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

Abstract

The sarafotoxins and endothelins are ϳ25-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. The endothelins (ET)3 are a family of 21-25-residue peptides that fall into two major categories, endothelins and sarafotoxins (Srt). The names are derived from their biological sources, endothelial cells, and the venoms of snakes of the genus Atractaspis (in Hebrew, Saraf Ein Gedi). Three ETs are found in humans, and an array of Srts are present in the venoms of different Atractaspis sp. ET family complexity arises from a variety of mechanisms; expression from multiple genes, proteolytic processing, and possibly RNA editing (1-4). NMR and crystallographic studies reveal that these peptides contain a bicyclic two-disulfide (Cys 1 -Cys 15 , Cys 3 -Cys 11 ) framework with a specific tertiary structure. The N-terminal residues form a ␤-strand followed by a ␤-turn at residue 5 (5); residues 9 -16 are mainly ␣-helical, whereas the C-terminal residues are flexible in solution (6). However, the crystal structure and protease susceptibility studies suggest that these C-terminal residues may associate with the bicyclic core as an ␣-helix (7, 8).ETs have many physiological effects including stimulation of cell proliferation, promotion of the expression of cell adhesion molecules and extracellular matrix components, activation of the inflammatory cascade, and the well characterized contraction of smooth muscle tissue (9). In humans, these biological acti...

Peptide-Based Selective Inhibitors of Matrix Metalloproteinase-Mediated Activities

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The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

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Grafting of functional motifs onto protein scaffolds identified by PDB screening – an efficient route to design optimizable protein binders

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