Structure-based drug discovery using trehalose-6-phosphate phosphatase, an infectious disease target

Abstract

Over 400 million people are currently affected by parasitic worm infections. While seldom fatal, the long-term consequences of helminthiases are significant and global burden is estimated at 22 million disability adjusted life years per annum. Amid growing concerns of zoonosis, treatment resistance and geographical expansion, there has been a call for the development of novel anti-parasitic strategies. To minimise potential treatment side effects, common approaches target proteins that are essential for parasite survival but absent from the host genome. One such target is the essential enzyme trehalose-6-phosphate phosphatase (TPP), the final enzyme in the trehalose biosynthesis pathway. TPP is essential for roundworm survival, a validated target for the treatment of tuberculosis and also shows promise as a vaccine against the nematode Brugia malayi, the causative agent of lymphatic filariasis. This work focuses on the TPP enzymes of the parasitic nematodes Toxocara canis and Ancylostoma ceylanicum as the centre of a target-based approach to identify novel TPP inhibitors with potential as early drug discovery leads. Structure-based sequence analysis of the TPP enzymes of a range of pathogens supported the proposal of three topological groups with structural differences and revealed species-specific variation in what is otherwise considered a highly conserved enzyme family. To account for interspecies variation during downstream drug discovery work, the TPPs of Mycobacterium tuberculosis, Stenotrophomonas maltophilia and Pseudomonas aeruginosa were added to the study, forming a panel of nematode and bacterial TPPs. Enzymatic characterisation of these enzymes revealed that they are kinetically suited to their physiological function and largely employ superstoichiometric burst kinetics, which suggests a role for conformational change during catalysis. To identify novel scaffolds for TPP inhibition, 5,452 compounds were screened as potential TPP ligands. Of these, 222 compounds (4% selection rate) were selected and tested for TPP inhibition in vitro. This work culminated in the identification of a series of novel TPP inhibitors based on three chemical scaffolds, with low-micromolar inhibition constants and structures amenable to further development. The specificity of these compounds suggests that some may provide avenues for cross-species control of a range of parasitic diseases, while others may be leveraged to target only the nematode enzymes. One compound was found to be a species-specific suicide inhibitor of the nematode TPPs and has potential applications as a control or probe molecule in future TPP assay development. Two additional compounds were found to inhibit TPP via a mixed-type ‘competitive noncompetitive’ mechanism, which suggests the presence of an additional binding site distal to the active site. Based on structure-activity analysis, avenues for future rational design of TPP inhibitors are proposed and it is hoped that this work will support the development of new treatments for parasitic disease.