Essential tyrosine residues in 3-ketosteroid-Δ1-dehydrogenase from Rhodococcus rhodochrous

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系Tetranitromethane treatment of 3-ketosteroid-Δ1-dehydrogenase of Rhodococcus rhodechrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0.014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid

Expression of CysLT2 receptors in asthma lung, and their possible role in bronchoconstriction

AbstractBackgroundThe expression and functional role of CysLT2 receptors in asthma have not been clarified. In this study, we evaluated CysLT2 receptors expression, and effects of CysLT2-and CysLT1/2-receptor antagonists on antigen-induced bronchoconstriction using isolated lung tissues from both asthma and non-asthma subjects.MethodsCysLT1 and CysLT2 receptors expression in asthma and non-asthma lung tissue preparations was examined in immunohistochemistry experiments, and their functional roles in antigen-induced bronchoconstriction were assessed using ONO-6950, a dual CysLT1/2-receptor antagonist, montelukast, a CysLT1 receptor antagonist, and BayCysLT2RA, a CysLT2 receptor-specific antagonist.ResultsCysLT1 receptors were expressed on the bronchial smooth muscle and epithelium, and on alveolar leukocytes in 5 in 5 non-asthma subjects and 2 in 2 asthma subjects. On the other hand, although degrees of CysLT2 receptors expression were variable among the 5 non-asthma subjects, the expression in the asthma lung was detected on bronchial smooth muscle, epithelium and alveolar leukocytes in 2 in 2 asthma subjects. In the non-asthma specimens, antagonism of CysLT2 receptors did not affect antigen-induced bronchial contractions, even after pretreatment with the CysLT1-receptor specific antagonist, montelukast. However, in the bronchus isolated from one of the 2 asthma subjects, antagonism of CysLT2 receptors suppressed contractions, and dual antagonism of CysLT1 and CysLT2 receptors resulted in additive inhibitory effect on anaphylactic contractions.ConclusionsCysLT2 receptors were expressed in lung specimens isolated from asthma subjects. Activation of CysLT2 receptors may contribute to antigen-induced bronchoconstriction in certain asthma population

Discovery of Gemilukast (ONO-6950), a Dual CysLT₁ and CysLT₂ Antagonist As a Therapeutic Agent for Asthma

An orally active dual CysLT₁ and CysLT₂ antagonist possessing a distinctive structure which consists of triple bond and dicarboxylic acid moieties is described. Gemilukast (ONO-6950) was generated via isomerization of the core indole and the incorporation of a triple bond into a lead compound. Gemilukast exhibited antagonist activities with IC₅₀ values of 1.7 and 25 nM against human CysLT₁ and human CysLT₂, respectively, and potent efficacy at an oral dose of 0.1 mg/kg given 24 h before LTD₄ challenge in a CysLT₁-dependent guinea pig asthmatic model. In addition, gemilukast dose-dependently reduced LTC₄-induced bronchoconstriction in both CysLT₁- and CysLT₂-dependent guinea pig asthmatic models, and it reduced antigen-induced constriction of isolated human bronchi. Gemilukast is currently being evaluated in phase II trials for the treatment of asthma

Discovery of Gemilukast (ONO-6950), a Dual CysLT₁ and CysLT₂ Antagonist As a Therapeutic Agent for Asthma

An orally active dual CysLT₁ and CysLT₂ antagonist possessing a distinctive structure which consists of triple bond and dicarboxylic acid moieties is described. Gemilukast (ONO-6950) was generated via isomerization of the core indole and the incorporation of a triple bond into a lead compound. Gemilukast exhibited antagonist activities with IC₅₀ values of 1.7 and 25 nM against human CysLT₁ and human CysLT₂, respectively, and potent efficacy at an oral dose of 0.1 mg/kg given 24 h before LTD₄ challenge in a CysLT₁-dependent guinea pig asthmatic model. In addition, gemilukast dose-dependently reduced LTC₄-induced bronchoconstriction in both CysLT₁- and CysLT₂-dependent guinea pig asthmatic models, and it reduced antigen-induced constriction of isolated human bronchi. Gemilukast is currently being evaluated in phase II trials for the treatment of asthma