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BioAssay: AID 2794

qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Rabbit FBPA Selectivity

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(1477)
 
 
Active(59)
 
 
Inactive(1264)
 
 
Inconclusive(154)
 
 
 Tested Substances
 Tested Substances
All(1478)
 
 
Active(59)
 
 
Inactive(1265)
 
 
Inconclusive(154)
 
 
AID: 2794
Data Source: NCGC (FBPA826)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-04-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 59
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
2472qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreenScreeningdepositor-specified cross reference
2785qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Giardia lamblia growth inhibitionConfirmatorysame project related to Summary assay
2786qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Mammalian cellular toxicityConfirmatorysame project related to Summary assay
2787qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Primary screen confirmationConfirmatorysame project related to Summary assay
2795qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreen confirmationConfirmatorysame project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

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 addresses whether hits in the primary screen are selective for Class I FBPA, by using rabbit FBPA enzyme in the protocol.
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 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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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 0.0003244270 uM (0.000324427μM**)% Activity at given concentration.Float%
15Activity at 0.0009732809 uM (0.000973281μM**)% Activity at given concentration.Float%
16Activity at 0.00292 uM (0.00291984μM**)% Activity at given concentration.Float%
17Activity at 0.00876 uM (0.00875953μM**)% Activity at given concentration.Float%
18Activity at 0.013 uM (0.0125438μM**)% Activity at given concentration.Float%
19Activity at 0.025 uM (0.0250876μM**)% Activity at given concentration.Float%
20Activity at 0.028 uM (0.0277449μM**)% Activity at given concentration.Float%
21Activity at 0.056 uM (0.0560447μM**)% Activity at given concentration.Float%
22Activity at 0.080 uM (0.0799409μM**)% Activity at given concentration.Float%
23Activity at 0.112 uM (0.112249μM**)% Activity at given concentration.Float%
24Activity at 0.226 uM (0.226245μM**)% Activity at given concentration.Float%
25Activity at 0.448 uM (0.448358μM**)% Activity at given concentration.Float%
26Activity at 0.703 uM (0.703399μM**)% Activity at given concentration.Float%
27Activity at 0.897 uM (0.896716μM**)% Activity at given concentration.Float%
28Activity at 1.793 uM (1.79343μM**)% Activity at given concentration.Float%
29Activity at 2.132 uM (2.13187μM**)% Activity at given concentration.Float%
30Activity at 3.587 uM (3.58686μM**)% Activity at given concentration.Float%
31Activity at 7.032 uM (7.03212μM**)% Activity at given concentration.Float%
32Activity at 12.49 uM (12.4946μM**)% Activity at given concentration.Float%
33Activity at 15.10 uM (15.1016μM**)% Activity at given concentration.Float%
34Activity at 28.69 uM (28.6949μM**)% Activity at given concentration.Float%
35Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
36Activity at 57.50 uM (57.5μM**)% 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)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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