Biophysical characterization and in silico analysis of natural and synthetic compounds targeting Listeria monocytogenes HtrA protease.

HtrA protein is a member of a serine protease family with dual functions as a protease and molecular chaperone. It is a virulence factor in many bacteria, including the food-borne pathogen Listeria monocytogenes (Lm), which induces listeriosis in humans. Hence, inhibitors of LmHtrA protease have great importance in the control of infection. Many natural compounds have been used in the inhibition studies of proteases; here, we have performed the inhibition studies of LmHtrA with 31 compounds from different origins. The spectrophotometric assays revealed that plant compounds are promising inhibitors of LmHtrA protease activity compared to other tested peptides and synthetic compounds. The green tea catechin, EGCG has been identified as an inhibitor of protease activity of LmHtrA with a low IC₅₀ value of 0.754 ± 0.2 μM. The substrate cleavage analysis by SDS-PAGE and SPR experiments corroborates the spectrophotometric results by exhibiting protease inhibition and showing the micromolar affinity of EGCG with LmHtrA, respectively. The interaction between rLmHtrA and EGCG was investigated by fluorescence spectroscopy. The binding constant and the number of binding sites were determined as 1.86 × 10⁽⁵⁾ M^(-1) and 1.2, respectively. The molecular docking and dynamics results of LmHtrA-inhibitor complexes have provided new insights into the inhibition mechanism of LmHtrA compared with other serine proteases. The findings of this study may open up new avenues for the development of natural compound-based derivatives of LmHtrA inhibitors that might be more potent and less harmful to humans.