NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

Grant: 1 R03 MH092153-01
PI Name: Malcolm Walkinshaw, University of Edinburgh

NCGC Assay Overview:

Various species of the protozoan family Trypanosomatidae are responsible for a range of serious human diseases in tropical and subtropical areas of the world. The subspecies Trypanosoma brucei is one of three known to cause sleeping sickness in sub-Saharan Africa, significantly contributing to the millions of people worldwide who are infected by these parasites and endangering hundreds of millions more. Many of the disorders caused by trypanosomatids are fatal if left untreated, but most currently used drugs are inefficient and toxic.
Of many possible drug targets in trypanosomatid parasites, the carbohydrate metabolism pathway is seen as potentially one of the most selective, as T. brucei, when in the bloodstream of its mammalian host, is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply [1]. Oxidative metabolism involving the mitochondrial tricarboxylic acid cycle and oxidative phosphorylation are repressed in these parasites, and recent RNA interference (RNAi) experiments have shown that even partial depletion of certain individual glycolytic enzymes can lead to the death of cultured parasites [2]. One such glycolytic enzyme, phosphofructokinase (PFK), catalyzes the formation of fructose-1,6-bisphosphate (F1,6BP) and ADP from fructose-6-phosphate (F6P) and ATP, and in many metabolic circumstances makes an important contribution to the control of flux through the glycolytic pathway. As a specific glycolytic target, PFK is particularly attractive as it catalyzes the first irreversible step in glycolysis, and structural and kinetic studies have shown very substantial and essential differences from corresponding host enzymes [3], allowing for the discovery of parasite-selective inhibitors.
The goal of this screen is to find small-molecule inhibitors of T. brucei PFK using a biochemical assay that measures PFK activity as a function of ADP production. A luminescence-based assay for T. brucei PFK activity has been developed which uses luciferase to detect production of ADP. Traditional luciferase-based nucleotide detection reagents rely on ATP production as a direct substrate for the luciferase reaction; however, PFK catalyzes the transfer of a phosphate from ATP to F6P, resulting in the net depletion of ATP. To directly measure the ADP product of the PFK reaction, we used the ADP-Glo detection kit (Promega), which utilizes two separate reactions to 1) deplete all remaining uncatalyzed ATP and 2) convert all remaining ADP to ATP, which is then detected through a traditional luciferase-coupled reaction. Compounds were screened as a concentration-titration series that ranged from 57 uM to 0.7 nM.

Please refer to AIDs 485367 and 485368 for assay details.

This in an ongoing project, we will make the update accordingly once a probe is identified.

Grant Number: 1 R03 MH092153-01