Development of IDH1/2 inhibitors (CTD2project) Measured in Biochemical System Using Plate Reader - 2107-01_Inhibitor_SinglePoint_HTS_Activity
Isocitrate dehydrogenase IDH1, IDH1(R132H), dehydrogenase, a-ketoglutarate, tumorigenesis, human glioma, HTS ..more
BioActive Compounds: 123
Isocitrate dehydrogenase IDH1, IDH1(R132H), dehydrogenase, a-ketoglutarate, tumorigenesis, human glioma, HTS
The R132H mutation in IDH1 encoding cytosolic NADP+-dependent isocitrate dehydrogenase 1 is very frequent in human glioma diffuse astrocytoma and oligodendroglioma, common in AML. So far, all IDH1 mutations in gliomas affect codon 132 and more than 90% of the mutations are of the R132H type. IDH1 mutated protein hydrolyzes alpha-ketoglutarate and produces 2-hydroxyglutarate (2-HG).We are specificly seeking for IDH1(R132H) selective inhibitors. A diaphorase coupled assay will be used to capture the NADPH reduction for IDH1 R132H alpha-ketoglutarate reductase activity. For primary screen, 8ug/ml IDH1(R132H) was incubated with 10 uM of compounds in the presence of 20 uM of NADP and 400uM alpha-ketoglutarate for 60 minutes at ambient temperature in 1536 well plates. The IDH1/R132H mutant reductase activity was measured through diaphorase coupling with NADPH. Resazurin fluorescence were recorded with Envision plate reader(PerkinElmer). No enzyme wells as positive control were included in each plate and used to scale the data in conjunction with in-plate DMSO control.
Inhibition of IDH activity will show increase of rezarurin fluorescence signal
Preparation of Mater mixes:
Enzyme Mix: IDH1-R132H stock is diluted in assay buffer to 16 ug/ml
Substrate Mix: prepared in assay buffer, 40 uM NADPH (Sigma, N0411), 0.8 mM alpha-ketoglutarate (alpha-ketoglutaric acid disodium salt dihydrate, Sigma, 75892).
Detection Mix: prepared in assay buffer, 1 ug/ml rat diaphorase (Sigma, D1190), 50 uM resazurin sodium salt (Sigma, 199303).
Add 2.5 ul buffer to positive control wells of the ARPs(assay ready plates) using combi nl.
Add 2.5 ul enzyme mix to assay wells
Incubate the assay plates at RT for 20 min.
Add 2.5 ul substrate mix to all wells on plates by conbi nl to initiate the reaction
Incubate at RT for 60 min reaction.
Add 2.5 ul detection mix to all wells on plates to quench the reaction.
Read on Envision (Ex 535/Em 595) after 30min incubation at RT
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 30.
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 75.
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)