The constant evolution of methicillin-resistant Staphylococcus aureus (MRSA), a predominant pathogen in purulent skin and soft tissue infections, has contributed to notable morbidity and mortality across hospital and community globally [[1], [2], [3], [4], [5]]. MRSA has exhibited broad resistance to almost all classes of antibiotics, including vancomycin, historically considered as the last-line defense against MRSA. Additionally, the prevalence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) is associated with poor clinical outcomes [[6], [7], [8], [9]]. To fight against these infections, a lot of efforts have been focused on the improvement of the existing antibiotics through structural hybridization, antibody-antibiotic conjugates, and advanced targeted drug delivery systems to circumvent resistance mechanism [10]. Meanwhile, parallel research explores novel therapeutic approaches targeting bacterial cell division machinery (e.g., FtsZ inhibitors) and biofilm disruption strategies [11,12]. A continued need for the implementation of alternative MRSA interventions featuring novel structural scaffolds and innovative mechanisms is of great urgent.

DNA polymerase IIIC (DNA pol IIIC), a conserved and essential enzyme governing chromosomal replication in low G + C-content Gram-positive bacteria (genomic GC <50 %), represents a promising antibacterial target due to its phylogenetic exclusivity. This enzyme is ubiquitously present in clinically relevant genera, such as Streptococcus spp., Enterococcus spp., Staphylococcus spp. [13]. In view of the minimal homology of with mammalian polymerase, DNA pol IIIC offers a critical therapeutic advantage by attenuating nonspecific toxicity profiles in human cells. Ibezapolstat, as a DNA pol IIIC inhibitor, has achieved beneficial effects in its phase II clinical trials in patients with Clostridioides difficile infection [14]. Therefore, DNA pol IIIC has been considered as a high-value target against multidrug-resistant Gram-positive bacteria with the advantage of rare drug-resistance.

In our previous studies, we identified a novel cycloberberine (CBBR) scaffold, derived from natural medicine berberine (BBR), as a pharmacophore with intrinsic anti-MRSA activity, by carrying out systematic anti-MRSA structure-activity relationship studies focusing on the variations on the C7, C8 and C13 positions [[15], [16], [17], [18], [19], [20]]. Among the reported derivatives, 8-acetoxycycloberbeirne (1, Fig. 1) gave the lowest minimum inhibitory concentration (MIC) values of 1–8 μg/mL against methicillin-susceptible S. aureus (MSSA) and MRSA, outperforming cycloberberine ether/amine/amide derivatives (MIC range: 2 to >64 μg/mL). To further explore the importance of the C8 ester moiety on activity, 44 CBBR ester derivatives were synthesized and evaluated for their antimicrobial activity against Gram-positive strains, taking 8-acetoxycycloberbeirne (1) as a lead. Then, the in vivo anti-MRSA efficacy of the key compound in murine infection model was assayed, and its direct targeting on DNA pol IIIC was disclosed and validated.