ABSTRACT
Biologics can be an improvement to small molecule drugs, providing high specificity for an identified target, lowering toxicity and limiting side effects. To achieve effective delivery, the biologic must have sufficient time to reach the target tissue. A prolonged half-life in the circulating environment is desired, but often serum stability is limited by proteases. Proteolysis in the serum causes degradation and inactivation as the biologic is fragmented and more rapidly cleared from the body. To improve the circulating half-life, large, hydrophilic polymers may be conjugated or stable fusion tags may be engineered to increase the effective size of the peptide and to hinder degradation by proteases. Improved resistance to proteases is essential for effective delivery. Here, a proof of concept study is presented using a metal-binding tripeptide tag known as the claMP Tag to create an inline conjugate and the ability of the tag to inhibit proteolysis was examined.
Subject(s)
Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Engineering/methods , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Densitometry , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Hydrogen-Ion Concentration , Maltose-Binding Proteins/chemistry , Maltose-Binding Proteins/genetics , Maltose-Binding Proteins/metabolism , Nickel/chemistry , Nickel/metabolism , Oligopeptides/genetics , Protein Stability , Proteolysis , Recombinant Fusion Proteins/geneticsABSTRACT
The mechanism of matrix metalloproteinase-8 (MMP-8) inhibition was investigated using ellipsometric measurements of the interaction of MMP-8 with a surface bound peptide inhibitor, tether-metal abstraction peptide (MAP), bound to self-assembled monolayer films. MMP-8 is a collagenase whose activity and dysregulation have been implicated in a number of disease states, including cancer metastasis, diabetic neuropathy, and degradation of biomedical reconstructions, including dental restorations. Regulation of activity of MMP-8 and other matrix metalloproteinases is thus a significant, but challenging, therapeutic target. Strong inhibition of MMP-8 activity has recently been achieved via the small metal binding peptide tether-MAP. Here, the authors elucidate the mechanism of this inhibition and demonstrate that it occurs through the direct interaction of the MAP Tag and the Zn(2+) binding site in the MMP-8 active site. This enhanced understanding of the mechanism of inhibition will allow the design of more potent inhibitors as well as assays important for monitoring critical MMP levels in disease states.
