Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics.

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.

Bioconversion of cinnamic acid derivatives by Schizophyllum commune.

To investigate the production of useful phenols from plant resources, we examined the metabolism of cinnamic acid derivatives by a wood-rotting fungus, Schizophyllum commune. Four cinnamic acid derivatives (cinnamic, p-coumaric, ferulic, and sinapic acids) were tested as substrates. Two main reactions, reduction and cleavage of the side chain, were observed. Reduction of the side chain was confirmed in cinnamic acid and p-coumaric acid metabolism. The side chain cleavage occurred in p-coumaric acid and ferulic acid metabolism but the initial reactions of these acids differed. Sinapic acid was not metabolized by S. commune. p-Hydroxybenzaldehyde accumulation was observed in the culture to which p-coumaric acid was added. This suggests that S. commune is a useful agent for transforming p-coumaric acid into p-hydroxybenzaldehyde.

[Studies of the Various Chronic Kidney Failure Rat Models and Hemodialysis Mini-pig Model for the Evaluation of Anti-hyperphosphatemia Drugs].

Animal models of chronic kidney failure (CKF) have been developed for the pharmacodynamic evaluation of various phosphate binders that are used clinically to treat hyperphosphatemia in patients with chronic kidney disease. However, these models represent different disease states and severities, depending on the experimental conditions and are not clearly defined for pharmacological evaluation. In addition, experimental models have not yet been established for artificial dialysis. The purpose of this study was to confirm the utility of the various rat models of CKF and the mini-pig model of hemodialysis as models of hyperphosphatemia for pharmacodynamic evaluation. Various rat models of pre-dialysis CKF (oral adenine dosing, 5/6 resection, and ligation nephrectomy model) were evaluated through determinations of serum and urinary parameters (osmolality, creatinine, and phosphorus), pathological observations of kidney, and the phosphorus-absorbing properties of lanthanum carbonate (La) formulations. The rat and mini-pig models were compared based on each evaluation index. In the oral adenine dosing model, serum phosphorus increased markedly and the area under the serum phosphorus concentration-time curve (phosphorus AUC) decreased in a dose-dependent manner with the administration of La formulations. In contrast, a significant decrease in serum phosphorus AUC, a prolongation of the dialysis interval, and an improvement in dialysis efficiency were observed after administration of La formulations to the mini-pig hemodialysis model. Furthermore, the results of bioequivalence studies between two La formulations (Fosrenol and SW670, a generic formulation) suggested that the rat and mini-pig models are useful and precise as pre-dialysis and dialysis models, respectively.

Formation of 4-vinyl guaiacol as an intermediate in bioconversion of ferulic acid by Schizophyllum commune.

In order to utilize phenolic compounds in unused biomass resources, the metabolic pathway of ferulic acid by way of a white-rot fungus, Schizophyllum commune, was investigated. Ferulic acid was immediately degraded, and the formation of 4-vinyl guaiacol was confirmed by GC-MS analysis. The metabolic test of ferulic acid and its degradation products indicated that S. commune converted ferulic acid into 4-vinyl guaiacol by decarboxylation. This was then oxidized to vanillin and vanillic acid. This result indicates that S. commune distinguished ferulic acid from lignins and metabolized it specifically.

Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques to measure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrP(Sc)) was comparable to that of enzyme-linked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis.