Publications by authors named "Ichiji Namatame"

In recent years, there has been an increasing demand for the detection of rare cells in drug discovery research, such as cells that have differentiated off-purpose or are required for immunogenicity evaluation. Since detection and quantification limits depend on the robustness of the experiment, inter-human differences in technique have a significant impact on the performance of the assay system. Here, we integrated flow cytometry into a cell experiment platform, Screening Station, to construct a robust assay system, examined each step of the flow cytometric pretreatment using Jurkat cells, and finally evaluated the overall assay performance.

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In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure.

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Due to their physiological relevance, cell-based assays using human-induced pluripotent stem cell (iPSC)-derived cells are a promising in vitro pharmacological evaluation system for drug candidates. However, cell-based assays involve complex processes such as long-term culture, real-time and continuous observation of living cells, and detection of many cellular events. Automating multi-sample processing through these assays will enhance reproducibility by limiting human error and reduce researchers' valuable time spent conducting these experiments.

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Metabolic programs are rewired in cancer cells to support survival and tumor growth. Among these, recent studies have demonstrated that glutamate-oxaloacetate transaminase 1 (GOT1) plays key roles in maintaining redox homeostasis and proliferation of pancreatic ductal adenocarcinomas (PDA). This suggests that small molecule inhibitors of GOT1 could have utility for the treatment of PDA.

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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.

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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.

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In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.

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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.

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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.

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Neglected tropical diseases (NTDs) are an extremely important issue facing global health care. To improve "access to health" where people are unable to access adequate medical care due to poverty and weak healthcare systems, we have established two consortiums: the NTD drug discovery research consortium, and the pediatric praziquantel consortium. The NTD drug discovery research consortium, which involves six institutions from industry, government, and academia, as well as an international non-profit organization, is committed to developing anti-protozoan active compounds for three NTDs (Leishmaniasis, Chagas disease, and African sleeping sickness).

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Trypanosoma cruzi causes Chagas disease, a severe disease affecting 8-10 million people in Latin America. While nifurtimox and benznidazole are used to treat this disease, their efficacy is limited and adverse effects are observed. New therapeutic targets and novel drugs are therefore urgently required.

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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.

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As previously reported, triacsin C, a selective inhibitor of acyl-CoA synthetase, inhibited the synthesis of cholesteryl ester and triacylglycerol in mouse peritoneal macrophages, leading to a reduction of lipid droplets. Therefore, the in vivo efficacy was studied. Low-density lipoprotein receptor-knockout (LDLR-/-) mice were fed a high cholesterol diet (0.

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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.

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Fungal beauveriolide III (BeauIII, 1b), a cyclodepsipeptide inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) and showing antiatherogenic activity in mouse models, consists of L-Phe, L-Ala, D-allo-Ile, and 3-hydroxy-4-methyloctanoic acid (HMA) moieties, but the stereochemistry of the HMA part has not until now been fully defined. To determine it, four HMA stereoisomers were synthesized and labeled with (S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethane sulfonate (AP-OTf), a chiral fluorescent reagent. The derivatives were separated by HPLC and compared with the natural HMA derivative, which was thereby identified as (3S,4S)HMA in BeauIII.

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We developed a polymeric 2-DE chip system. The chip consisted of an IEF region, an SDS-PAGE region, a valveless connection port, and a sample introduction port. A "junction structure" as a valveless connection port, which allowed separating and connecting the first- and second-dimensional gels, was fabricated between their regions.

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High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE.

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During screening for inhibitors of lipid droplet accumulation in mouse peritoneal macrophages, two coumarins identified as decursin and decursinol angelate were isolated from the roots of Angelicae gigantis. The cellular molecular target of these inhibitors in macrophages was studied. Decursin and decursinol angelate inhibited cholesteryl ester (CE) synthesis with IC50 values of 9.

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Sespendole was isolated as an inhibitor of lipid droplet formation in macrophages from the culture broth of a fungal strain Pseudobotrytis terrestris FKA-25. The compound inhibited the synthesis of cholesteryl ester and triacylglycerol by mouse macrophages with IC50 values of 4.0 and 3.

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Synthesis of beauveriolide III (1b), which is an inhibitor of lipid droplet accumulation in macrophages, was achieved by solid-phase assembly of linear depsipeptide using a 2-chlorotrityl linker followed by solution-phase cyclization. On the basis of this strategy, a combinatorial library of beauveriolide analogues was carried out by radio frequency-encoded combinatorial chemistry. After automated purification using preparative reversed-phase HPLC, the library was tested for inhibitory activity of CE synthesis in macrophages to determine structure-activity relationships of beauveriolides.

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Four triterpenoids 1-4 isolated from the fruit body of Torametes orientaris were found to inhibit lipid droplet accumulation in macrophages. From the biochemical analysis, compounds 2 and 3 inhibited selectively cholesteryl ester synthesis in macrophages, while compounds 1 and 4 showed inhibition of both cholesteryl ester and triacylglycerol syntheses.

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Cancer chemotherapy for hypoxic tumor cells is thought to be an important issue, since hypoxia is related to tumor growth, apoptosis, angiogenesis and metastasis. Here, the bioactivities of xanthohumol (XN), a diacylglycerol acyltransferase inhibitor, against hypoxic cells were investigated. At first, the inhibitory effects of XN on the formation of lipid droplets in the cytoplasm were evaluated in hypoxia.

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Tetracyclic pyrans (+)-chloropuupehenone (1) and (+)-chloropuupehenol (5) and its C8-R-isomer (+)-3 were synthesized via a one-pot condensation of 1-chloro-2-lithio-3,5,6-tris(tert-butyldimethylsilyloxy)benzene (8) with (4aS,8aS)-3,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethylnaphthalene-1-carboxaldehyde (7). The major condensation product, (4aS,6aR,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (4), after desilylation provided tetracyclic pyran (+)-(4aS,6aR,12bS)-2H-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene-9,10-diol (3). At a dosage of 42 mg/rat over 8 h, pyran diol 3 inhibited the intestinal absorption of cholesterol by 71% in rats.

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Five new beauveriolides were isolated from the acetone extracts of Beauveria sp. FO-6979 mycelia fermented in amino acid-supplemented media. The structures were elucidated by spectroscopic studies including NMR experiments and chemical degradation.

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