Secondary LDH Assay for Activators of Human Reticulocyte Pyruvate Kinase: for Probe SAR
Wael M. Rabeh, Lyudmila Nedyalkova and Hee-Wan Park [Structural Genomics Consortium, 100 College St. Toronto, Ontario, Canada] ..more
BioActive Compound: 1
NIH Chemical Genomics Center [NCGC]
Wael M. Rabeh, Lyudmila Nedyalkova and Hee-Wan Park [Structural Genomics Consortium, 100 College St. Toronto, Ontario, Canada]
NCGC Assay Overview:
Human pyruvate kinase reticulocyte (hPK-R) enzyme was supplied as a highly purified (>95% pure) preparation from Structural Genomics Consortium in Toronto (Ontario, Canada) and a secondary assay was used to evaluate compounds. This assay couples the formation of pyruvate from hPK-R using lactate dehydrogenase (LDH) and NADH. The depletion of NADH is followed in a fluorescent-based kinetic assay that follows the formation of pyruvate to lactate, as catalyzed LDH. Pyruvate kinase substrates, PEP and ADP, were present in the assay at 0.1 mM and 4 mM respectively. In this kinetic assay initial rates were determined by following the fluorescence of NADH depletion.
NCGC Assay Protocol Summary:
Three uL of substrate mix (final concentration, 50 mM Tris-Cl pH 8.0, 200 mM KCl, 15 mM MgCl2, 0.1 mM PEP, 4.0 mM ADP, and 0.2 mM NADH) was dispensed into black-solid 1536 well plates. 23 nL of compounds were delivered by a pin tool and 1 uL of enzyme mix (final concentrations, 10 nM hPK-R and 1 uM of LDH) was added. Plates were immediately placed in ViewLux (Perkin Elmer) and NADH fluorescence was determined at 30 second exposure intervals for between 3 and 6 minutes. Data were normalized to the uninhibited and EC100 activation using known activators such as fructose-bis-phosphate.
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 activators are ranked higher than compounds that showed apparent inhibition.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)