Zymographic Evaluation of Plasminogen Activators and Plasminogen Activator Inhibitors

Ramsby, Melinda L.

doi:10.1016/s0065-2423(04)38004-2

Cited by 3 publications

(3 citation statements)

References 136 publications

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“…The band is assigned as a 100% intensity of MMP9 being expressed by a 4T1 cell. In the presence of an MMP inhibitor, the intensity should be reduced, indicating the inhibition of such an inhibitor in MMP9 gelatinolytic activity …”

Section: Resultsmentioning

confidence: 99%

“…In the presence of an MMP inhibitor, the intensity should be reduced, indicating the inhibition of such an inhibitor in MMP9 gelatinolytic activity. 41 Figure 2b presents a zymogram of MMP9 expressed by 4T1 cells consisting of five bands: untreated cell 1; untreated cell 2; and cells treated by 2, 7, and 9 at a 10 μM concentration. It is clear that the band intensity of the untreated 4T1 cells is different than that of the 4T1 cells treated with those three active compounds, indicating that 2, 7, and 9 have inhibited the activity of MMP9.…”

Section: ■ Introductionmentioning

confidence: 99%

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Arylamide as Potential Selective Inhibitor for Matrix Metalloproteinase 9 (MMP9): Design, Synthesis, Biological Evaluation, and Molecular Modeling

Hariono

Nuwarda

Yusuf

et al. 2019

J. Chem. Inf. Model.

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Previous studies have reported that compounds bearing an arylamide linked to a heterocyclic planar ring have successfully inhibited the hemopexin-like domain (PEX9) of matrix metalloproteinase 9 (MMP9). PEX9 has been suggested to be more selectively targeted than MMP9’s catalytic domain in a degrading extracellular matrix under some pathologic conditions, especially in cancer. In this study, we aim to synthesize and evaluate 10 arylamide compounds as MMP9 inhibitors through an enzymatic assay as well as a cellular assay. The mechanism of inhibition for the most active compounds was investigated via molecular dynamics simulation (MD). Molecular docking was performed using AutoDock4.0 with PEX9 as the protein model to predict the binding of the designed compounds. The synthesis was carried out by reacting aniline derivatives with 3-bromopropanoyl chloride using pyridine as the catalyst at room temperature. The MMP9 assay was conducted using the FRET-based MMP9 kits protocol and gelatin zymography assay. The cytotoxicity assay was done using the MTT method, and the MD simulation was performed using AMBER16. Assay on MMP9 demonstrated activities of three compounds (2, 7, and 9) with more than 50% inhibition. Further inhibition on MMP9 expressed by 4T1 showed that two compounds (7 and 9) inhibited its gelatinolytic activity more than 50%. The cytotoxicity assay against 4T1 cells results in the inhibition of the cell growth with an EC50 of 125 μM and 132 μM for 7 and 9, respectively. The MD simulation explained a stable interaction of 7 and 9 in PEX9 at 100 ns with a free energy of binding of −8.03 kcal/mol and −6.41 kcal/mol, respectively. Arylamides have potential effects as selective MMP9 inhibitors in inhibiting breast cancer cell progression.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Arylamide as Potential Selective Inhibitor for Matrix Metalloproteinase 9 (MMP9): Design, Synthesis, Biological Evaluation, and Molecular Modeling

Hariono

Nuwarda

Yusuf

et al. 2019

J. Chem. Inf. Model.

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“…Overlay zymography is particularly useful in the analysis of complex proteolytic systems such as the plasminogen activator/plasminogen activator inhibitor (PA/PAI, ) as a variety of components (cofactors, cosubstrates, and inhibitors) can be incorporated in the indicator support and tested independently after the electrophoresis. Putative or protease class specific substrates and inhibitors can also be easily screened by this technique.…”

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confidence: 99%

Retaining in‐gel zymographic activity of cysteine proteases via a cysteine‐supplemented running buffer

2016

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Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins.

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Computational modeling of culture media for enhanced production of fibrinolytic enzyme from marine bacterium Fictibacillus sp. strain SKA27 and in vitro evaluation of fibrinolytic activity

2019

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Zymographic Evaluation of Plasminogen Activators and Plasminogen Activator Inhibitors

Cited by 3 publications

References 136 publications

Arylamide as Potential Selective Inhibitor for Matrix Metalloproteinase 9 (MMP9): Design, Synthesis, Biological Evaluation, and Molecular Modeling

Arylamide as Potential Selective Inhibitor for Matrix Metalloproteinase 9 (MMP9): Design, Synthesis, Biological Evaluation, and Molecular Modeling

Retaining in‐gel zymographic activity of cysteine proteases via a cysteine‐supplemented running buffer

Computational modeling of culture media for enhanced production of fibrinolytic enzyme from marine bacterium Fictibacillus sp. strain SKA27 and in vitro evaluation of fibrinolytic activity

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