Amplex red 10 acetyl 3 7 dihydroxyphenoxazine

Recombinant human fumarate hydratase (with 6 histidine tag in the N-terminal of protein), dithiothreitol (DTT) and HBSS solution were purchased from Thermofisher Scientific (Waltham, MA). L-malate dehydrogenase from pig heart, diaphorase from clostridium kluyveri, β-NAD (NAD+), resazurin, Brij 35, sodium fumarate dibasic, malic acid and horse radish peroxidase (HRP) were purchased from Sigma (St. Louis, MO). Fumarate hydratase-IN-2 was purchased from MedChem Express (Monmouth Junction, NJ). Compound 8 was purchase from Enamine (Monmouth Junction, NJ). Amplex Red (10-Acetyl-3,7-dihydroxyphenoxazine) was purchased from Cayman Chemical (Ann Arbor, MI). Six libraries were screened, including Sytravon (a retired Pharma screening collection that contains a diversity of novel small molecules, NCATS), NPACT (NCATS Pharmacologically Active Chemical Toolbox, NCATS), NPC (NCATS Pharmaceutical Collection, NCATS),¹⁶ MIPE (Mechanism Interrogation Plate, NCATS), Kinase inhibitor library (an in-house collection of kinase inhibitors, NCATS) and LOPAC (1,280 Pharmacologically Active Compounds Sigma-Aldrich, St. Louis, MO) libraries.

Bacterial (Streptomyces chromofuscus), peanut, and cabbage PLD was purchased from Enzo and sigma-aldrich respectively. Amplex Red 10-Acetyl-3,7-dihydroxyphenoxazine (Cayman chemical). Horseradish peroxidase (VWR), choline oxidase (VWR). All lipids including 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC), 1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine (TopFluorPC), 1-(dipyrrometheneboron difluoride) undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (TopFluorLPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC or 18:1 PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPEtOH), and 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DOPEtOH) were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform at stock concentrations of 10 or 25 mg/mL except C8-PC and TopFluor lipids which were purchased as powder. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was from Tocris Bioscience and made at a stock concentration of 1mM in ethanol. Valinomycin was also purchased from Tocris Bioscience and made at a stock concentration of 200μM in ethanol. The detergent n-dodecyl-β-D-maltoside (DDM) was purchased from Anatrace (D310S) and made at 200mM. ACMA (9-amino-6-chloro-2-methoxyacridine), was purchased from Life Technologies and made at a stock concentration of 2mM in ethanol.