Inhibitors of Mycobacterium tuberculosis UDP-galactopyranose mutase (UGM) enzyme - High throughput screening using Fluorescent polarization assay Measured in Biochemical System Using Plate Reader - 2105-01_Inhibitor_SinglePoint_HTS_Activity_Set6
Uridine Diphosphate (UDP), UDP-Galactopyranose Mutase (UGM), Inhibitor Screening, Fluorescent Polarization (FP) assay, Mycobacterium tuberculosis ..more
BioActive Compounds: 194
Uridine Diphosphate (UDP), UDP-Galactopyranose Mutase (UGM), Inhibitor Screening, Fluorescent Polarization (FP) assay, Mycobacterium tuberculosis
This is a FP assay to measure binding between UGM from M.tuberculosis and UDP. UGM is a recombinant protein with a N-terminal 6xHistidine tag expressed in and purified from E.coli bacteria. UDP is conjugated to a fluororescein for use as tracer in FP assay. Emission of UDP-Fluorescein after excitation with plane-polarized light is measured. When UDP-fluorescein is bound to UGM, the bound complex tumbles slowly in solution than the unbound free UDP-fluorescein. Differences in the amount of polarized emission from the free and UGM bound UDP-fluorescein is used to quantify the binding between UGM and UDP.
Small molecule inhibitors that compete with UDP-fluorescein for binding to UGM protein will be identified.
Reagents and materials:
1. UGM protein
UGM from M.tuberculosis is expressed in E.coli as 6xHis tagged protein, purified and loaded with flavin in 50 mM potassium phosphate pH7.0 and 150 mM NaCl buffer. The protein is sterile filtered and stored at 4'C during the screening.
This fluorescent tracer compound is chemically synthesized by linking fluorescein to beta-phosphate of UDP using a 6-carbon linker.
3. Positive control compound
This is an aminothiazole molecule which inhibits UGM and UDP.fluorescein binding with 7-10 uM IC50
4. Assay buffer - 50 mM Potassium phosphate pH 7.0
Assay buffer is prepared from a 20X concentrated stock solution. The 20X stock solution (1 M) is prepared by dissolving 47.6315 grams of Potassium phosphate monobasic (Sigma Cat # P8416, Molecular weight 136.09) in 350 mL of sterile distilled water; pH was adjusted to 7.0 and sterile filtered.
5. BSA 6% stock solution
Prepare 6% BSA stock in MilliQ water and filter through 0.2 uM filter to sterilize the stock solution
6. Assay Ready Plates (ARPs)
ARPs are prepared by dispensing 7.5 nL of chemical compounds per well at 10 mM concentration in 1536-well High Base Aurora Black plates
1. Prepare UGM solution in assay buffer at 3X concentration (150nM) from 5.9 uM stock
2. Prepare UDP.fluorescein solution at 3X concentration (60nM) from 680 uM stock in assay buffer containing 3X BSA (0.06%)
3. Prepare positive control compound in assay buffer at 3X concentration (150nM) from 100 mM stock
4. Dispense 2.5 ul of positive control compound to wells marked for positive control and 2.5 ul of assay buffer to all other wells in the 1536-well Assay Ready Plates (ARPs) using Beckman BioRapTR
5. Dispense 2.5 ul of UGM to all the wells using Combi nL (Thermo Scientific)
6. Dispense 2.5 ul of UDP.fluorescein to all the wells using another Combi nL
7. Spin the plates for 30 sec at 1000 rpm
8.Incubate the plates for 30 min at room temperature
9. Read the plates in ViewLux (Perkin Elmer)
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 (v7.0.3):
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.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
This was set as equal to the mean of the normalized 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 10.
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 100.
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 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
** Test Concentration.
Data Table (Concise)