Ichiji Namatame

4-Hydroxypyridazin-3(2H)-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 N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-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(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-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(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Fully automated two-dimensional electrophoresis system for high-throughput protein analysis.

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

In silico, in vitro, X-ray crystallography, and integrated strategies for discovering spermidine synthase inhibitors for Chagas disease

Chagas disease results from infection by Trypanosoma cruzi and is a neglected tropical disease (NTD). Although some treatment drugs are available, their use is associated with severe problems, including adverse effects and limited effectiveness during the chronic disease phase. To develop a novel anti-Chagas drug, we virtually screened 4.8 million small molecules against spermidine synthase (SpdSyn) as the target protein using our super computer “TSUBAME2.5” and conducted in vitro enzyme assays to determine the half-maximal inhibitory concentration values. We identified four hit compounds that inhibit T. cruzi SpdSyn (TcSpdSyn) by in silico and in vitro screening. We also determined the TcSpdSyn–hit compound complex structure using X-ray crystallography, which shows that the hit compound binds to the putrescine-binding site and interacts with Asp171 through a salt bridge.

Crystal structures of FMN‐bound and FMN‐free forms of dihydroorotate dehydrogenase from Trypanosoma brucei

Dihydroorotate dehydrogenase (DHODH) is a flavin‐binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three‐dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for neglected tropical diseases, especially because these enzymes possess significantly different structural and functional properties from the membrane‐associated human enzyme. The existing crystal structures of the flavin mononucleotide (FMN)‐bound DHODHs reveal a number of interactions stabilizing FMN. However, to understand the binding mechanism correctly, it is necessary to compare the structures of the FMN‐bound and FMN‐free forms, because the protein moiety of the former is not necessarily the same as the latter. Here, we prepared the FMN‐free DHODH of Trypanosoma brucei using an Escherichia coli overexpression system. Although this apoform lacks enzymatic activity, simple incubation with FMN activated the enzyme. It was stable enough to be crystallized, enabling us to determine its structure by X‐ray crystallography at 1.6 Å resolution. We also determined the FMN‐bound form at 1.8 Å resolution. Although the two structures have essentially the same scaffold, we observed flipping of a peptide‐bond plane in the vicinity of the FMN‐binding site, accompanied by an alternative hydrogen‐bonding pattern. Comparisons of B factors of the protein main chain revealed that binding of FMN decreased flexibility of most of the residues at the FMN‐binding site, but increased flexibility of a lid‐like loop structure over the active center. This increase was ascribed to a conformational change in an FMN‐contacting residue, Asn195, which induced a rearrangement of a hydrogen‐bond network of the residues comprising the lid.

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.

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi 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 Trypanosoma cruzi, 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.