular energetics is deleteriously disrupted in MetH’s absence, supporting methionine synthase's value as an antifungal drug target.   As conditions encountered, and consequently fungal metabolic requirements, may vary significantly throughout infection it is crucial to validate targets in established infections. To ascertain the relevance of MetH for fungal growth in various developmental stages we took advantage of the metH_tetOFF mutant, which expresses metH under the control of the regulatable tetOFF system, whereby the addition of doxycycline (Dox) shuts down expression of the gene. Modulation of metH expression in vitro revealed that it is required for growth of conidia, germlings and hyphae in a fungistatic manner. The genetic model was optimised for use in murine and Galleria mellonella models of established invasive aspergillosis. These models showed that downregulation of metH reduces A. fumigatus virulence in established infections at a level comparable to downregulating expression of the target of the azoles, the lanosterol 14-a-demethylase, making methionine synthase an attractive antifungal drug target.   Following validation of MetH as a promising antifungal target thi

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Bromley (Supervisor), Elaine Bignell (Supervisor) & Jorge Amich Elias (Supervisor)