Studies were performed in SPF facilities at the University of Edinburgh under licence PPL 70/8102 and at the University of Glasgow under PPL 60/4442. 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. and is transmitted by the bite of CXCL12 an infected tsetse travel1. The subspecies in eastern and southern Africa2,3. HAT has two stages: the first, haemolymphatic stage includes nonspecific symptoms, such as headache and bouts of fever. The second stage occurs when the parasite has invaded the central nervous system (CNS), leading to progressive mental deterioration and ultimately death. There are five registered drugs currently used to treat HAT; all have a number of drawbacks, including severe side effects associated with significant toxicity/mortality or prolonged and complex dosing regimens, including a requirement for intravenous administration4. A new oral drug, fexinidazole has recently been given approval for use in the clinic (https://www.dndi.org/diseases-projects/portfolio/). The bloodstream form (BSF) of has evolved to rely on the high (5?mM) levels of glucose available in host blood as fuel. In this stage of its life cycle, the parasite mitochondrion is usually highly compromised and cannot carry out oxidative phosphorylation and exclusively uses glycolysis as the sole source of ATP. Our hypothesis was therefore that this glycolytic pathway would be a suitable target for small molecule anti-HAT drugs. As proof of concept, RNA interference-mediated knockdown experiments showed that even a 50% decrease in glycolytic flux is sufficient to kill the parasite in vitro5. phosphofructokinase (TbPFK) is located in peroxisome-related organelles called glycosomes6 and carries out the third step in the glycolytic pathway, phosphorylating fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (F16BP) (Fig.?1). Low sequence identity of ~20% with the three human isoforms (hPFK-M, hPFK-L and hPFK-P) despite sharing very SC-144 similar active sites7 supported the choice of this target. Open in a separate windows Fig. 1 Glycolysis in bloodstream form PFK and showed up to fivefold better potency compared with or parasites in in vitro culture with poor EC50 values of at best ~20?M, presumably due to poor uptake by the parasites10. Open in a separate windows Fig. 2 Optimisation of the CTCB series of inhibitors of TbPFK.IC50 values (M) for inhibition of phosphofructokinase. EC50 values (M) for in vitro parasite killing of the bloodstream form of strain Lister 427 (see Supplementary Methods?2.1 and 4.2). L.E. ligand efficiency. IC50 values are based on at least three impartial measurements (biological replicates). EC50 values were initially decided from two technical replicates. Estimated standard deviations (ESDs) for selected compounds were decided using biological SC-144 replicate studies (in an in vitro killing assay and also tested in an enzyme inhibition assay against TbPFK. The of ?9.57?kcal?mol?1 comprises contribution of ?4.03?kcal?mol?1. The with SC-144 EC50 values between 150 and 250?nM, showing even slightly better potency than against the Tb427 and TbGVR35 laboratory strains (Supplementary Table?4). Structural, enzymatic and binding studies of TbPFK characterise the inhibitory mechanism. Trypanosomatid PFKs have been characterised by X-ray crystallography and two conformational says have been identified that fit with a classical description of an allosteric enzyme that transitions from an inactive T-state conformation to an active R-state conformation13. Comparison of the T-state and R-state structures show that activation of TbPFK requires a large movement of the crucial catalytic residues Asp229 and Asp231 (Fig.?3 and Supplementary Movie?1). The carboxyl groups of the two Asp residues hydrogen bond with the F6P substrate and also coordinate a catalytic magnesium ion, facilitating transfer of a phosphoryl group from ATP. The mobile activating loop is usually locked in its active R-state by the side chain of Leu232, which sits on the same mobile loop and fits into the allosteric drug-binding pocket (Fig.?3 and Supplementary Movie?1). The mode of action for the CTCB family of inhibitors is usually to lock the tetramer in the inactive T-state, with the activation loop held remote from the substrate molecules. Enzyme kinetic studies confirmed that this CTCB compounds are not competitive against either ATP or F6P. TbPFK inhibition was studied using an enzyme assay, in which production of ADP by TbPFK was coupled to the reactions of pyruvate kinase and lactate dehydrogenase: the conversion of pyruvate to lactate and NADH to NAD+, is usually monitored by reduction of UV absorbance at 340?nm. The SC-144 MichaelisCMenten plots (Supplementary Fig.?2 and Supplementary Table?1) show inhibitory behaviour for the compound SC-144 CTCB-405, which is typical for the compound series. The reduction of.
Studies were performed in SPF facilities at the University of Edinburgh under licence PPL 70/8102 and at the University of Glasgow under PPL 60/4442