Hinokiflavone Attenuates the Virulence of Methicillin-Resistant Staphylococcus aureus by Targeting Caseinolytic Protease P

Drug-resistant bacteria was the third leading cause of death worldwide in 2019, which sounds like a cautionary note for global public health. Therefore, developing novel strategies to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections is the need of the hour. Caseinolytic protease P (ClpP) represents pivotal microbial degradation machinery in MRSA involved in bacterial homeostasis and pathogenicity, considered an ideal target for combating S. aureus infections. Herein, we identified a natural compound, hinokiflavone, that inhibited the activity of ClpP of MRSA strain USA300 with an IC50 of 34.36 mu g/mL. Further assays showed that hinokiflavone reduced the virulence of S. aureus by inhibiting multiple virulence factors expression. Results obtained from cellular thermal transfer assay (CETSA), thermal shift assay (TSA), local surface plasmon resonance (LSPR) and molecular docking (MD) assay enunciated that hinokiflavone directly bonded to ClpP with confirmed docking sites, including SER-22, LYS-26 and ARG-28. In vivo, the evaluation of anti-infective activity showed that hinokiflavone in combination with vancomycin effectively protected mice from MRSA-induced fatal pneumonia, which was more potent than vancomycin alone. As mentioned above, hinokiflavone, as an inhibitor of ClpP, could be further developed into a promising adjuvant against S. aureus infections.