Program

Poster Presentations

Day 4, May 18（Fri.）　　Poster

4P-07　　PDF

Activity-based protein profiling for identification of GPR119 agonist, DS-8500a hydrolyzing enzyme in human liver microsomes

(¹DS RD Novare, ²Daiichi-Sankyo)
^oMayumi Hayashi¹, Yuji Ogura¹, Akiko Watanabe², Chie Makino², Naoya Wada¹, Kazuishi Kubota¹

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.