2-Hydroxychalcone is a gonococcal-specific antimicrobial with activity against elongation factor Tu and butanediol dehydrogenase.

AIMS: This study explored botanical extracts to guide discovery of alternative antimicrobials with activity against the multidrug-resistant bacterial pathogen Neisseria gonorrhoeae.

MATERIALS AND

METHODS

In vitro antimicrobial activity was analyzed by agar dilution method and time-kill assays, while in vivo activity was evaluated in a mouse infection model. The antimicrobial target was identified by pull-down assays and validated by interaction studies with wild-type and targeted mutant proteins.

KEY FINDINGS: The extract of Chinese Populus spp. propolis (CPP) displayed the most potent antigonococcal activity. Subsequently, galangin was identified as the most active compound in CPP. However, galangin showed no in vivo activity in the mouse infection model. Screening for active structural analogues of galangin resulted in the identification of 2-hydroxychalcone (2-HC), which showed a minimum inhibitory concentration (MIC) of 8-16 μM, and enhanced gonococcal clearance in the infection model. Pull-down experiments identified EF-Tu and butanediol dehydrogenase (Bdh) as putative 2-HC targets. Further binding analysis of 2-HC with recombinant purified proteins showed an equilibrium dissociation constant (KD) of 1.28 μM for EF-Tu and 5.97 μM for Bdh. Finally, 2-HC binding pockets on EF-Tu and Bdh were identified by molecular docking studies showing that 2-HC interacted with Glu260 of EF-Tu and Ser273 of Bdh, which was validated by mutagenesis studies. For Bdh, we furthermore demonstrated that 2-HC impacted its enzyme activity with and IC50 of 44-64 μM, resulting in perturbed NAD+/NADH ratios.

SIGNIFICANCE: 2-HC displays a bimodal antigonococcal mechanism, making it an interesting candidate for further development as antigonococcal therapy.