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BioAssay: AID 2451

qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia

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(287747)
 
 
Active(2033)
 
 
Inactive(273078)
 
 
Inconclusive(12878)
 
 
 Tested Substances
 Tested Substances
All(291269)
 
 
Active(2061)
 
 
Inactive(276158)
 
 
Inconclusive(13050)
 
 
AID: 2451
Data Source: NCGC (FBPA821)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-02
Modify Date: 2010-04-28

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 2033
Depositor Specified Assays
AIDNameTypeComment
2464qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Summarysummary
2472qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreenscreening
2794qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Rabbit FBPA Selectivityconfirmatory
2795qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreen confirmationconfirmatory
2785qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Giardia lamblia growth inhibitionconfirmatory
2787qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Primary screen confirmationconfirmatory
2786qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Mammalian cellular toxicityconfirmatory
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.
Protocol
NCGC Assay Protocol:

Step,Parameter,Value,Description
1,Enzyme reagent,2 uL,40 nM FBPA
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 uL of FBPA enzyme. Bottle kept at 4C.
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 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.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.00164 uM (0.001645μM**)% Activity at given concentration.Float%
15Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
16Activity at 0.00825 uM (0.00825μM**)% Activity at given concentration.Float%
17Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
18Activity at 0.041 uM (0.04115μM**)% Activity at given concentration.Float%
19Activity at 0.093 uM (0.0930036μM**)% Activity at given concentration.Float%
20Activity at 0.133 uM (0.132721μM**)% Activity at given concentration.Float%
21Activity at 0.201 uM (0.201088μM**)% Activity at given concentration.Float%
22Activity at 0.417 uM (0.417333μM**)% Activity at given concentration.Float%
23Activity at 0.510 uM (0.509728μM**)% Activity at given concentration.Float%
24Activity at 0.845 uM (0.845038μM**)% Activity at given concentration.Float%
25Activity at 1.285 uM (1.28469μM**)% Activity at given concentration.Float%
26Activity at 2.352 uM (2.3523μM**)% Activity at given concentration.Float%
27Activity at 3.436 uM (3.43627μM**)% Activity at given concentration.Float%
28Activity at 5.179 uM (5.17857μM**)% Activity at given concentration.Float%
29Activity at 10.67 uM (10.6746μM**)% Activity at given concentration.Float%
30Activity at 12.94 uM (12.9392μM**)% Activity at given concentration.Float%
31Activity at 21.68 uM (21.6814μM**)% Activity at given concentration.Float%
32Activity at 32.15 uM (32.1492μM**)% Activity at given concentration.Float%
33Activity at 59.56 uM (59.5568μM**)% Activity at given concentration.Float%
34Activity at 89.21 uM (89.2095μM**)% Activity at given concentration.Float%
35Activity at 136.3 uM (136.318μM**)% Activity at given concentration.Float%
36Activity at 187.5 uM (187.5μM**)% Activity at given concentration.Float%
37Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

Data Table (Concise)
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