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

Janelle L Lauer-Fields,Mare Cudic,Shuo Wei,Frank Mari,Gregg B Fields,Keith Brew

doi:10.1074/jbc.m611612200

Janelle L Lauer-Fields, Mare Cudic + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m611612200

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Abstract

The sarafotoxins and endothelins are approximately 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.

Highlights

The crystallographic structure of the tissue inhibitors of metalloproteinases (TIMPs)-1/ matrix metalloproteinases (MMPs)-3 complex indicated that ϳ75% of the intermolecular contacts of the TIMP-1 component with MMP-3 are made by the residues surrounding the Cys1–Cys70 disulfide bond, residues 1–5 and 66 –70 (14)
Hydrophobicity, which is often associated with high affinity binding to MMPs, can lead to membrane association and lower in vivo availability, whereas the presence of a potent zinc-binding moiety in most inhibitors may contribute to binding to zinc metalloproteins other than MMPs with the potential for deleterious side effects
Members of the endothelin family of peptides have well defined tertiary structures stabilized by two disulfide bonds

Summary

Introduction

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 activities are mediated via the endothelin receptors, ETA and ETB. Similar contacts were observed subsequently in the structure of complexes of TIMP-2 with MMP-14 (15) and MMP-13 (16) and the complex of N-TIMP-1 with MMP-1 (17) To develop their potential as therapeutic agents while minimizing deleterious side effects, N-TIMPs have been successfully engineered by mutation of their MMP interaction sites to enhance their inhibitory selectivity for different metalloproteinases (18 –20). There are many advantages associated with engineering a smaller peptide inhibitor of metalloproteinases if a TIMP-like scaffold can be identified In relation to this possibility, we noticed a topological similarity between ETs and the core of the MMP interaction site of TIMP (see Fig. 1).

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