| dc.description.abstract | The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the
‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis
respectively. In their human infective form in the bloodstream all three trypanosomatid
parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the
third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the
phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6-
bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is
essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set
of structural and regulatory differences compared to the mammalian host enzyme. These
differences, coupled with the availability of trypanosomatid PFK crystal structures present an
opportunity for the structure-based design of specific inhibitors against the enzyme.
Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and
Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and
their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’)
were designed against T. brucei PFK with the aim of developing novel treatments against Human
African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical
characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery
program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to
trypanosomatid PFKs. We show that the compounds are specific; neither competing with the
natural substrates ATP or F6P nor inhibiting the human PFK enzyme.
We describe the development and testing of highly potent and specific low molecular weight
PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage
I HAT in mice models. We describe the tight, 1:1 binding of these compounds with
trypanosomatid PFKs, and the thermodynamic characteristics of binding through various
biophysical assays. We also show the unprecedented characterisation of the reverse PFK
reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry
out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and
F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric
regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the
forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and
inhibition by the drug-like compounds against trypanosomatid PFKs. | en |
