Hydroquinine, a Cinchona Alkaloid, Exhibits Antibacterial Activity Against Pathogenic Bacteria by Targeting the Arginine/Ornithine Antiporter (AOA) in the ADI Pathway

Hydroquinine, a cinchona alkaloid, has demonstrated antibacterial activity against multidrug-resistant pathogens, with previous transcriptomic data implicating the arginine deiminase (ADI) pathway. However, its potential molecular target engagement remains to be fully characterized. This study aimed to investigate potential molecular targets of hydroquinine within the ADI pathway utilizing an Escherichia coli BL21 heterologous expression model. We integrated computational molecular docking as a hypothesis-generating tool, in silico-guided site-directed mutagenesis, and continuous broth microdilution growth kinetics profiling. Phenotypic profiling of the ADI-pathway transformants revealed that heterologous expression of the arginine/ornithine antiporter (AOA, encoded by arcD) was associated with growth tolerance under sub-inhibitory hydroquinine pressure. Serving strictly to nominate candidate binding residues, molecular docking predicted that the quinoline and quinuclidine moieties of hydroquinine may interact with residues Trp301 and Glu150 within the predicted AOA binding region. Consistent with these computational predictions, site-directed mutagenesis supported the contribution of these residues to the observed hydroquinine-associated phenotype. E. coli BL21 variants harboring the W301V and E150I arcD mutations lost the observed growth tolerance and exhibited significantly reduced maximum specific growth rates under sub-inhibitory hydroquinine stress. Furthermore, baseline growth defects in untreated mutants indicated potential fitness costs associated with these substitutions. In conclusion, this study provides preliminary insights into AOA as a potential target within a surrogate heterologous host, highlighting bacterial transport systems as candidate targets for future investigation of cinchona alkaloid-based antibacterial development.