An NMR Biochemical Assay for Fragment-Based Drug Discovery: Evaluation of an Inhibitor Activity on Spermidine Synthase of Trypanosoma cruzi

Although NMR in fragment-based drug discovery is utilized almost exclusively to evaluate physical binding between molecules, it should be also a powerful tool for biochemical assay, evaluating inhibitory effect of compounds on enzymatic activity. Time-dependent spectral change in real-time monitoring or inhibitor concentration-dependent spectral change after constant-time reaction was processed by factor analysis, by which reaction rate or IC₅₀ value was obtained. Applications to spermidine synthase of Trypanosoma cruzi, which causes Chagas disease, are described

4‑Hydroxypyridazin-3(2<i>H</i>)‑one Derivatives as Novel d‑Amino Acid Oxidase Inhibitors

d-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the <i>N</i>-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3­(2<i>H</i>)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2­(1<i>H</i>)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)­pyridine-2­(1<i>H</i>)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3­(2<i>H</i>)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2­(1<i>H</i>)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)­ethyl]-4-hydroxypyridazin-3­(2<i>H</i>)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on <i>Trypanosoma cruzi</i> and Human Enzymes

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of <i>Trypanosoma cruzi</i>, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on <i>Trypanosoma cruzi</i> and Human Enzymes

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of <i>Trypanosoma cruzi</i>, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity