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BioAssay: AID 588333

Confirmatory Assay to Find Inhibitors of T. brucei phosphofructokinase: T. brucei in vitro Parasite Assay

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
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
AID: 588333
Data Source: NCGC (PFK809)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-08-25
Hold-until Date: 2012-08-25
Modify Date: 2012-08-26

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 5
Depositor Specified Assays
AIDNameTypeComment
488768qHTS Assay to Find Inhibitors of T. brucei Phosphofructokinase: SummarysummarySummary AID
Description:
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.

Selected active compounds were additionally tested in a well-validated in vitro parasite assay to determine the relative potencies of lead compounds against live T. brucei cultures.

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

MLPCN Grant: MH092153-01
Assay Submitter (PI): Malcom Walkinshaw, University of Edinburgh
Protocol
Dilutions of T. brucei brucei were incubated in modified Iscove's cell culture medium (supplemented with 10% FBS, 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate and 0.16 mM thymidine) with titrations of compound for 72 hours. The cell density is adapted so that after 72 hours of incubation, the cells are at the end of the logarithmic growth phase (~5x104 tryps/mL). General morphology and motility were recorded after incubation, and Alamar Blue dye was added as a fluorescent marker of viability (ex530/em590) and read on a fluorescent plate reader following 4 hrs of incubation at 37 degrees C. The results of this assay were used to prioritize medicinal chemistry efforts in conjunction with the human cell toxicity assay to give a measure of relative parasite/host toxicity ratio.
Comment
Compounds are considered "active" if their E50 is below 200 uM; compounds are considered "inactive" if no EC50 could be observed.

An Activity score of 80 is assigned to compounds with an EC50 below 40 uM; an activity score of 50 is assigned to compounds with an EC40 above 40 uM. An activity score of 0 is assigned to all compounds that are inactive.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Phenotype String
2EC50 (uM) FloatμM
Additional Information
Grant Number: MH092153

Data Table (Concise)
Classification
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