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

Abstract

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.

Bioguided Fractionation of Local Plants against Matrix Metalloproteinase9 and Its Cytotoxicity against Breast Cancer Cell Models: In Silico and In Vitro Study

“…To determine the cytotoxicity of each extract on 4T1, T47D, and Vero cells, the MTT assay was employed. 4T1 is a triple-negative breast cancer cell line from Mus musculus [ 54 ] that contains PEX9 showing 61% homology with PDB 1ITV upon Blast Analysis [ 55 ]. T47D is a cell model to characterize the progesterone-specific effect of a luminal A subtype of breast cancer [ 56 ], whereas Vero cell is a non-tumorigenic cell from the kidney tissue of African green monkey [ 57 ].…”

The Pharmacological Tails of Matrix Metalloproteinases and Their Inhibitors

“…Peptides generated to mimic the outer beta strand of blade I or IV resulted in decreased levels of MMP9 dimers and also a reduction cell migration [33]. MMP-9 can also increase angiogenesis [38]. Using an allosteric inhibitor to the PEX domain, Hariono et al [38] demonstrated that inhibition of ECM proteolysis, which decreases release of vascular endothelial growth factor (VEGF) from within the ECM, significantly reduces binding of VEGF to its membrane receptor and subsequently, decreases angiogenesis.…”

“…MMP-9 can also increase angiogenesis [38]. Using an allosteric inhibitor to the PEX domain, Hariono et al [38] demonstrated that inhibition of ECM proteolysis, which decreases release of vascular endothelial growth factor (VEGF) from within the ECM, significantly reduces binding of VEGF to its membrane receptor and subsequently, decreases angiogenesis. broad-spectrum compounds [42].…”

“…Strategies to inhibit MMP functions with small molecules has been explored ( Table 2) [ 33,38,40,41,45]. In silico analysis of MMP9 in which molecular docking programs were utilized to map potential ligand binding sites in the PEX domain at the dimerization interface were successful at identifying compound that interfered with MMP9 homodimerization and blocked a downstream signaling pathway critical for MMP9 mediated cell migration and invasion [46].…”

Discovery of potent and specific inhibitors targeting the active site of MMP-9 from the engineered SPINK2 library

“…Other attempts have been made to target exosite domains, such as the HPX domain, which are less conserved. Several exosite inhibitors abrogated cancer cell migration through specific prevention of the association of pro-MMP-9 with both α4β1 integrin and CD44 without modulating the catalytic activity of MMP-9 [21][22][23]. Although exosite recognition could contribute to improving the specificity of active-site inhibitors, it remains difficult to obtain specific active-site inhibitors against a single MMP.…”