External ID: LDHA

Protocol: The assay was optimized to 1536-well plates. Briefly, 3 muL of human lactate dehydrogenase 5 (#A38558H, Meridian Life Science, Inc., Memphis, TN) in LDH assay buffer (200 mM Tris HCl pH 7.4, 100 microM EDTA and 0.01% Tween-20) was added to a black solid bottom 1536-well assay plate (Greiner Bio-One) using a BioRAPTR FRD dispenser (Beckman Coulter, Brea, CA). A 1536-well pintool dispenser outfitted with 20 nL pins (Wako Automation, San Diego, CA) was used to transfer 23 nL of DMSO-solubilized compound (both library and vehicle controls) to each 1536-well assay plate. Following compound transfer, 1 muL of substrate solution containing NADH and sodium pyruvate (Sigma-Aldrich, St. Louis, MO) in LDH assay buffer was dispensed via BioRAPTR FRD to initiate the reaction. Final concentrations in the 4 microL reaction volume were 2 nM LDHA enzyme, 0.06 mM NADH and 0.2 mM sodium pyruvate. Following a 5 minute incubation period at room temperature, 1 muL of detection reagent (C. kluyveri diaphorase (Sigma-Aldrich) and resazurin sodium salt (Sigma-Aldrich) in LDH assay buffer) was added to a total volume of 5 muL. Final concentrations of detection reagents were 0.133 mg/mL diaphorase and 37 muM resazurin. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (ex540, em590 nm) at 0 and 20 minutes. Fluorescence was normalized using enzyme-free and DMSO-treated control wells on each plate.

Comment: PubChem score indicates % inhibition at 229 uM or 114 uM of the compound (Max_response). Active compounds with Max_response greater than -100%, PUBCHEM_ACTIVITY_SCORE is 100. Active compounds with Max_response between -80% and -100%, PUBCHEM_ACTIVITY_SCORE is 80. Active compounds with Max_response between -60% and -80%, PUBCHEM_ACTIVITY_SCORE is 60. Active compounds with Max_response between -30% and -60%, PUBCHEM_ACTIVITY_SCORE is 40. Inconclusive Compounds with Max_response between -25% and -30% have PUBCHEM_ACTIVITY_SCORE 20. For all inactive compounds with Max_response greater than -25%, PUBCHEM_ACTIVITY_SCORE is 0

External ID: CHEMBL2061058

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2012
Volume: 55
Issue: 7
First Page: 3101
Last Page: 3112
DOI: 10.1021/jm201537d

Target ChEMBL ID: CHEMBL5619
ChEMBL Target Name: DNA-(apurinic or apyrimidinic site) lyase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned