AID	Name	Type	Comment
2451	qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia	confirmatory
2464	qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Summary	summary
2472	qHTS Assay for Inhibitors of Fructose-1,6-bisphosphate Aldolase from Giardia Lamblia: Coupling assay counterscreen	screening

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 can inhibit growth of giardia lamblia trophozoites.

Trophozoites of the G. lamblia isolate WB (33) were grown at pH 7.0 in modified TYI-S-33 medium. Detachment of trophozoites for preparation of inocula was achieved by chilling the cultures on ice for 20 min.

NCGC Assay protocol:

2uL/well media was dispensed per well into 1536-well white solid-bottom plates using a Multidrop Combi dispenser (Thermo Scientific). Subsequently, 23 nL/well compound solutions or DMSO controls were dispensed to the assay plates containing media via a pintool workstation (Kalypsys, San Diego) to avoid the potential contamination to the compound source plates. Unless otherwise noted, Giardia trophozoids suspended in media were dispensed as 100 cells/well in 4uL volumes. The plates were covered with plastic low-evaporation lids, individually sealed in Type A Bio-Bags and incubated at 37C for 48 hours. Following incubation, 4uL/well of ATPlite reagent was dispensed with a Multidrop Combi dispenser. The assay plates were briefly centrifuged at 1000 RPM and luminescence signal was detected on a ViewLux plate reader (PerkinElmer) after 20 min incubation at room temperature.

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

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.

Grant Number: MH085699-01