Ellagitannins (Ellagic Acid, Urolithin A, Urolithin B) Inhibit the Catalytic Activity of Human Recombinant Metalloproteinase 9.

Background: Ellagitannins are well-recognized for their antioxidant, chemopreventive, anti-inflammatory, and neuroprotective efficacy. Due to their poor absorption and extensive catabolism, it is proposed that urolithins, as ellagic acid (EA) metabolites, are the real active molecules exerting these biological functions.

Objectives: This research evaluated the inhibitory effects of EA, urolithin A (Uro A), and urolithin B (Uro B) on the activity of recombinant human matrix metalloproteinase 9 (rhMMP-9). Dysregulation of MMP-9 activity is directly involved in various pathologies; therefore, inhibition of this enzyme has clinical importance.

Methods: The rhMMP-9 activity was measured by a standard protease assay with casein as the substrate in the presence and absence of natural compounds, and the corresponding kinetic parameters were calculated. Interaction affinity between the enzyme and each of the ellagitannins studied was determined by the surface plasmon resonance (SPR) method. Molecular docking was performed using the C-terminally truncated human pro-MMP-9 structure as the receptor protein (PDB ID 1L6J) to predict ligand-receptor interaction and visualize the in vitro results.

Results: The rhMMP-9 assay showed that EA, Uro A, and Uro B demonstrated inhibitory activity with IC values of 17.14 µM, 33.29 µM, and 13.17 µM, respectively. Kinetic interaction parameters calculated using SPR analysis showed the lowest KD for Uro B (4.3 × 10 M), compatible with its IC. KD values calculated were 11.3 × 10 M for EA and 6.7 × 10 M for Uro A. A mixed type of inhibition with a non-competitive-uncompetitive pattern for Uro A and Uro B and a competitive-non-competitive pattern for EA was revealed.

Conclusions: Our results showed the promising inhibitory potential of EA, Uro B, and Uro A to affect the catalytic activity of the MMP-9 enzyme and also confirmed the fibronectin domain as a potential site for drug design against MMP-9.