Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2by Redox Cycling in Reducing Environments

We have screened the Library of Pharmacologically Active Compounds (LOPAC) and the National Institutes of Health (NIH) Small Molecule Repository (SMR) libraries in a horseradish peroxidase-phenol red (HRP-PR) H 2 O 2 detection assay to identify redox cycling compounds (RCCs) capable of generating H 2 O 2 in buffers containing dithiothreitol (DTT).Two RCCs were identifi ed in the LOPAC set, the ortho-naphthoquinone β-lapachone and the paranaphthoquinone NSC 95397.Thirty-seven (0.02%) concentrationdependent RCCs were identifi ed from 195,826 compounds in the NIH SMR library; 3 singleton structures, 9 ortho-quinones, 2 para-quinones, 4 pyrimidotriazinediones, 15 arylsulfonamides, 2 nitrothiophene-2-carboxylates, and 2 tolyl hydrazides.Sixty percent of the ortho-quinones and 80% of the pyrimidotriazinediones in the library were confi rmed as RCCs.In contrast, only 3.9% of the para-quinones were confi rmed as RCCs.Fifteen of the 251 arylsulfonamides in the library were confi rmed as RCCs, and since we screened 17,868 compounds with a sulfonamide functional group we conclude that the redox cycling activity of the arylsulfonamide RCCs is due to peripheral reactive enone, aromatic, or heterocyclic functions.Cross-target queries of the University of Pittsburgh Drug Discovery Institute (UPDDI) and PubChem databases revealed that the RCCs exhibited promiscuous bioactivity profi les and have populated both screening databases with signifi cantly higher numbers of active fl ags than non-RCCs.RCCs were promiscuously active against protein targets known to be susceptible to oxidation, but were also active in cell growth inhibition assays, and against other targets thought to be insensitive to oxidation.Profi ling compound libraries or the hits from screening campaigns in the HRP-PR H 2 O 2 detection assay signifi cantly reduce the timelines and resources required to identify and eliminate promiscuous nuisance RCCs from the candidates for lead optimization.