C. difficile toxins: HTS for inhibitors of TcdB glycohydrolase activity Measured in Biochemical System Using Plate Reader - 7074-01_Inhibitor_SinglePoint_HTS_Activity
Keywords: Clostridium difficile, toxin, virulence factors, TcdB, glycohydrolase, glucosyltransferase, uridine 5'-diphosphoglucuronic acid trisodium salt, glucose ..more
BioActive Compounds: 837
Keywords: Clostridium difficile, toxin, virulence factors, TcdB, glycohydrolase, glucosyltransferase, uridine 5'-diphosphoglucuronic acid trisodium salt, glucose
Assay Overview: The goal of this assay is to identify inhibitors to the C. difficile toxin B (TcdB), specifically to residues 1-549 that encodes the glycohydrolase and glucosyltransferase substrate binding and enzymatic domains. Recombinant TcdB (expressed and purified from Bacillus megaterium) was assayed for glycohydrolase activity in the presence of substrate uridine 5'-diphosphoglucuronic acid trisodium salt (UDPG). Glucose is released from the reaction and is detected by fluorescence using a commercially available coupled glucose oxidase/peroxidase kit (EnzyChrome Glucose Assay Kit, BioAssay Systems).
Expected Outcome: Compounds that demonstrate inhibition of TcdB will show a decrease in fluorescent signal, compared to a neutral (DMSO) control and positive control, a non-hydrolysable substrate. Only compounds showing a decrease of signal greater than 3 standard deviations of the mean of the neutral control will be considered positive.
10X assay buffer (500 mM HEPES pH 8.0, 2M KCl, 0.1% Triton X-100, 1% BSA (added at time of use))
1mM Uridine 5'-diphosphoglucose disodium salt hydrate (UDPG) in water (substrate)
1mM Uridine 5'-diphosphoglucuronic acid (UA) in water (positive control, non-hydrolysable substrate)
50mM MnCl2 in water
1mg/ml purified TcdB (1-549), provided by D. Haslam, Washington University School of Medicine, St. Louis
EnzyChrom Glucose assay Kit (BioAssay Systems, Hayward CA)
All reagents purchased from Sigma (St. Louis, MO) unless otherwise noted
1.For each 1536-well plate, 10ml of 1X assay components is prepared for dispensing on a liquid handler:
a.1 ml 10X buffer + 10mg BSA
b.1 ml 50mM MnCl2 (final = 5 mM MnCl2)
c.8 ml water
d.100 ml 1mg/ml TcdB (final = 10 mg/ml)
2.For each plate, 1ml per of 1mM UDPG and 1mM UA (positive control) are set up on a liquid handler.
3.The EnzyChrome Reagents (Enzyme & Dye)-each at 1:100 in water are prepared and set up on a liquid handler
1.A liquid handler was used to dispense 4.5 ul 1X buffer to each well of pre-made assay ready plates (ARPs)
2.Another liquid handler was then used to add 0.5 ml of UDPG (final = 100uM) to all wells except positive control wells, and 0.5 microl of UA (final = 100uM) is added to positive control wells (based on defined plate pattern)
3.Plates were incubated for 30 min at 37oC
4.After incubation, 1.25 ml of each 1X EnzyChrome reagent was added to each well
5.Plates were returned to the incubator for 30 min at 37 degrees C
6.Fluorescence was read (ex525/em598, 0.5s exposure)
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: The plate pattern correction algorithm 'Assay Pattern (additive)' in Genedata (v10.0.2) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 24%.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
** Test Concentration.
Data Table (Concise)