Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors

The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzy me (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for t he treatment of cardiovascular diseases. Such an approach requires a Fast. simple and selective assay to measure ECE activity. allowing rapid screenin g of inhibitors. We describe here two new ECE substrates based on the conce pt of intramolecularly quenched fluorescence' which may fulfill this aim. O ne. S-1 [Pya(21)-Nop(22)-bET-1(19-35)], is the (19-35) fragment of the natu ral peptide big-ET-1(1-38), which is modified by introducing the fluorescen t amino acid, pyrenylalanine (Pya), in position 21 and a quencher. p-nitrop henylalanine (Nop), in position 22. The second substrate (S,) is a small pe ptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-Nh(2), from a biased subs trate peptide library. The recombinant, hECE 1c, cleaved both Pya(21)-Nop(2 2)-bET-1(19-35) and the natural substrate selectively between residues 21 a nd 22, whereas cleavage occurred between alanine and phenylalanine in the s mall peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. The se assays, which can be used directly on tissue homogenates, are the most s ensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors.