qHTS Validation Assay to Find Inhibitors of Phosphoglycerate Kinase
throughout the tropical and subtropical world. Sleeping sickness, caused by two subspecies of Trypanosoma brucei, is endemic to areas of sub-Saharan Africa, while Chagas disease, a manifestation of Trypanosoma cruzi infection, is prevalent throughout Central and South America. In addition, many species of Leishmania are responsible for a range of cutaneous and visceral diseases, causing ulcers, more ..
BioActive Compounds: 19
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH093212-01
PI Name: Malcolm Walkinshaw, University of Edinburgh
NCGC Assay Overview:
Several species of the Trypanosomatidae family of parasitic protozoans cause a spectrum of diseases
throughout the tropical and subtropical world. Sleeping sickness, caused by two subspecies of Trypanosoma brucei, is endemic to areas of sub-Saharan Africa, while Chagas disease, a manifestation of Trypanosoma cruzi infection, is prevalent throughout Central and South America. In addition, many species of Leishmania are responsible for a range of cutaneous and visceral diseases, causing ulcers, destructive lesions and kalaazar, a lethal infection of the viscera. Collectively, these organisms infect 30 million people worldwide (~500 million live in endemic areas), and account for ~130,000 deaths annually. Given their debilitating and prevalent nature, trypanosomatid parasites impose a heavy medical, economic and social burden on entire populations, primarily in the developing world. The problem is further exacerbated by the fact that existing treatments have progressed little in the past 50 years, and current drugs show high toxicity and poor efficacy.
This project will build on previous work that has demonstrated carbohydrate metabolism to be a strong drug target in trypanosomatids . Blood-stream stages of T. brucei are entirely dependent on glycolysis as a source of ATP, and the tricarboxylic acid cycle and oxidative phosphorylation are both repressed. Glycolysis in axenically cultured T. cruzi, representing the intracellular pathogenic stage, is also essential for these parasites . Moreover, the glycolytic pathway in trypanosomatids is organized in a unique manner, with the first seven of the ten enzymes sequestered inside organelles called glycosomes , with the consequence that many enzymes differ significantly from their host counterparts. Previous studies have shown that the glycolytic enzyme, phosphoglycerate kinase (PGK), is a viable target for drugs to kill these parasites. Known inhibitors of TbPGK (glycosomal isoenzyme) include the small molecules suramin , Ib1 , a bisubstrate analog , as well as a series of adenosine derivatives ; several such compounds were demonstrated to effectively stunt the growth of T. brucei and T. cruzi parasites in culture .
NCGC Assay Protocol Summary:
A stepwise description of the 1536-well assay is shown in Table 2. Briefly, 3ul of substrate/PGK buffer (columns 1,2,5-48) are dispensed into Greiner white solid bottom 1536-well assay plates; columns 3-4 receive only TEA buffer as 0x PGK controls. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10nl slotted pins, V&P Scientific, San Diego, CA). Following addition of compound, 1ul of GAPDH (100nM final concentration, all columns) is added to initiate the reaction (this upstream coupling enzyme is necessary for in-well production of PGK substrate, 1,3-bisphosphoglycerate (1,3-BPG), which is unstable and not commercially available). The plates are then incubated at room temperature for 60 minutes. Kinase-Glo Plus reagent is added (2ul) and plates incubated for 10 minutes, before being transferred to a ViewLux high-throughput CCD imager (PerkinElmer) wherein single end-point measurements of luminescence are acquired using a clear filter. All reagents are diluted in an assay buffer consisting of 100mM TEA (pH 7.6), 5mM MgCl2 and 0.01% Tween-20.
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 inhibitors are ranked higher than compounds that showed apparent activation.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)