Biochemical characterisation and essentiality of proteins involved in myo-inositol metabolism from Trypanosoma cruzi

Abstract

myo-Inositol is a precursor to the essential membrane lipid, phosphatidylinositol (PI). In Trypanosoma cruzi, PI may form inositol phosphorylceramide (IPC) and glycosylphosphatidylinositol (GPI)-anchored molecules/glycoproteins, which coat the parasite’s cell-surface for essential parasite-host interactions.

In T. cruzi, myo-inositol is putatively de novo synthesised and extracellularly scavenged. The project’s aim was characterising the putative inositol-3-phosphate synthase (TcINO1) from the de novo synthesis pathway and the myo-inositol transporter (TcMIT) from the extracellular uptake pathway.

Each protein was recombinantly expressed in E. coli. This demonstrated TcMIT is a multi-transmembrane (TM) domain protein with 12 likely TM domains, suggesting TcMIT might be an H⁺ symporter. Recombinant TcINO1 expression and purification allowed TcINO1 confirmation as a bona fide INO1 with Michaelis-Menten kinetics.

Heterocyclic fragment libraries were screened for compounds interacting with recombinantly purified TcINO1. Several Otava library compounds stabilised TcINO1, which demonstrated initial positive homotropic cooperativity.

TcINO1 and TcMIT were genetically manipulated in T. cruzi. This revealed de novo synthesised myo-inositol may be primarily utilised for alkyl acyl PI and incorporated into GPI-anchors, whilst extracellular myo-inositol might be mainly used for bulk diacyl PI for membranes or incorporated into IPC. Decreased exogenous myo-inositol may be compensated by de novo synthesis; however, the converse was not observed.

T. cruzi TcINO1 and TcMIT genetic manipulations demonstrated TcINO1 is cytosolic whilst TcMIT is plasma membrane bound. It also genetically validated both genes as essential T. cruzi genes.

Otava library compounds stabilising recombinantly purified TcINO1 were tested in parental strain T. cruzi. The three most potent compounds altered EC₅₀s in genetically altered TcINO1 T. cruzi cell lines. This strongly suggests TcINO1 is a chemically validated Chagas’ disease therapeutic target. The best compound had an EC₅₀ (EC₅₀ = 6.11 ± 0.31 μM) on par with Nifurtimox, the current Chagas’ disease therapy, and a high selectivity index, making it preferable over Nifurtimox.

Date of Award	2 Jul 2025
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Terry K Smith (Supervisor)