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J. Biol. Chem., Vol. 282, Issue 37, 26948-26955, September 14, 2007

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Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors^*

Janelle L. Lauer-Fields, Mare Cudic, Shuo Wei¹, Frank Mari, Gregg B. Fields, and Keith Brew²

From the College of Biomedical Science and Department of Chemistry & Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431

The sarafotoxins and endothelins are 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K_i values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.

Received for publication, December 19, 2006 , and in revised form, July 5, 2007.

^* This work was supported by National Institutes of Health Grants AR 40994 (to K. B.) and CA 98799 (to G. B. F.) and by a grant from the American Federation for Aging Research (to J. L. L.-F.). This work was also supported in part by National Institutes of Health Grant P41 RR-001081. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Cell Biology, University of Virginia Health Sciences Center, P.O. Box 800732, Charlottesville, Virginia 22908.

² To whom correspondence should be addressed: College of Biomedical Science, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431. Tel.: 561-297-0407; Fax: 561-297-2221; E-mail: kbrew{at}fau.edu.

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