Biochemical and biophysical studies of Trypanosoma cruzi phosphofructokinase as a target against Chagas disease

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• 作者：Dr Peter M Fernandes ; BMBCh^a ; ^{peter.fernandes@ed.ac.uk" class="auth_mail" title="E-mail the corresponding author} ; James Kinkead ; BSc^a ; Iain McNae ; PhD^a ; Prof Paul Michels ; PhD^a ; Prof Malcolm Walkinshaw ; PhD^a
• 刊名：The Lancet
• 出版年：2016
• 出版时间：25 February 2016
• 年：2016
• 卷：387
• 期：supp_S1
• 页码：S43
• 全文大小：62 K

文摘

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a chronic infection affecting the sympathetic nervous system; no effective non-toxic treatments are available. Inhibitors of trypanosomatid glycolytic enzymes are promising therapeutic targets, as shown by research on T brucei phosphofructokinase (PFK). The aim of this study was to investigate the structure, function, and regulation of T cruzi PFK, to provide insights into the suitability of this enzyme as a drug target.

Methods

BL21(DE3) Escherichia coli cells were transformed with pET28a plasmid containing cDNA for T cruzi PFK with N-terminal hexahistidine tag, and induced with isopropyl-β-D-thiogalactopyranoside. Pellets were lysed by liquid homogenisation and purified through immobilised metal ion affinity chromatography and size exclusion chromatography. Kinetic values and inhibition were determined with an auxiliary enzyme-linked assay with pyruvate kinase and lactate dehydrogenase. Crystallisation was done via the hanging drop method in 24-well plates at 18°C.

Findings

T cruzi PFK was fully purified and verified with mass spectrometry (Mascot score of 241). Dynamic light scattering indicated a homogenous sample of molecular weight 239 kDa. Apo-enzyme and holo-enzyme crystals were produced (cofactors ATP or AMP). Maximum specific activity (V_max) was 58·9 μmol/min per mg. ATP binding was hyperbolic (Michaelis-Menten kinetics) with K_mATP 52·9 μM. F6P binding was sigmoidal (allosteric sigmoidal kinetics) with K_halfF6P 429 μM. GTP was an alternative substrate but showed reduced activity and affinity compared with ATP. AMP was an allosteric activator, reducing K_mATP to 15 μM. F16BP was inhibitory above 2·5 mM, but no other metabolites altered activity. Novel small molecule inhibitors demonstrated sub-50 nanomolar IC50 values for T cruzi PFK, and were inactive against human PFK.

Interpretation

The biophysical results indicate that T cruzi PFK is tetrameric, similar to other PFKs. Compared with T brucei PFK, T cruzi PFK has a higher affinity for ATP and F6P and similar V_max. The relative lack of allosteric regulators can be explained by the localisation of T cruzi PFK within glycosomes. The physiological significance of GTP use is doubtful given the lack of a glycosomal mechanism for regenerating GTP from GDP. Novel inhibitors bound allosterically, showing that they have great promise as candidate drugs.

Funding

Wellcome Trust.