Yumiko Muroga

Species Differences in Angiotensin II Generation and Degradation by Mast Cell Chymases

Abstract Although chymases are known to exhibit species differences in regard to angiotensin (Ang) II generation and degradation, their properties have never been compared under the same experimental conditions. We analyzed the processing of Ang I by chymases of a variety of species (human chymase, dog chymase, hamster chymase-1, rat mast cell protease-1 [rMCP-1], mouse mast cell protease-4 [mMCP-4]) at physiological ionic strength and under neutral pH conditions. Human chymase generated Ang II from Ang I without further degradation, whereas the chymases of other species generated Ang II, followed by degradation at the Tyr4-Ile5 site in a time-dependent manner. Kinetic analysis showed that in terms of Ang II generating activity (analyzed by cleavage of the Phe8-His9 bond using the model peptide Ang, Ile5-His6-Pro7-Phe8-His9-Leu10), the chymases ranked as follows:dog > human > hamster > mouse > rat (kcat/Km: 18, 11, 0.69, 0.059, 0.030 μ M− 1min− 1), and that in terms of Ang II degrading activity (i.e., cleavage of the Tyr4-Ile5 bond of Ang II), the order was hamster > rat > mouse > dog (kcat/Km: 5.4, 4.8, 0.39, 0.29 μ M−1min−1). These results suggest species differences in the contribution of chymases to local Ang II generation and degradation.

Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series.

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC(50) 3.2 nM; MCP-1-Induced Chemotaxis IC(50) 0.83 nM; Ca(2+) Flux IC(50) 7.5 nM].

Crystal structure of a complex of human chymase with its benzimidazole derived inhibitor

The crystal structure of human chymase complexed with a novel benzimidazole inhibitor, TJK002, was determined at 2.8 Å resolution. The present study shows that the benzimidazole ring of the inhibitor takes the stable stacking interaction with the protonated His57 in the catalytic domain of human chymase.

Generation and characterization of new monoclonal antibodies against human chymase.

We have succeeded in producing monoclonal antibodies directed against a wide variety of epitopes of human chymase by using two different immunogens: a recombinant human chymase-heparin mixture, and chymase alone. Hybridomas were screened by ELISA, and 7 clones were selected based on antibody titers. Epitopes were localized by Western blotting with a C-terminal-deletion series of chymase-GST fusion proteins, and it was possible to use the antibodies for Western blotting and immunohistochemistry. Dot-blot analysis for species specificity revealed that the MAbs bound canine chymase as well as human chymase, and that two of them also bound rodent chymases. These results indicate that the antibodies can be used for various immunological analyses in further investigations of chymase.