Tb PFK orthogonal confirmatory assay using ATP depletion (Kinase-Glo Plus) as an alternative measure of Tb PFK activity: Hit Validation
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. ..more
BioActive Compounds: 490
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 . 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 . 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 , allowing for the discovery of parasite-selective inhibitors.
The primary qHTS data of T. brucei PFK and ADP-Glo confirmatory data are available in PubChem (AIDs: 485367 and 485368). A luminescent secondary assay was also performed that used luciferase-based nucleotide detection (Kinase-Glo Plus, Promega) similar to the qHTS, but which used ATP (rather than ADP) concentration as an alternate measure of T. brucei PFK activity. The assay measures substrate depletion (ATP) rather than product formation (ADP), coupled to an identical manner of luciferase-based signal detection. As such, compounds which truly act on T. brucei PFK produce a change in signal in the opposite direction of the primary qHTS assay, while compounds which affect the final luciferase reaction produce signal changes in identical directions. Compounds were screened as a concentration-titration series that ranged from 57uM to 0.7nM.
1. Verlinde, C.L., et al., Glycolysis as a target for the design of new anti-trypanosome drugs. Drug Resist Updat, 2001. 4(1): p. 50-65.
2. Albert, M.A., et al., Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei. J Biol Chem, 2005. 280(31): p. 28306-15.
3. McNae, I.W., et al., The crystal structure of ATP-bound phosphofructokinase from Trypanosoma brucei reveals conformational transitions different from those of other phosphofructokinases. J Mol Biol, 2009. 385(5): p. 1519-33.
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH092153-01
PI Name: Dr. Malcolm Walkinshaw
Substrate buffer was dispensed into white, solid 1536-well plates at 3ul/well in 0.1M triethanolamine (TEA) buffer, pH 8.0, containing final concentrations of 5 mM MgCl2, 0.01% Tween20, 0.5 mM fructose-6-phosphate (F6P) and 0.1mM ATP. Then, 23nl of compounds or DMSO were delivered to each well using a pin tool. One ul T. brucei PFK (0.1M TEA, 0.01% Tween20, 0.1% bovine serum albumin (BSA) and 1.25nM PFK, final concentrations) was then dispensed, and plates were incubated at room temperature for 45 min. The luminescent detection reagent, Kinase-Glo Plus (Promega), was then added at 2ul/well and incubated for ten minutes at room temperature. The plates were measured on a ViewLux plate reader for luminescent signal using a clear filter with a one second exposure. The %Activity was determined from the corrected luminescence values. As no specific T. brucei PFK inhibitors have been identified in the literature, 1x (1.25nM) and 0x PFK enzyme controls (untreated) were included to normalize %Activity of identified inhibitors; 0x enzyme values corresponded to 100%Activity (full inhibition), while 1x PFK enzyme values were used to normalize 0% Activity (no inhibition). The Kinase-Glo Plus protocol varies from the primary screen in that it measures ATP, rather than ADP, concentration, and thus provides a measure of substrate depletion, rather than of product formation.
Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent increases in luminescence, concordant with a decrease in PFK activity and ATP depletion. Inversely, active activators showed a concentration-dependent decrease in luminescence signal below 1x PFK control levels, suggestive of an increase in ATP depletion. Inactive compounds showed no effect on luminescence signal.
Keywords: T. brucei, phosphofructokinase, PFK, glycolysis, luminescence, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)