qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreen confirmation
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 ..
BioActive Compounds: 84
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 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. Hits from the primary screen was run in this assay to confirm their inactivity against the coupling assay.
NCGC Assay Protocol:
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)
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.
4,FRD or Kalypsys dispenser, 2 uL of reaction mixture. Bottle kept at 4C and covered in foil to protect from light.
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
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)