title: Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2.
abstract: Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.
authors.affiliation: Department of Pharmacology, Texas Biotechnology Corporation, 7000 Fannin Street, Houston 77030, USA.
authors.forename: Zhong-Hua
authors.initials: ZH
authors.lastname: Yan
authors.forename: Kai-Jun
authors.initials: KJ
authors.lastname: Ren
authors.forename: Yafan
authors.initials: Y
authors.lastname: Wang
authors.forename: Sharon
authors.initials: S
authors.lastname: Chen
authors.forename: Tommy A
authors.initials: TA
authors.lastname: Brock
authors.forename: Ajay A
authors.initials: AA
authors.lastname: Rege
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D002268:Carboxypeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D009569:Nitric Oxide / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A; D010649:Phenylalanine / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010455:Peptides; D009569:Nitric Oxide; D014443:Tyrosine; D010649:Phenylalanine; D002268:Carboxypeptidases; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2