Structure-based screening and in vitro validation of potentially druggable molecules against Vibrio parahaemolyticus...

Thermostable Direct Hemolysin (TDH) protein is a key virulence factor of Vibrio parahaemolyticus, responsible for hemolysis, cardiotoxicity, and enterotoxicity. TDH is naturally synthesized as a soluble monomer and subsequently oligomerizes into a tetrameric pore-forming complex, making it a crucial target for therapeutic intervention. This study aimed to identify novel drug candidates from ZINC15 and DrugBank databases using in silico virtual screening, guided by Lipinski's Rule of Five. Seven promising compounds were shortlisted after initial screening based on binding affinity, hydrogen bonding interactions, and electrostatic complementarity. Among the seven candidates, five were selected for in vitro validation. Of these, two compounds, ZINC000009698202 and ZINC219968783, significantly inhibited the hemolytic activity of the V. parahaemolyticus culture supernatant. However, prolonged incubation with ZINC000009698202 resulted in the agglutination of Red Blood Cells (RBCs), limiting its suitability as a therapeutic agent. Therefore, ZINC219968783 was selected for further studies to assess its ability to inhibit the hemolytic activity of the recombinant TDH (rTDH) protein expressed in E. coli. Overall, this study highlights ZINC219968783 as a promising druggable candidate capable of mitigating the pathogenicity of V. parahaemolyticus by inhibiting the function of the TDH protein. These findings offer valuable insights into the development of potential antibiotic alternatives for controlling V. parahaemolyticus-associated infections.