Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice

I.w Mcnae ,Scott P Webster,Martin K Walker,Simon Pettit,Adrian J Highton,Andrew J Keats,Antonio Vong,Elizabeth A Blackburn,Chris Swain,Jeremy C Mottram,Martin A Wear,Carol Austin,Jacqueline Dornan,Peter M Fernandes ,Ryan Ritchie,Li-Hsuan Yen,Divya Malik,James Kinkead,Paul A.m Michels ,Elmarie Myburgh,Nicholas A Gray ,Malcolm D Walkinshaw

doi:10.1038/s41467-021-21273-6

Abstract

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Highlights

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, known as sleeping sickness
Human African trypanosomiasis (HAT), known as sleeping sickness is caused by two subspecies of the parasitic protist Trypanosoma brucei and is transmitted by the bite of an infected tsetse fly[1]
We show how an X-ray structure-based design approach has led from an initial high-throughput screening hit to the synthesis of a series of a highly specific inhibitors against the trypanosome PFK that block glycolysis in the parasite, but have no effect on the human enzyme

Summary

Introduction

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, known as sleeping sickness. Each compound was tested in vitro in an enzyme inhibition assay and in a parasite killing assay (see ‘Methods’ and Supplementary Fig. 1) leading to the identification of CTCB-12, which potently inhibited TbPFK with an IC50 value of 0.83 μM and high ligand efficiency of 0.51 (Fig. 2).

Results

Conclusion