The use of homology modeling and site-directed mutagenesis in the identification of selective inhibitors of endothelin-converting enzyme-2 Abstract: Endothelin-converting enzyme-2 (ECE-2a) is a member of M13 family of zinc metallopeptidases. The characteristic features of ECE-2 such as neuroendocrine distribution and acidic pH optimum suggest an important role in the regulation of peptide levels in vivo. In order to better understand the structure of the active site and to aid in the development of specific inhibitors of this peptidase, we generated a three-dimensional molecular model of ECE-2 by homology modeling using the crystal structure of its close homolog, neprilysin (EC 3.4.24.11) as a template. On the bases of predictions made from the molecular model we mutated two residues, Trp 148 and Tyr 563 in the S2' subsite of the catalytic cleft of ECE-2 and examined the kinetic properties of these mutated recombinant enzymes. We find that mutation of Tyr 563 of ECE-2 significantly affects the catalytic activity and inhibitor binding of the enzyme as predicted by the model. We next used the molecular model to virtually screen a 13000 compound library of small drug-like molecules and the top-scoring compounds were tested for their ability to inhibit ECE-2 in biochemical assay. This led to the identification of three compounds that inhibited ECE-2 with high affinity and exhibited increased specificity for ECE-2 as compared to neprilysin. Thus the model provides a new useful tool to probe the active site of ECE-2 and design new selective inhibitors of this enzyme.