Comprehensive analysis of the antibacterial activity of 5,8-dihydroxy-1,4-naphthoquinone derivatives against methicillin-resistant Staphylococcus aureus

Considering growing concern with antibacterial resistance, the antimicrobial properties of naphthoquinones have recently garnered increasing interest. While much attention has been given to 1,4-naphthoquinone and its derivatives, the antibacterial properties of 5,8-dihydroxy-1,4-naphthoquinone derivatives remain underexplored. Therefore, in this study, we conducted comprehensive in vitro and in vivo analyses of the antibacterial activity of 35 naturally sourced and chemically synthesized derivatives of 5,8-dihydroxy-1,4-naphthoquinone. Kirby-Bauer antibiotic testing led to the identification of 3 compounds with activity against methicillin-resistant Staphylococcus aureus (MRSA), and one of these (PNP-02) exhibited activity similar to that of vancomycin according to minimum inhibitory concentration, minimum bactericidal concentration, and time-kill assays. Microscopic and biochemical assays demonstrated that PNP-02 exerts detrimental effects on the cell wall and cell membrane of MRSA. Mechanistic studies involving proteomic sequencing analyses and focused Western blotting, and RT-qPCR assays revealed that PNP-02 disrupts the integrity of the cell membrane by inhibiting the arginine biosynthesis and pyrimidine metabolism pathways, thereby enhancing membrane permeability and inducing bacterial death. In vivo, PNP-02 was found to exert an antibacterial effect like that of vancomycin in a mouse model of skin wound healing. The compound was found to exert low toxicity to cultured human cells and in hemolysis assays and to be stable when incubated in serum. Thus, our results indicated that compound PNP-02 has promising bioactivity against MRSA and suggested that it is a potential new antibacterial agent.