title: Identification of tissue-restricted bioreaction suitable for in vivo targeting by fluorescent substrate library-based enzyme discovery.
abstract: Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yoshioka
authors.affiliation: PRESTO, Japan Science and Technology Agency (JST) , Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nakada
authors.forename: Jun
authors.initials: J
authors.lastname: Onagi
authors.forename: Yugo
authors.initials: Y
authors.lastname: Kuriki
authors.forename: Mitsuyasu
authors.initials: M
authors.lastname: Kawaguchi
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.affiliation: CREST, Japan Agency for Medical Research and Development (AMED) , Chiyoda-ku, Tokyo 100-0004, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D000813:Anilides / Q000737:chemistry / Q000378:metabolism*; D043182:Carboxylesterase / Q000378:metabolism*; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005453:Fluorescence; D006801:Humans; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000813:Anilides; D003374:Coumarins; C517204:CES2 protein, human; D043182:Carboxylesterase; C028743:7-amino-4-methylcoumarin