AID	Name	Type	Probe	Comment
1631	qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase	confirmatory
2095	qHTS Assay for Activators of Human Muscle isoform 2 Pyruvate Kinase: Summary	summary	6
2620	Secondary LDH Assay for Activators of Human Pyruvate Kinase M1 Isoform: for Probe SAR	confirmatory
2653	Secondary Assay for Activators of Human Pyruvate Kinase M2 isoform - Cell Titer Glo Cytotoxicity for Probe SAR	confirmatory
2266	qHTS Assay for Inhibitors of Leishmania Mexicana Pyruvate Kinase (LmPK): Summary	summary
2625	Secondary LDH Assay for Activators of Human Liver Pyruvate Kinase: for Probe SAR	confirmatory
2576	Secondary LDH Assay for Activators of Human Pyruvate Kinase M2 isoform: for Probe SAR	confirmatory

NIH Chemical Genomics Center [NCGC]
Matthew G. Vander Heiden, M.D., Ph.D. [Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115]
MLPCN Grant: 1 R03 MH085679-01

NCGC Assay Overview:
Human pyruvate kinase muscle 2 (hPK-M2) enzyme was supplied as a highly purified (>95% pure) preparation from Harvard Medical School and assayed for its ability to generate ATP from ADP using phosphoenolpyruvate (PEP) as a substrate. ATP generation was detected in a coupled reaction by luciferase-mediated luminescence, an ATP-dependent process. Pyruvate kinase substrates, PEP and ADP, were present in the assay at Km and approximately 10-fold below Km respectively. The enzyme was assayed at an intermediate level of activity to screen for both inhibitors and activators.

NCGC Assay Protocol Summary:
Three uL of substrate mix (at r.t.) in assay buffer (50 mM Imidazole pH7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween 20, 0.05% BSA) was dispensed into white solid bottom 1,536 well microtiter plates so that the final concentrations of substrates in the assay were 0.1 mM ADP and 0.5 mM PEP. 23 nL of compound were delivered by a pin tool and 1 uL of enzyme mix (final concentration of 0.1 nM) in assay buffer (4 degree Celsius) was added. Plates were incubated at room temperature for 1 hour. Two uL of detection mix (Kinase-Glo, Promega; at 4 degree Celsius protected from light) was added and luminescence read by a ViewLux (Perkin Elmer) at 1 second exposure/plate. Data were normalized to the uninhibited (column 3) and AC100 inhibition (no enzyme in column 2). To monitor activation, the first column contained a titration of the activator, NCGC00031955-03 (16-point 1:2 dilutions in duplicate starting at 57 uM) and the first 16 rows of column 4 contained 57 uM of NCGC00031955-03.

Compound Ranking:

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 inhibitors are ranked higher than compounds that showed apparent activation.
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.

Grant Number: 1 R03 MH085679-01