Turbidometric Biochemical Primary HTS to identify inhibitors of ERp5 Measured in Biochemical System Using Plate Reader - 7002-01_Inhibitor_SinglePoint_HTS_Activity
Keywords: PDIA6 protein disulfide isomerase family A, member 6 , ERp5, arterial thrombosis, inhibitors of ERp5, anti-thrombotic, insulin aggregation assay ..more
BioActive Compounds: 178
Keywords: PDIA6 protein disulfide isomerase family A, member 6 , ERp5, arterial thrombosis, inhibitors of ERp5, anti-thrombotic, insulin aggregation assay
An assay was set up to monitor and identify inhibitors of the ERp5 mediated aggregation of insulin. The assay measures aggregation of insulin chains by monitoring absorbance at 650 nm. ERp5 is a member of the thiol isomerase family and cleaves disulfide bonds in the beta chain of insulin causing them to aggregate. When ERp5 is inhibited there is a decrease in aggregation and therefore a decrease in absorbance.
Expected Outcome: Active hits are determined as compounds that decrease absorbance by at least 25% of the signal reduction observed in the mean of positive control wells.
Recombinant ERp5 produced in E.coli was received from collaborator. Batch 04.06.12 was received in large tubes (16 tubes with 7 ml/tube) with concentration of 4.4 mg/ml.
100 mM Potassium Phosphate, pH 7.0 with 2 mM EDTA
Prepare 1M stock solutions of K2HPO4 (dibasic- 176 g/L)) and KH2PO4 (monobasic 136.09 g/L)
to prepare 1L of assay buffer add:
61.5 ml dibasic 1M solution
38.5 ml monobasic 1M solution
4 ml EDTA (0.5 M stock from Sigma cat # E7889, lot # 021M8719)
896 ml sterile H2O
Prepare Bovine Insulin:
Bovine insulin from SIGMA Cat # I6634 Lot # SLBD2712V: Insulin from Bovine Pancreas. Insulin is stored at - 20 C as lyophilized powder. Reconstitute 100 mg with 10 ml of 0.1 N HCL. Dilute 1:8 with assay buffer for a final concentration of 213 uM. Once reconstituted insulin may be stored at -20 C and thawed once.
ERp5 batch 04.06.12 at 4.4 mg/ml stock solution. Dilute 1:92 in diluted insulin from step above (final concentration 48 ug/ml = 1uM). This is referred to as insulin/ERp5 mix.
1M Stock solution in sterile H20. Dilute 1:178 for a working solution of 5.6 mM (this will be diluted 1:10 in the assay well for final concentration of 350 uM).
Positive Control from TCI America Cat # P008 Lot # FHJ01 9,10-Phenanthrenequinone CAS # 84-11-7 MW 208.21
Prepare 10 mM stock of 9,10-Phenanthrenequinone (2.08 mg/ml DMSO)
Dilute 10 mM stock in DMSO to 200 uM by diluting 1:50
This will be diluted 1:10 in well for a final concentration of 20 uM.
Set up Reagents:
One bottle on bioraptr will hold 9,10-Phenanthrenequinone positive control.
Remaining bottles will contain Insulin/ ERp5 reaction mixture (213 uM insulin with 1 uM ERp5).
Combi NL will deliver DTT.
Dispense 8.0 ul of insulin/ERp5 mix to all wells using the BioraptR.
Dispense 1.0 uL 200 uM 9,10-Phenanthrenequinone to positive control wells using BioraptR.
Move plate to combiNL and deliver 1.0 ul DTT to all wells.
Spin plates in centrifuge 2 min 1000 RPM to break bubbles.
Incubate plate 90 minutes RT.
Read plates on Envision using absorbance (650 nm) protocol on the Envision.
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.
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 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) 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 25.
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 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
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)