日本質量分析学会 第66回質量分析総合討論会

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ポスター発表

第4日 5月18日(金)  ポスター会場

Activity-based protein profilingを用いた、ヒト肝ミクロソーム中のGPR119アゴニスト, DS-8500a加水分解酵素の同定

(1第一三共RDノバーレ2第一三共)
o林真由美1小倉裕次1渡邉亜紀子2牧野智恵2和田直也1久保田一石1

DS-8500a is a GPR119 agonist and developed for the treatment of type 2 diabetes mellitus. It was revealed that a hydrolyzed form of DS-8500a was a major metabolite in human plasma. Identification of DS-8500a hydrolyzing enzyme is important to understand interspecies difference of drug metabolism for the clinical development of the drug.
Preliminary results of an inhibitor screening of DS-8500a hydrolysis using liver microsomes have suggested that a serine hydrolase plays a major role in hydrolysis processes of DS-8500a. Moreover, interspecies difference in DS-8500a hydrolysis was observed.
In this study, we investigated DS-8500a hydrolyzing enzyme by activity-based protein profiling (ABPP). ABPP utilizes active site-directed probes to profile the functional state of enzymes in whole proteomes. Fluorophosphonate probes were developed to profile the serine hydrolase activity in biological samples. We coupled competitive ABPP with a mass spectrometry (MS)-based quantification method using Tandem Mass Tag (TMT) labeling to identify a major DS-8500a hydrolyzing enzyme.
As a result, we identified several proteins including fatty acid amide hydrolase 2 (FAAH2) as candidates. We showed that exogenously expressed FAAH2 robustly exhibited the DS-8500a hydrolysis activity. The DS-8500a hydrolysis activity of human liver microsomes was more than 50% blocked by a FAAH1/2 inhibitor. These data indicate that FAAH2 is a major DS-8500a hydrolase in human liver microsomes.