Jennifer S. Laurence

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 Zn2+ 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.

Matrix metalloproteinases (MMPs) are a class of zinc and calcium‐dependent endopeptidases responsible for degrading extracellular matrix (ECM) components. Their activity is critical for both normal biological function and pathological processes (Dejonckheere et al., Cytokine Growth Factor Rev 2011;22:73–81). In dental restorations, the release and subsequent acid activation of MMPs contributes to premature failure. In particular, MMP‐8 accelerates degradation by cleaving the collagen matrix within the dentin substrate in incompletely infiltrated aged bonded dentin (Buzalaf et al., Adv Dent Res 2012;24:72–76), hastening the need for replacement of restorations. Therefore, development of a dental adhesive that better resists MMP‐8 activity is of significant interest. We hypothesize that modification of the polymer surface with an inhibitor would disable MMP‐8 activity. Here, we identify the metal abstraction peptide (MAP) as an inhibitor of MMP‐8 and demonstrate that tethering MAP to methacrylate polymers effectively inhibits catalysis. Our findings indicate complete inhibition of MMP‐8 is achievable using a grafting approach. This strategy has potential to improve longevity of dental adhesives and other polymers and enable rational design of a new generation of biocompatible materials. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 324–331, 2015.

Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

The Extracellular 1 (EC1) domain of E-cadherin has been shown to be important for cadherin–cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell–cell adhesion located at the adherens junction of the biological barriers (i.e., intestinal mucosa and the blood–brain barrier (BBB)). Cadherin peptides can modulate cadherin interactions to improve drug delivery through the BBB. However, the mechanism of modulating the E-cadherin interactions by cadherin peptides has not been fully elucidated. To provide a basis for subsequent examination of the structure and peptide-binding properties of the EC1 domain of human E-cadherin using solution NMR spectroscopy, the ¹H, ¹³C and ¹⁵N backbone resonance of the uniformly labeled-EC1 were assigned and the secondary structure was determined based on the chemical shift values. These resonance assignments are essential for assessing protein–ligand interactions and are reported here.

Metabolic activity of the oral microbiota leads to acidification of the microenvironment and promotes demineralization of tooth structure at the margin of composite restorations. The pathogenic impact of the biofilm at the margin of the composite restoration could be reduced by engineering novel dentin adhesives that neutralize the acidic microenvironment. Integrating basic moieties into methacrylate derivatives has the potential to buffer against acid-induced degradation, and we are investigating basic monomers for this purpose. These monomers must be compatible with existing formulations, which are hydrophobic and marginally miscible with water. As such, co-solvent systems may be required to enable analysis of monomer function and chemical properties. Here the authors present an approach for examining the neutralizing capacity of basic methacrylate monomers in a water/ethanol co-solvent system using nuclear magnetic resonance (NMR) spectroscopy. NMR is an excellent tool for monitoring the impact of co-solvent effects on pKa and buffering capacity of basic monomers because chemical shift is extremely sensitive to small changes that most other methods cannot detect. Because lactic acid (LA) is produced by oral bacteria and is prevalent in this microenvironment, it was used to analyze the effectiveness of basic monomers to neutralize acid. The ¹³C chemical shift of the carbonyl in LA was monitored as a function of ethanol and monomer concentration and each was correlated with pH to determine the functional buffering range. This study shows that the buffering capacity of even very poorly water-soluble monomers can be analyzed using NMR.