Computational analysis of the metal selectivity of matrix metalloproteinase 8.

Matrix metalloproteinase (MMP) is a class of metalloenzyme that cleaves peptide bonds in extracellular matrices. Their functions are important in both health and disease of animals. Here using quantum mechanics simulations of the MMP8 protein, the coordination chemistry of different metal cofactors is examined. Structural comparisons reveal that Jhan-Teller effects induced by Cu(II) coordination distorts the wild-type MMP8 active site corresponding to a significant reduction in activity observed in previous experiments. In addition, further analysis suggests that a histidine to glutamine mutation at residue number 197 can potentially allow the MMP8 protein to utilize Cu(II) in reactions. Simulations also demonstrates the requirement of a conformational change in the ligand before enzymatic cleavage. The insights provided here will assist future protein engineering efforts utilizing the MMP8 protein.

Discovery of Sulfated Small Molecule Inhibitors of Matrix Metalloproteinase-8.

Elevated matrix metalloproteinase-8 (MMP-8) activity contributes to the etiology of many diseases, including atherosclerosis, pulmonary fibrosis, and sepsis. Yet, very few small molecule inhibitors of MMP-8 have been identified. We reasoned that the synthetic non-sugar mimetics of glycosaminoglycans may inhibit MMP-8 because natural glycosaminoglycans are known to modulate the functions of various MMPs. The screening a library of 58 synthetic, sulfated mimetics consisting of a dozen scaffolds led to the identification of only two scaffolds, including sulfated benzofurans and sulfated quinazolinones, as promising inhibitors of MMP-8. Interestingly, the sulfated quinazolinones displayed full antagonism of MMP-8 and sulfated benzofuran appeared to show partial antagonism. Of the two, sulfated quinazolinones exhibited a >10-fold selectivity for MMP-8 over MMP-9, a closely related metalloproteinase. Molecular modeling suggested the plausible occupancy of the S1' pocket on MMP-8 as the distinguishing feature of the interaction. Overall, this work provides the first proof that the sulfated mimetics of glycosaminoglycans could lead to potent, selective, and catalytic activity-tunable, small molecular inhibitors of MMP-8.

Enzymatic inhibitory activity of Ficus deltoidea leaf extract on matrix metalloproteinase-2, 8 and 9.

Dysregulation of matrix metalloproteinases (MMPs) activity is known in many pathological conditions with which most of the conditions are related to elevate MMPs activities. Ficus deltoidea (FD) is a plant known for its therapeutic properties. In order to evaluate the therapeutic potential of FD leaf extract, we study the enzymatic inhibition properties of FD leaf extract and its major bioactive compounds (vitexin and isovitexin) on a panel of MMPs (MMP-2, MMP-8 and MMP-9) using experimental and computational approaches. FD leaf extract and its major bioactive compounds showed pronounced inhibition activity towards the MMPs tested. Computational docking analysis revealed that vitexin and isovitexin bind to the active site of the three tested MMPs. We also evaluated the cytotoxicity and cell migration inhibition activity of FD leaf extract in the endothelial EA.hy 926 cell line. Conclusively, this study provided additional information on the potential of FD leaf extract for therapeutical application.

Effect of matrix metalloproteinase 8 inhibitor and chlorhexidine on the cytotoxicity, oxidative stress and cytokine level of MDPC-23.

OBJECTIVE: This study investigated the effect of matrix metalloproteinase-8 inhibitor I (MMP8-I) and chlorhexidine (CHX) on the viability, oxidative stress and cytokine secretion of MDPC-23 under short-term (30min) and long-term (3 days) culture. METHODS: MDPC-23 were treated with MMP8-I or CHX for 30min, 1day, 2days and 3days to detect the proliferation by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. In the following assays, MDPC-23 treated with 0.0003% CHX were referred to CHX group, treated with 8µM MMP8-I were MMP8-I group. Cells without additional treatment were regarded as control group. The cell cycle, reactive oxygen species (ROS) level, and apoptosis were assessed by flow cytometry. The cytokine level was quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: In 30min, CHX at concentrations higher than 0.0003% dilution inhibited cell proliferation when compared to the control group. MMP8-I (0.1-500µM) showed no obvious cytotoxicity to MDPC-23, and MMP8-I (1000µM) inhibited cell proliferation. In 3 days, CHX (0.0003%) significantly inhibited cell growth, while MMP8-I (8µM) had no cytotoxicity. In the CHX group, the S phase population was decreased, and cellular ROS were increased in 3 days. In the MMP8-I group, the change of S phase population and cellular ROS was not significant compared with the control group. Cell apoptosis was not elevated in the MMP8-I group, while the apoptosis rate was increased in the CHX group both in 30min and 3 days. In 30min, CHX treatment significantly increased the secretion of interleukin (IL)-1ß and IL-8, but slightly increased the secretion of IL-10, while MMP8-I caused no change in cytokines. In 3 days, CHX treatment significantly increased the secretion of IL-1ß, IL-6, and IL-8, and inhibited the secretion of IL-10. MMP8-I treatment caused the increase of IL-6. SIGNIFICANCE: Compared with CHX, MMP8-I at low concentration did not result in cytotoxicity, oxidative stress, or the disorder of immune response.

Endotoxin-induced changes of type VII collagen- cleaving matrix metalloproteinases in lamellar tissue of extracorporeally perfused equine limbs.

OBJECTIVE To investigate the effect of lipopolysaccharide (LPS) on type VII collagen- cleaving matrix metalloproteinases (MMPs) in the lamellar tissue of extracorporeally perfused equine limbs. SAMPLE 10 right forelimbs and 3 left forelimbs collected from 10 adult horses after slaughter at a licensed abattoir. PROCEDURES Extracorporeal perfusion of the isolated equine limbs was performed for 10 hours under physiologic conditions (control-perfused limbs; n = 5) and with the addition of 80 ng of LPS/L of perfusate (LPS-perfused limbs; 5). Lamellar tissue specimens were then collected from the dorsal aspect of the hooves. Additionally, corresponding control specimens were collected from the 3 nonperfused left forelimbs. Immunohistochemical analysis was performed on paraffin-embedded tissue blocks with antibodies against total (latent and active) MMP-1, MMP-2, MMP-8, and MMP-9 as well as antibody against active MMP-9. Intensity of immunohistochemical staining was scored, and stain distribution in the lamellar tissue was noted. RESULTS Staining intensity of total and active MMP-9 was significantly increased in LPS-perfused versus control-perfused limbs. No such difference was identified for MMP-1, MMP-2, and MMP-8. CONCLUSIONS AND CLINICAL RELEVANCE Of the 4 MMPs that are capable of degrading type VII collagen, MMP-9 was the only one for which production increased in the lamellar tissue of isolated equine limbs perfused with versus without a clinically relevant concentration of LPS. These results suggested that MMP-9 may be involved in initiation of pathological changes in lamellar tissue in endotoxin-induced laminitis, whereas MMP-1, MMP-2, and MMP-8 may be less relevant.

Effect of matrix metalloproteinase 8 inhibitor on resin-dentin bonds.

OBJECTIVES: This study investigated the effect of matrix metalloproteinase 8 (MMP-8) on resin-dentin bonds, assessed the mechanical properties of the interfaces over time, and discussed the potential application of MMP-8 inhibitor I (MMP8-I) as a specific MMP-8 inhibitor to be incorporated into dental adhesives. METHODS: The activation and inhibition of MMP-8 was detected by colorimetric assay. After 1 day, 6 months and 1 year of storage of Control, MMP8-I, and chlorhexidine (CHX) groups, the microtensile bond strengths (µTBS) were used to evaluate the bond strength and failure mode distributions, and nanoleakage analysis was used to evaluate the minor scattered silver particles. RESULTS: Colorimetric assay showed that the activated MMP-8 was enhanced by adhesive procedures, while it was inhibited by the additional treatment of MMP8-I or CHX. Compared with the Control and CHX groups, the MMP8-I group had significantly higher bond strength and the hybrid layer from the MMP8-I-treated dentin exhibited structural integrity of the collagen network and decreased silver nitrate penetration after 1 year of storage. SIGNIFICANCE: MMP-8 inhibition I protects against the degradation of resin-dentin bonds over time, which is better than broad-scale enzyme inhibitor CHX. It shows that MMP8-I may be used in dentistry for preventing collagen degradation within hybrid layers to extend the longevity of resin-dentin bonds.

An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.

Collagenolytic Matrix Metalloproteinase Structure-Function Relationships: Insights From Molecular Dynamics Studies.

Several members of the zinc-dependent matrix metalloproteinase (MMP) family catalyze collagen degradation. Experimental data reveal a collaboration between different MMP domains in order to achieve efficient collagenolysis. Molecular dynamics (MD) simulations have been utilized to provide atomistic details of the collagenolytic process. The triple-helical structure of collagen exhibits local regions of flexibility, with modulation of interchain salt bridges and water bridges contributing to accessibility of individual chains by the enzyme. In turn, the hemopexin-like (HPX) domain of the MMP initially binds the triple helix and facilitates the presentation of individual strands to active site in the catalytic (CAT) domain. Extensive positive and negative correlated motions are observed between the CAT and HPX domains when collagen is bound. Ultimately, the MD simulation studies have complemented structural (NMR spectroscopy, X-ray crystallography) and kinetic analyses to provide a more detailed mechanistic view of MMP-catalyzed collagenolysis.

Synthesis, biological activity and molecular modeling study of new Schiff bases incorporated with indole moiety.

A new series of heterocyclic Schiff bases 2-9 containing indole moiety were synthesized by facile and efficient condensation of indole-3/2/5-carboxaldehyde (1a/1b/1c) with different aromatic and heterocyclic primary amines using conventional and/or microwave irradiation methods. The structures of the obtained compounds were assigned by sophisticated spectroscopic and spectrometric techniques (1D-NMR, 2D-NMR and MS). The synthesized compounds were screened for their cytotoxicity and antibacterial activities. In vitro cytotoxicity screening revealed that compound 5 exhibited moderate activity against KB-3-1 cell line (IC50=57.7 µM) while 5-indolylimino derivative 7 indicated close to the activity (IC50=19.6 µM) in comparison with the positive control (+)-Griseofulvin (IC50=19.2 µM), while the tested compounds 5, 6b, 7 and 9 revealed good or moderate antibacterial activity. In addition, molecular docking study of Schiff bases 2-9 was performed by Molecular Operating Environment (MOE 2014.09) program on the matrix metalloproteinase-8 (MMP-8) (Protein Data Bank (PDB) ID: 1MNC) in an attempt to explore their mode of action as anticancer drugs.

[Effects of serum starvation on cell cycle synchronization in primary human umbilical vein endothelial cells].

OBJECTIVE: To investigate the optimal starvation conditions of human umbilical vein endothelial cells (HUVECs) and establish a highly efficient and stable method for separating HUVECs. METHODS: HUVECs harvested from human umbilical cords by digestion with 0.1% collagenase II for 15 min were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth factor (ECGS) and 1% penicillin/streptomycin solution(P/S) at 37 degrees celsius; in 5% CO2. The cells were observed for cell morphology under an inverted microscope and identified with immunofluorescence assay. The purity of HUVECs was detected using flow cytometry (FCM). The cell cycles of HUVECs cultured in the presence of 0, 0.1%, 0.5%, and 1% FBS for 0, 6, 12, 18, and 24 h were analyzed with flow cytometry. RESULTS: s The purity of HUVECs harvested by digestion with 0.1% collagenase II reached 99.67%. The primary HUVECs showed a cobblestone or volute appearance in vitro. Immunocytochemistry showed that HUVECs highly expressed VIII-related antigen. Cell culture in the presence of different concentrations of FBS for 6 h resulted in 70% G0/G1 phase cells, which increased to 80%-90% at 12 h of cell culture, and further to around 95% at 18 and 24 h. CONCLUSION: Digestion with 0.1% collagenase II can obtain high-purity primary HUVECs. Culturing HUVECs in serum-free medium for 12 h can result in a high purity (over 80%) of G0/G1 phase cells.

Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in Escherichia coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

Combinatorial Screening Identifies Novel Promiscuous Matrix Metalloproteinase Activities that Lead to Inhibition of the Therapeutic Target IL-13.

The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease.

Isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34(Low)CD31(Pos)VEGFR-3(Pos)PODOPLANIN(Pos) LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Evaluation of a new standardized enzymatic isolation protocol for human umbilical cord-derived stem cells.

The umbilical cord (UC) represents an important source of mesenchymal stem cells (MSC). These human UC-derived MSC (UC-MSC) have already been isolated using a protocol based on the migratory and plastic adhesive properties of MSC (UC-MSC-Mig). The UC-MSC-Mig isolation method, however, is difficult to standardize. Therefore, we developed an enzymatic isolation protocol (UC-MSC-Enz) to overcome the above mentioned disadvantages. First, we investigated the UC-MSC-Enz for their MSC properties. We found that UC-MSC-Enz express the MSC markers CD73, CD90 and CD105 and are able to differentiate into osteoblasts, adipocytes and chondroblasts fulfilling the MSC criteria of the International Society for Cellular Therapy. Previously we found that UC-MSC-Mig are unique among MSCs due to their significant expression of several hepatic (progenitor) markers. Therefore, we also investigated the expression of hepatic transcription factors and other hepatic markers in UC-MSC-Enz at both the mRNA and protein level. We found that the expression of hepatic transcription factors (GATA4, GATA6, SOX9 and SOX17) and hepatic markers (AFP, DPP4, CX43, DKK1, DSG2, KRT18 and KRT19) in UC-MSC-Enz was not significantly different from those of UC-MSC-Mig. Consequently, this optimized enzyme-based method represents a fast, robust and standardized way to isolate UC-MSC for a broad range of applications.

Grafting MAP peptide to dental polymer inhibits MMP-8 activity.

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.

Association between -799C/T single nucleotide polymorphism of the MMP­8 promoter region and thoracic aortic dissection.

Thoracic aortic dissection (TAD) is a life-threatening vascular condition, in which matrix metalloproteinases (MMPs) are involved. Since the key determinants underlying MMP action remain elusive, the present study investigated the correlation between single nucleotide polymorphisms (SNPs) in the promoter region of the MMP­8 gene and a predisposition to TAD, by comparing genotypes of TAD patients and healthy controls. From 154 TAD patients and 148 healthy individuals, DNA samples were obtained from venous blood, and genotyping was performed by a combination of polymerase chain reaction and automatic sequencing to detect SNPs in the MMP­8 promoter. Data were analyzed and odds ratios (OR) and 95% confidence intervals (CI) were calculated. P<0.05 was considered to indicate a statistically significant result. Two SNPs, -799C/T and -767A/T, were identified in the MMP­8 promoter. Distribution of the -767A/T genotype was not significantly different between the patients and healthy controls. The -799C/C genotype was utilized as a match control, and significant differences in the genotypic distribution were observed between the patients with TAD and the controls. Furthermore, it was identified that the distribution of the ­799C/T+T/T and -799C/C genotypes between the TAD and control populations was significantly different. The frequency of T allele distribution was higher in the TAD group (27%) than in the control group (13.5%). The genotype distribution followed the Hardy-Weinberg equilibrium. In the present study, it was concluded that the ­799C/T polymorphism in the promoter region of MMP­8 may be associated with the development of TAD and that the T allele may increase patient predisposition to the disease.

Combined structure- and ligand-based pharmacophore modeling and molecular dynamics simulation studies to identify selective inhibitors of MMP-8.

Matrix metalloproteinase-8 (MMP-8) is the key mediator in initiating type I collagen degradation and is associated with rheumatoid arthritis. In the present study, a pharmacophore hypothesis was developed based on selective non zinc binding inhibitors of MMP-8. The pharmacophore hypothesis was refined manually and validated by observing structures and the interactions of MMP-8 inhibitors. The refined pharmacophore model was able to discriminate the non-zinc binding inhibitors of MMP-8 with respect to other inhibitors. Hence this study proposes a combined structure- and ligand-based pharmacophore model that is suitable for retrieving the novel inhibitors of MMP-8. The pharmacophore hypothesis AADRH was used as query for retrieving potential compounds from the Zinc database and hits were selected based on the catalytic selective amino acid residues of Arg 222, and Tyr 227. We identified six compounds as potent inhibitors and their selectivity profile were checked against different subtypes of MMPs using the cross-docking method. Molecular dynamics results indicated that ZINC 00673680 forms a stable interaction with the key amino acid residues and avoids the zinc atom with a distance of 5.49 Å. Our computational study might be useful for further development of selective MMP-8 inhibitors.

[Effect of quaternary ammonium methacrylates incorporation into a dental adhesive on the resistance of enzymatic degradation of resin-dentine interfaces].

OBJECTIVE: To evaluate the effect of quaternary ammonium methacrylates incorporation into a dental adhesive on the resistance of enzymatic degradation of resin-dentine interfaces. METHODS: Thirty caries-free extracted human third molars were randomly divided into three groups (n = 10): 2-methacryloxylethyl dodecylmethyl ammonium bromide (MAE-DB) incorporated adhesive served as the experimental group, pre-treatment with chlorhexidine (CHX) served as a positive control, Adper(TM) Single Bond 2 served as a negative control. The resin-dentin interfaces were prepared using total etch bond system. After storage in distilled water at 37 °C for 24 h, the bonded teeth were vertically sectioned into beams. Beams were respectively immersed in artificial saliva containing 2 g/L MMP-8 at 37 °C for 0 h, 24 h and 120 h (n = 30). Micro-tensile bond strength, failure modes and nanoleakage were examined. RESULTS: There were no significantly differences of micro-tensile bond strength between groups before hydrolysis (P > 0.05). After the enzymatic hydrolysis of 24 h and 120 h, the micro-tensile bond strength of MAE-DB groups [(31.13 ± 8.77) MPa, (24.14 ± 6.64) MPa] were significantly higher than that of the negative control groups [(25.63 ± 6.90) MPa, (15.22 ± 6.57) MPa] (P < 0.05). Most of the failures were found in the base part of the hybrid layer in the negative control group, while failures occurred through the top of the hybrid layer in CHX and MAE-DB groups after the enzymatic hydrolysis.Specimens from each immediate group showed minor silver deposits in hybrid layer. After beams being enzymatic hydrolyzed for 120 h, it was shown that the silver nitrate uptake in the negative control group were significantly different from those in the CHX and MAE-DB groups (P < 0.05). CONCLUSIONS: Dental adhesive incorporation of MAE-DB could improve the anti-degrade ability of resin-dentin interfaces.