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BioAssay: AID 2560

Rescreen of TbHK1 primary actives - DPI cherry picked compounds

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(212)
 
 
Active(29)
 
 
Inactive(181)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(212)
 
 
Active(29)
 
 
Inactive(181)
 
 
Inconclusive(2)
 
 
AID: 2560
Data Source: University of Pittsburgh Molecular Library Screening Center (MH082340-TbHK1 rescreen of DPI cherry picked compounds)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-03-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 29
Depositor Specified Assays
AIDNameTypeProbeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1screening
2230Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first seriesconfirmatory
1632HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinationsconfirmatory
492951Human Glck Counter Screen Assayconfirmatory
2600Identification of Inhibitors of Trypanosoma Brucei Hexokinases - summary assaysummary1
449725IMR-90 (cell viability counter screen)confirmatory
Description:
Excerpt from MH0882340 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.
Protocol
Basic High Throughput Screening Assay protocol

The basic procedure for the TbHK1 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 (final compound concentration 10 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.

This assay protocol was used to retest the primary actives obtained from DPI. It was performed in duplicate using a single concentration (10 uM). Compounds active in this assay and inactive in the G6PDH counterscreen progressed to IC50 determinations.
Comment
Activity Criteria, Secondary Assay Plan, Hit and Lead Criteria

Based on the data generated in the PMLSC assay protocol review document we recommend an active criterion for the TBHK1 assay of DPI cherrypicked TbHK1 primary actives >/= 50% inhibition.
PUBCHEM_ACTIVITY_OUTCOME

If HTS % Inhibition>=50 in both assay runs then PUBCHEM_ACTIVITY_SCORE=40 and PUBCHEM_ACTIVITY_OUTCOME=2

If HTS % Inhibition <50 in both assay runs then PUBCHEM_ACTIVITY_SCORE=0 and PUBCHEM_ACTIVITY_OUTCOME=1

If HTS % Inhibition >=50 in one assay run and <50 in the other assay run then PUBCHEM_ACTIVITY_SCORE=60 and PUBCHEM_ACTIVITY_OUTCOME=3

Definition of Hit criteria: "Actives" in this assay format will progress to further testing (i.e., IC50 determinations) as long as they do not register as interfering with the G6PDH counterscreen.
Rapid HTS screen >/= 50% at a screening concentration of 10 uM
Structural confirmation by LCMS

Secondary Assay Testing Paradigm
Confirm inhibition using the primary assay format for IC50 determinations
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1HTS raw data_run 1 (10μM**)Raw HTS data (run 1)FloatμM
2HTS raw data_run 2 (10μM**)Raw HTS data (run 2)FloatμM
3HTS % Inhibition_run 1 Percent inhibition of signal readout based on assay plate max and min controls (run 1)Float
4HTS % Inhibition_run 2 Percent inhibition of signal readout based on assay plate max and min controls (run 2)Float
5Assay plate Z-factor_run 1 Assay plate Z-factor (run 1)Float
6Assay plate Z-factor_run 2Assay plate Z-factor (run 2)Float
7Assay plate S:B_run 1 Assay plate signal to background (1)Float
8Assay plate S:B_run 2 Assay plate signal to background (2)Float
9HTS Assay Date_run 1 Date the HTS assay was performed (run 1)String
10HTS Assay Date_run 2Date the HTS assay was performed (run 2)String

** Test Concentration.
Additional Information
Grant Number: MH082340

Data Table (Concise)
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