Inhibition of Proteases by Mussel-Inspired Monomer

Abstract

Objectives: To evaluate the inhibitory effect of N-(3,4-dihydroxyphenethyl)methacrylamide (DMA) on the soluble and matrix-bound proteases. Methods: The inhibitory effects of DMA (1, 5, and 10 mM) dissolved in 50% ethanol/water on soluble collagenase and cysteine cathepsins (B and K) were evaluated using fluorometric assay kits. The effect of DMA on endogenous matrix metalloproteinases (MMPs) was examined by in situ zymography, and the fluorescence intensity was calculated by Imaris software. The 50% ethanol/water solution served as the vehicle control and 1 mM Chlorhexidine (CHX) served as the positive control. The optical density values of collagenase and cysteine cathepsins treated by different groups were analyzed by one-way ANOVA followed by Tukey test (α=0.05). The fluorescence intensity of in situ zymography was analyzed by Kruskal-Wallis one-way ANOVA followed by Dunn’s test (α=0.05). Results: The inhibition of proteases by DMA increased in a dose-dependent manner. The 10 mM DMA inactivated more than 95% of soluble collagenase, which was similar to 1 mM CHX (p>0.05), but was significantly higher than other treated groups (p<0.05). For the inactivation of cysteine cathepsin B, 10 mM DMA reached nearly 70% inhibition rate, which was significantly higher than 1 mM CHX (57.64%) (p<0.05). But for cysteine cathepsin K, the inhibitory effect of 1 mM CHX (94.81%) was significantly higher than 10 mM DMA (87.60%) (p<0.05). In situ zymography revealed that similar to 1 mM CHX, both 5 mM and 10 mM DMA significantly inactivated the matrix-bound MMPs, with a dramatic reduction of the fluorescence intensity compared with other groups (p<0.05). Conclusions: The 10 mM DMA achieved a comparable effect as 1 mM CHX on the inactivation of collagenase and matrix-bound MMPs; while its inhibitory effect is higher on cysteine cathepsins B, but lower on cysteine cathepsins K compared to 1 mM CHX.