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BioAssay: AID 2579

G6DPH counterscreen for TbHK1 inhibitors - Analogues series

Trypanosoma brucei, the digenic protozoan parasite that causes African sleeping sickness in man, annually infects ~500,000 people in sub-Saharan Africa, leading to 50,000-70,000 deaths per year. Glucose metabolism is essential for the parasite, with the pathogenic lifestage of the parasite, the bloodstream form (BSF), acquiring energy exclusively through glycolysis. ..more
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 Tested Compounds
 Tested Compounds
All(181)
 
 
Inactive(181)
 
 
 Tested Substances
 Tested Substances
All(182)
 
 
Inactive(182)
 
 
AID: 2579
Data Source: University of Pittsburgh Molecular Library Screening Center (MH082340-G6PDH counterscreen for TbHK1 inhibitors-First anal..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-03-16
Modify Date: 2010-12-09

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeProbeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1Screening depositor-specified cross reference: HTS assay for inhibitors of Trypanosoma brucei hexokinase 1 - primary screening data
1632HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinationsConfirmatory depositor-specified cross reference: HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinations (confirmatory)
2230Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first seriesConfirmatory depositor-specified cross reference: Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1 - analogue - first series
2600Identification of Inhibitors of Trypanosoma Brucei Hexokinases - summary assaySummary1 depositor-specified cross reference
449725IMR-90 (cell viability counter screen)Confirmatory depositor-specified cross reference
492951Human Glck Counter Screen AssayConfirmatory depositor-specified cross reference
2516G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compoundsOther same project related to Summary assay
2560Rescreen of TbHK1 primary actives - DPI cherry picked compoundsOther same project related to Summary assay
Description:
Excerpt from MH082340 application (Dr. James Morris, Clemson University)

Trypanosoma brucei, the digenic protozoan parasite that causes African sleeping sickness in man, annually infects ~500,000 people in sub-Saharan Africa, leading to 50,000-70,000 deaths per year. Glucose metabolism is essential for the parasite, with the pathogenic lifestage of the parasite, the bloodstream form (BSF), acquiring energy exclusively through glycolysis.

Hexokinase (HK), the first enzyme in glycolysis, catalyses the transfer of the phosphoryl group of ATP to glucose yielding glucose-6-phosphate. Several lines of experimental evidence confirm that HK activity is essential to T. brucei. First, RNA interference (RNAi) of HK in BSF parasites is lethal (see below and (Albert et al., 2005)). Also, attempts to generate knockouts have been unsuccessful (below and (Albert et al., 2005)). Last, specific inhibitors of TbHK activity have been developed that are trypanocidal, albeit at high concentrations (Trinquier et al., 1995; Willson et al., 2002).

T. brucei expresses two nearly identical HKs, TbHK1 and 2, from genes found in tandem on chromosome 10. Interestingly, the polypeptides are 98% identical. TbHK1 and 2 are distinct from mammalian HKs, however, sharing only 30-33% sequence identity. The biochemical differences between TbHKs and human HK suggest that TbHKs could be therapeutic targets. Indeed, it has been suggested that the possibility of developing specific inhibitors for TbHKs is far from remote (Opperdoes and Michels, 2001), and now our ability to generate active recombinant protein makes identifying long sought-after inhibitors a possibility.

Thus, a simple "mix and read" absorption-based assay was adapted to HTS format by the University of Pittsburgh Molecular Library Screening Center (PMLSC, a part of the Molecular Library Screening Center Network (MLSCN)) and was used to screen the MLSCN compound library for inhibitors of the enzyme. The TbHK1 assay was used to screen the NIH-SMR and the data has been posted on Pubchem. All primary actives were then counter screened using the G6DPH counterscreen (coupled assay) to remove chemotypes that interfere with the assay format.
Protocol
G6PDH counter-screening assay protocol
The basic screening procedure for the G6PDH HTS assay follows a stepwise addition of reaction mixture components (as follows):

1. 15 uL of a 30 uM concentration of test compound is added to appropriate wells (20 point concentration range - 20 uM highest concentration)
2. 15 uL of a G6P and NAD+ mixture is added for a final concentration of 0.2 mM and 0.6 mM respectively.
3. 15 uL of G6PDH enzyme is added per well (final concentration = 0.006 mUnits/uL).
4. Reaction incubates for 1 hour at room temperature.
5. 5 uL EDTA is added to each well (final concentration = 50 mM).

This assay was used as a counterscreen for the TbHK1 small molecule inhibitor analogues generated by the University of Kansas. These compounds were also rescreened in the TbHK1 dose response assays. This data reported within this assay indicates as to whether the analogues synthesized interfere with the assay format.
Comment
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when the mean IC50s is > 25 uM
2 - Substance is considered active when the mean IC50s is < 25uM

PUBCHEM_ACTIVITY_SCORE
20 - Compounds that were inactive in all triplicate 20-pt dose response assays with a mean IC50 > 25 uM.
40 - Compounds that were active in one of the triplicate 20-pt dose response assays with an IC50 >25 uM but exhibited an IC50 < 25 uM in the other runs.
60 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 15to 25 uM range
70 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 10 to 15 uM range
80 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 5 to 10 uM range
90 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 0 to 5 uM range
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierThis qualifier is intended to be interpreted with the TID called "IC50 _Mean". If qualifier is "=" than "IC50 _Mean" equals to the value in that column, if qualifier is ">" than "IC50 _Mean" is bigger than that value (Run 1)String
2IC50_Mean *Mean IC50 for the TbHK1 analogue derived from three replicate runs in the G6PDH counterscreening assayFloatμM
3Qualifier_IC50_Run1 This qualifier is intended to be interpreted with the TID called "IC50 _Run1". If qualifier is "=" than "IC50 _Run1" equals to the value in that column, if qualifier is ">" than "IC50 _Run1" is bigger than that value (Run 1)String
4Qualifier_IC50_Run2 This qualifier is intended to be interpreted with the TID called "IC50 _Run2". If qualifier is "=" than "IC50 _Run2" equals to the value in that column, if qualifier is ">" than "IC50 _Run2" is bigger than that value (Run 2)String
5Qualifier_IC50_Run3 This qualifier is intended to be interpreted with the TID called "IC50 _Run3". If qualifier is "=" than "IC50 _Run3" equals to the value in that column, if qualifier is ">" than "Run3" is bigger than that value (Run 3)String
6DR_IC50uM_Run1 IC50 determination for replicate 1 in the G6PDH counterscreening assayFloat
7DR_IC50uM_Run2 IC50 determination for replicate 2 in the G6PDH counterscreening assayFloat
8DR_IC50uM_Run3 IC50 determination for replicate 3 in the G6PDH counterscreening assayFloat
9DR_Hillslope_Run1 Dose response curve hillslope for replicate 1Float
10DR_Hillslope_Run2 Dose response curve hillslope for replicate 2Float
11DR_Hillslope_Run3 Dose response curve hillslope for replicate 3Float
12DR_Plate Mean Max Signal_Run1 Mean assay plate max for replicate 1Float
13DR_Plate Mean Max Signal_Run2 Mean assay plate max for replicate 2Float
14DR_Plate Mean Max Signal_Run3 Mean assay plate max for replicate 3Float
15DR_Plate Mean Min Signal_Run1 Mean assay plate min for replicate 1Float
16DR_Plate Mean Min Signal_Run2 Mean assay plate min for replicate 2Float
17DR_Plate Mean Min Signal_Run3 Mean assay plate min for replicate 3Float
18DR_plate Z'-factor_Run1 Assay plate Z factor for replicate 1Float
19DR_plate Z'-factor_Run2 Assay plate Z factor for replicate 2Float
20DR_plate Z'-factor_Run3 Assay plate Z factor for replicate 3Float
21DR_Run_date_run1 Assay date of replicate 1 String
22DR_Run_date_run2 Assay date of replicate 2 String
23DR_Run_date_run3Assay date of replicate 3 String

* Activity Concentration.
Additional Information
Grant Number: MH082340

Data Table (Concise)
Data Table ( Complete ):     View All Data
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