Fig 2: Lead and target MMP-2/9 inhibitors [28,49,50,54].

Fig 3: The inhibitory activity by IC50 of compounds (7f, 7a, and Sorafenib) on MMP-2.

Fig 4: Gelatin digestion and gene expression analysis of MMP-2 and MMP-9 inhibitors.(A) Zymogram showing MMP-2 inhibition in conditioned media from normal human fibroblast (NHF) and HaCaT cells treated with 50 nM candidate compounds during the developing phase. Significance was assessed using JMP software with the Kruskal-Wallis test, followed by post-hoc analysis using the Pairwise Wilcoxon Test (***p < 0.001, **p < 0.01). (B) Zymogram showing MMP-9 inhibition in HaCaT cell conditioned media treated with 50 nM candidate compounds, performed similarly to (a) but only in keratinocytes. (Significance was assessed using JMP software with the Kruskal-Wallis test, followed by post-hoc analysis using the Pairwise Wilcoxon Test (***p < 0.001, **p < 0.01). (C) Zymogram analysis of MMP-2 activity in conditioned media from NHF and HaCaT cells after 48-h treatment with 500 nM candidate compounds, demonstrating sustained inhibition. Significance was assessed using JMP software with the Kruskal-Wallis test, followed by post-hoc analysis using the Pairwise Wilcoxon Test (***p < 0.001, **p < 0.01). (D) Zymogram analysis of MMP-9 activity in HaCaT cells after 48-h treatment with 500 nM candidate compounds, following the same experimental setup as (C) but only in keratinocytes. Zymogram assays were conducted in duplicate. Significance was assessed using JMP software with the Kruskal-Wallis test, followed by post-hoc analysis using the Pairwise Wilcoxon Test (***p < 0.001, **p < 0.01). (E) Inhibition of fluorescent gelatin degradation by NHF cells cultured with candidate compounds at 1 μM for 4 days on a fluorescent-gelatin-coated plate, performed in triplicate. Significance was assessed using JMP software with the Kruskal-Wallis test, followed by post-hoc analysis using the Pairwise Wilcoxon Test (***p < 0.001, **p < 0.01). (F) Wound closure assay of HaCaT cells treated with candidate compounds at 5 μM for 40 hours, evaluating the impact on cell migration and wound healing. (G) Gene expression analysis of MMP-1 and (H) MMP-2 in NHF and HaCaT cells treated with candidate compounds at 5 μM for 24 hours, indicating no significant changes in expression levels. Statistical significance was assessed using the Kruskal-Wallis test followed by the Pairwise Wilcoxon Test (n = 3).

Fig 5: Structural analysis, design strategy, and synthesis of MMP-2 inhibitors.(A) Overall structure of MMP-1 (PDB ID: 1DTH) bound to a hydroxamate-based inhibitor; shown in front view (blue) and back view (grey) (B) Comparison of MMP-2 (PDB ID: 1QIB) and MMP-1 (PDB ID: 3SHI), highlighting essential binding domains, including the zinc-binding site and substrate-binding S1’ pocket, which are critical for selective inhibition. (C) Semi-rational design strategy for MMP-2 inhibitors, featuring hydroxamate-based zinc-binding groups (ZBGs) and selection of N-arylsulfonyl groups with varying lengths and bulkiness to enhance interactions within the hydrophobic S1' pocket of MMP-2.