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BioAssay: AID 2472

qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreen

The objective of this project is to obtain compounds that inhibit Giardia lamblia growth. G. lamblia is a human pathogen which afflicts impoverished nations; it is the most common cause of outbreaks of diarrhea in the United States. During the course of our study of potential drug targets in Giardia, we have identified the Class II (Zn2+ functions in electrophilic catalysis) more ..
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 Tested Compounds
 Tested Compounds
All(288496)
 
 
Active(1911)
 
 
Inactive(286649)
 
 
 Tested Substances
 Tested Substances
All(291445)
 
 
Active(1950)
 
 
Inactive(289495)
 
 
AID: 2472
Data Source: NCGC (FBPA823)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-04
Modify Date: 2010-04-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1911
Related Experiments
AIDNameTypeComment
2451qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia LambliaConfirmatorydepositor-specified cross reference
2464qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: SummarySummarydepositor-specified cross reference
2785qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Giardia lamblia growth inhibitionConfirmatorydepositor-specified cross reference
2786qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Mammalian cellular toxicityConfirmatorydepositor-specified cross reference
2787qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Primary screen confirmationConfirmatorydepositor-specified cross reference
2794qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Rabbit FBPA SelectivityConfirmatorydepositor-specified cross reference
2795qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreen confirmationConfirmatorydepositor-specified cross reference
Description:
MLPCN Grant: MH085699-01

Assay Provider: Osnat Herzberg, University of Maryland

NCGC Assay Overview:

The objective of this project is to obtain compounds that inhibit Giardia lamblia growth. G. lamblia is a human pathogen which afflicts impoverished nations; it is the most common cause of outbreaks of diarrhea in the United States. During the course of our study of potential drug targets in Giardia, we have identified the Class II (Zn2+ functions in electrophilic catalysis) fructose-1,6-bisphosphate aldolase (FBPA) as an excellent candidate for drug targeting. FBPA is a key glycolytic pathway enzyme, essential for G. lamblia survival. The human FBPA belongs to the Class I aldolases, which have a different substrate binding site structure and a radically different catalytic mechanism (employing a lysine-Schiff base intermediate). Therefore, affinity-based or mechanism-based inhibitors of the Giardia FBPA are expected not to interfere with the catalytic function of the human FBPA.

A glyceraldehyde-3-phosphate dehydrogenase/triose phosphate isomerase/NAD/arsenate coupled assay was adapted to the 1536-well HTP format for use in the primary screen. This was done by coupling it to a diaphorase/resazurin/resorufin reaction that can be monitored by fluorescence (excitation, 544 nm; emission, 590 nm). Compounds identified in this screen will be further evaluated by coupling the glycerol-3-phosphate/triose phosphate isomerase/NADH assay to a phenazine methosulfate/tetranitroblue tetrazolium color reaction. False positives due to the dehydrogenases and diaphorase inhibition will be eliminated, and selectivity assay will triage inhibitors of mammalian triose phosphate isomerase and the Class I FBPA. G. lamblia FBPA inhibitors identified by the in vitro analyses will be examined for growth inhibition of Giardia trophozoites, and for cytotoxicity in human cells.

This assay specifically addresses whether activity observed in the primary screen (AID 2451) is related to inhibition of the coupling assay used, as opposed to inhibition of aldolase. Actives in this assay are considered inconclusive for activity against aldolase.
Protocol
NCGC Assay Protocol:

Step,Parameter,Value,Description
1,Coupling enzyme reagent,2 uL,glyceraldehyde-3-phosphate
2,Compound,23 nL,3.68 nM-57.5 uM final concentration
3,Time,10 min,RT Incubation
4,Substrate/detection Reagent,2 uL,Substrate + enzyme coupling system + AmpLite Red kit
5,Time,20 min,RT Incubation
6,Detector,525 nm/598 nm (Fluorescence),ViewLux Fluorescence Read (Read 1)

Notes:
1,Medium-binding black solid-bottom Kalypsys plates. FRD or Kalypsys dispenser.
2,Compound Library (10 mM ,1:5 dilution). Pin-transferred for a [final] range of 3.68 nM-57.5 uM.
3,RT incubation
4,FRD or Kalypsys dispenser, 2 uL of reaction mixture. Bottle kept at 4C and covered in foil to protect from light.
5,RT incubation
6,ViewLux Protocol 1194; Excitation filter wheel A (525/20 Pol; BODIPY TMR FP), Emission filter wheel B (598/25 S; BODIPY TMR FP S).

Keywords: Fructose-bisphosphate aldolase, FBPA , Giardia lamblia , Giardia, HTS, Inhibitors
Comment
Compound Ranking:

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 20 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 19. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity at 58.21 uM (58.2112μM**)% Activity at given concentration.Float%

** Test Concentration.
Additional Information
Grant Number: MH085699-01

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