Screening of natural products targeting PBP3 and their synergistic effects with antibiotics in inhibiting Streptococcus pneumoniae

With the increasing resistance of Streptococcus pneumoniae (S. pneumoniae) to β-lactam antibiotics (BLAs), combination therapy has become an effective strategy to address this challenge. This study evaluated the inhibitory activity of ten natural medicines against S. pneumoniae using in vitro antibacterial assays. Seven herbs, including Galla chinensis, Coptidis rhizoma, Scutellaria baicalensis, Phellodendri chinensis cortex, Sophora flavescens, Salviae miltiorrhizae radix et rhizoma, and Houttuynia cordata Thunb, were found to be effective, with Coptidis rhizoma, Scutellaria baicalensis, and Phellodendri chinensis cortex showing the most significant activity. Additionally, surface plasmon resonance (SPR) technology was employed to couple the PBP3 target to a CM5 chip for target-guided fishing of potential active compounds and evaluation of their PBP3-binding capabilities. The ethanol extract of Scutellaria baicalensis exhibited the highest binding response, reaching 20,000 RU. The target-bound components were eluted and identified by UPLC-MS, confirming Oroxindin as the most potent target compound. Its dissociation constant (KD) with PBP3 was determined to be 5.049 × 10⁻⁷ mol/L, indicating stable binding and high affinity. Finally, it is noteworthy that in vitro antibacterial assays demonstrated a significant enhancement of antibiotic efficacy by Oroxindin: co-administration with cefquinome (CEF) reduced the minimum inhibitory concentration (MIC) for S. pneumoniae from 32 μg/mL to 1 μg/mL (a 32-fold increase in sensitivity), while co-administration with ampicillin (AMP) reduced the MIC from 32 μg/mL to 0.5 μg/mL (a 64-fold increase in sensitivity). This study identified and validated the sensitizing efficacy of Oroxindin in inhibiting S. pneumoniae, providing a new approach to addressing BLAs resistance and laying a foundation for mechanistic research and clinical translation of combination therapy strategies.