Homology modeling and site-directed mutagenesis to identify selective inhibitors of endothelin-converting enzyme-2

J Med Chem. 2008 Jun 26;51(12):3378-87. doi: 10.1021/jm7015478. Epub 2008 May 29.


Endothelin-converting enzyme-2 (ECE-2), a member of M13 family of zinc metallopeptidases, has previously been shown to process a number of neuropeptides including those derived from prodynorphin, proenkephalin, proSAAS, and amyloid precursor protein. ECE-2, unlike ECE-1, exhibits restricted neuroendocrine distribution and acidic pH optimum; it is consistent with a role in the regulation of neuropeptide levels in vivo. Here, we report the generation of a three-dimensional (3D) molecular model of ECE-2 using the crystal structure of neprilysin (EC as a template. On the basis of the predictions made from the molecular model, we mutated and tested two residues, Trp 148 and Tyr 563, in the catalytic site. The mutation of Tyr 563 was found to significantly affect the catalytic activity and inhibitor binding. The molecular model was used to virtually screen a small molecule library of 13 000 compounds. Among the top-scoring compounds three were found to inhibit ECE-2 with high affinity and exhibited specificity for ECE-2 compared to neprilysin. Thus, the model provides a new useful tool to probe the active site of ECE-2 and design additional selective inhibitors of this enzyme.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid Endopeptidases / antagonists & inhibitors*
  • Aspartic Acid Endopeptidases / chemistry*
  • Aspartic Acid Endopeptidases / genetics
  • Catalytic Domain
  • Conserved Sequence
  • Endothelin-Converting Enzymes
  • Glycopeptides / chemistry
  • Kinetics
  • Metalloendopeptidases / antagonists & inhibitors*
  • Metalloendopeptidases / chemistry*
  • Metalloendopeptidases / genetics
  • Models, Molecular*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Neprilysin / chemistry
  • Neprilysin / genetics
  • Protein Conformation
  • Quinolines / chemistry
  • Quinoxalines / chemistry
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Small Molecule Libraries
  • Structure-Activity Relationship
  • Tryptophan / genetics
  • Tyrosine / genetics


  • Glycopeptides
  • Quinolines
  • Quinoxalines
  • Recombinant Proteins
  • Small Molecule Libraries
  • Tyrosine
  • Tryptophan
  • Aspartic Acid Endopeptidases
  • Metalloendopeptidases
  • Neprilysin
  • Endothelin-Converting Enzymes
  • phosphoramidon