Mussel-inspired monomer – A new selective protease inhibitor against dentine collagen degradation

Highlights

• Anti-proteolytic ability of DMA increased in a dose-dependent manner except MMP-9.

• The binding mechanism of DMA toward proteases was simulated by molecular docking.

• CHX was a broad-spectrum inhibitor, while DMA selectively inhibited MMP-2 and − 8.

• DMA manifested comparable inhibitory effect as CHX on matrix-bound proteases.

Abstract

Objectives

To evaluate the inhibitory effect of a novel mussel-inspired monomer (N-(3,4-dihydroxyphenethyl)methacrylamide (DMA) on the soluble and matrix-bound proteases.

Methods

The inhibitory effect of DMA (0, 1, 5, and 10 mM) and 1 mM chlorhexidine (CHX) dissolved in 50% ethanol/water on soluble recombinant human matrix metalloproteinases (rhMMP-2, −8, and −9), as well as cysteine cathepsins (B and K) were evaluated using both fluorometric assay kits and molecular docking. The effect of CHX and DMA on matrix-bound proteases was examined by in situ zymography, and the fluorescence intensity and relative area were calculated by Image J software. All data obtained were analyzed by one-way ANOVA followed by Tukey test (α = 0.05).

Results

The anti-proteolytic ability of DMA increased in a dose-dependent manner except that of rhMMP-9. Inhibitory effect of 1 mM DMA against rhMMP-2, − 8, − 9, as well as cathepsin B and K was all significantly lower than 1 mM CHX (p < 0.05). The molecular docking analysis was in good agreement with the experimental results, that the binding energy of DMA was lower than CHX for all proteases. In situ zymography revealed that all DMA- and CHX-treated groups significantly inactivated the matrix-bound proteases, with a dramatic reduction of the fluorescence intensity and relative area compared with the control group (p < 0.05).

Significance

Under the prerequisite condition that the overall inhibitory performance on matrix-bound proteases was comparable by DMA and CHX, the more selective property of DMA could avoid inducing potential negative effects by suppressing MMP-9 when applied in dental treatment compared with CHX.

Introduction

The durability of resin-dentin bond relies on the integrity of the hybrid layer, a structure composed by the micro-interlocking of demineralized dentin collagen matrix with infiltrated adhesive monomers [1], [2]. However, since the hydrophobic adhesive monomers cannot fully occupy empty spaces around demineralized collagen fibrils and replace the remnant water [3], the uncoated collagen is prone to degradation by water hydrolysis [4] and endogenous protease action [5], [6]. Currently, two main categories of endogenous proteolytic enzymes are believed to be responsible for collagen degradation, including matrix metalloproteinases (MMPs) [7], [8], and cysteine cathepsins [9], [10].

MMPs are endogenous zinc- and calcium-dependent enzymes, which have been recognized as the major proteolytic enzymes for regulating extracellular matrix degradation [11]. Five MMPs have been found within the mineralized dentin matrix, including gelatinases MMP-2 and − 9 [12], collagenase MMP-8 [13], stromelysin MMP-3 [14], and MMP-20 [15]. These enzymes are mainly secreted by odontoblasts in their inactive form, and entrapped within the mineralized collagen matrix during odontogenesis [16], [17]. However, these pro-MMPs can be re-exposed and activated under acidic environment created by bacteria, phosphoric acid, and self-etch adhesives [18]. Low pH leads to cleavage of the prodomain and thus facilitates the functional activities of MMPs [19].

Cysteine cathepsins are members of C1 family of papain-like enzymes. There are in total 11 cysteine cathepsins present in human body [20], while both cathepsin B and cathepsin K have been reported to participate in collagen degradation in dentin matrix [10], [21]. Unlike cathepsin B which specifically targets cleavage in the non-helical telopeptide extensions of collagen, cathepsin K can also attack collagen at the triple helical region, and is therefore viewed as both a telopeptidase and a collagenase [22]. Similar to MMPs, cathepsins could also be activated by the application of phosphoric acid or self-etch adhesives in dentin bonding, but they are not stable at neutral or slightly alkaline pH [20].

Many studies have attempted to use protease inhibitors to suppress enzyme activities in the dentin matrix, so as to deter the degradation of collagen fibrils and prolong dentin bond durability [23], [24], [25]. Currently, the mechanism of MMP inhibitors used in dentistry can be classified into two categories, (1) chelation with zinc ion on the catalytic domain of MMPs; and (2) conformational change of the three-dimensional structure of MMPs [26]. Since the active site of all MMPs shares a highly-conserved catalytic domain which is composed by three histidine residues and the catalytic zinc ion, the strong zinc-binding inhibitors has the capability of inhibiting a broad-spectrum of MMPs [11]. However, experimental results demonstrated that the use of broad-spectrum MMP inhibitors may influence the normal process in dental development, resulting in an alternation in enamel and dentin formation as well as mineralization, and may cause severe systematic side effects (e.g., musculoskeletal syndrome and inflammation) [27], [28]. To date, the only broad-spectrum MMP inhibitor approved by US Food and Drug Administration (FDA) is Periostat® (doxycycline at sub-antimicrobial dose) for the treatment of periodontal disease [29], [30]. Therefore, researchers began to explore new inhibitors that have a relatively weak zinc binding ability and can induce the conformational change of MMPs with higher selectivity, such as the collagen cross-linkers. In the study done by Wang et al. [31], the researchers identified 19 potential inhibitors from 4000 natural compounds isolated from 100 medicinal plants using structure-based virtual screening. After further testing, two classes of natural compounds (caffeates and flavonoids) were found to have selective inhibition ability against MMP-2 and MMP-9 by occupying the specific pockets and inducing conformational change. Therefore, collagen cross-linkers have been widely incorporated in experimental adhesive systems to strengthen the mechanical properties of collagen fibrils, inhibit the activities of MMPs and cysteine cathepsins, and prolong the bonding durability of the hybrid layer [32].

However, the collagen cross-linkers are only simply mixed with dental adhesives in most published articles, which may gradually be released from the bonded interface and impair the longevity of resin restorations [33]. Besides, the free radical scavenging effect of the polyphenol cross-linkers may impede the polymerization of adhesives and impair the mechanical properties of the hybrid layer [34], [35]. Therefore, several studies attempted to combine the collagen cross-linker with polymerizable end, as a novel class of polymerizable collagen cross-linker monomers to be incorporated in adhesive systems [36], [37].

Inspired by this new concept, a mussel-derived monomer from marine environment, namely N-(3,4-dihydroxyphenethyl) methacrylamide (DMA) (Fig. 1), has been applied as a primer on dentin surface to prolong resin-dentin bond durability [38]. Theoretical saying, the carbon-carbon double bond of DMA could polymerize with adhesive monomers, the catechol group could cross-link with dentin collagen, while the amide bond is hydrolytically stable. Therefore, the experimental results supported that DMA could act as a “bridge” connecting the adhesive network and dentin collagen fibrils, unifying them as one whole structure to resist various external attacks from the harsh oral environment. However, the inhibitory effect of DMA on soluble and matrix-bound proteases has not been investigated. Therefore, the objective of current study was to evaluate the inhibitory effect of DMA on MMPs (−2, −8, −9) and cysteine cathepsins (B and K) in their soluble and matrix-bound forms via experimental and computational methods. The null hypotheses tested are that DMA has no inhibitory effect on (1) soluble proteases, and (2) matrix-bound proteases.