Bookmark and Share
BioAssay: AID 1632

HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinations

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
_
   
 Tested Compounds
 Tested Compounds
All(16)
 
 
Active(14)
 
 
Inactive(1)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(16)
 
 
Active(14)
 
 
Inactive(1)
 
 
Inconclusive(1)
 
 
AID: 1632
Data Source: University of Pittsburgh Molecular Library Screening Center (MH082340-IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-03-27
Modify Date: 2009-04-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 14
Related Experiments
AIDNameTypeProbeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1Screening depositor-specified cross reference
2230Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first seriesConfirmatory depositor-specified cross reference
2516G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compoundsOther depositor-specified cross reference
2560Rescreen of TbHK1 primary actives - DPI cherry picked compoundsOther depositor-specified cross reference
2579G6DPH counterscreen for TbHK1 inhibitors - Analogues seriesConfirmatory depositor-specified cross reference
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
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. This assay was used to determine confirm the inhibitory activity of the primary hits via IC50 determinations.
Protocol
Basic Assay protocol

The basic procedure for the TbHK1 assay follows a stepwise addition of reaction mixture components as follows:

1 15 uL of test compound is added to appropriate wells (final compound concentration range = 0-100 uM).

2 15 uL of a glucose + ATP + MgCl2 + NAD+ + G6PDH mixture is added with final concentrations of 0.5mM, 0.35mM, 1.5mM, 3mM, and 0.006mUnits/uL, respectively.

3 15 uL of TbHK1 enzyme is added per well (final 0.5ng/uL).

4 Reaction incubates for 2 hours at room temperature.

5 5 uL EDTA is added (final 50mM).

6 Data was captured at A340 and represents the increase in NADH
in the reaction mixture.
Comment

PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when the triplicate IC50s are > 100 uM
2 - Substance is considered active when the triplicate IC50s are < 100 uM
3 - Substance activity outcome is inconclusive when one of triplicate IC50s is > 100 uM

PUBCHEM_ACTIVITY_SCORE
20 - Compounds that were inactive in all triplicate 10-pt dose response assays with a mean IC50 > 100 uM.
40 - Compounds that were active in one of the triplicate 10-pt dose response assays with an IC50 >100 uM but exhibited an IC50 < 100 uM in the other runs.
60 - Compounds that were active in three 10-pt dose response assays with a mean IC50 in the 20 to 100 uM range
70 - Compounds that were active in three 10-pt dose response assays with a mean IC50 in the 10 to 20 uM range
80 - Compounds that were active in three 10-pt dose response assays with a mean IC50 in the 1 to 10 uM range
90 - Compounds that were active in three 10-pt dose response assays with a mean IC50 < 1 uM.
Result Definitions
Show more
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.String
2IC50_Mean*IC50 determination from a 10 point dose response assay (Runs 1-3)FloatμM
3Qualifier_IC50_Run1This qualifier is intended to be interpreted with the TID called "DR IC50 uM_Run1". If qualifier is "=" than "DR IC50 uM_Run1" equals to the value in that column, if qualifier is ">" than "DR IC50 uM_Run1" is bigger than that value (Run 1)String
4Qualifier_IC50_Run2This qualifier is intended to be interpreted with the TID called "DR IC50 uM_Run2". If qualifier is "=" than "DR IC50 uM_Run2" equals to the value in that column, if qualifier is ">" than "DR IC50 uM_Run2" is bigger than that value (Run 2)String
5Qualifier_IC50_Run3This qualifier is intended to be interpreted with the TID called "DR IC50 uM_Run3". If qualifier is "=" than "DR IC50 uM_Run3" equals to the value in that column, if qualifier is ">" than "DR IC50 uM_Run3" is bigger than that value (Run 3)String
6DR_IC50uM_Run1IC50 determination from a 10 point dose response assay (Run 1)Float
7DR_IC50uM_Run2IC50 determination from a 10 point dose response assay (Run 2)Float
8DR_IC50uM_Run3IC50 determination from a 10 point dose response assay (Run 3)Float
9DR_IC50_Hillslope_Run 1R2 value, the square of the linear correlation coefficient for a given fit cell (Run 1)490 nm and 520 nm) (n=32) (Run 1)Float
10DR_IC50_Hillslope_Run 2R2 value, the square of the linear correlation coefficient for a given fit cell (Run 2)490 nm and 520 nm) (n=32) (Run 2)Float
11DR_IC50_Hillslope_Run 3R2 value, the square of the linear correlation coefficient for a given fit cell (Run 3)490 nm and 520 nm) (n=32) (Run 3)Float
12DR_%inhibition@100 uM_Run1 (100μM**)Dose response (n=1) % inhibition of the assay at 100 uM compound calculated from the mean max and min plate controls (Run 1)Float
13DR_%inhibition@100 uM_Run2 (100μM**)Dose response (n=1) % inhibition of the assay at 100 uM compound calculated from the mean max and min plate controls (Run 2)Float
14DR_%inhibition@100 uM_Run3 (100μM**)Dose response (n=1) % inhibition of the assay at 100 uM compound calculated from the mean max and min plate controls (Run 3)Float
15DR_Plate Mean Max Signal_Run1Dose response plate Mean maximum assay signal window plate controls n=32 (Run 1)Float
16DR_Plate Mean Max Signal_Run2Dose response plate Mean maximum assay signal window plate controls n=32 (Run 2)Float
17DR_Plate Mean Max Signal_Run3Dose response plate Mean maximum assay signal window plate controls n=32 (Run 3)Float
18DR_Plate Mean Min Signal_Run1Dose response plate Mean minimum assay signal window plate controls n=24 (Run 1)Float
19DR_Plate Mean Min Signal_Run2Dose response plate Mean minimum assay signal window plate controls n=24 (Run 2)Float
20DR_Plate Mean Min Signal_Run3Dose response plate Mean minimum assay signal window plate controls n=24 (Run 3)Float
21DR_plate Z'-factor_Run_1HTS Z-factor statistic calculated from assay plate MAX and MIN (Run 1)Float
22DR_plate Z'-factor_Run_2HTS Z-factor statistic calculated from assay plate MAX and MIN (Run 2)Float
23DR_plate Z'-factor_Run_3HTS Z-factor statistic calculated from assay plate MAX and MIN (Run 3)Float
24DR_Run date_run1Date the HTS assay was performed (run 1)String
25DR_Run date_run2Date the HTS assay was performed (run 2)String
26DR_Run date_run3Date the HTS assay was performed (run 3)String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH082340

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