Optimized alkyne-modified pyridylpiperazine efflux pump inhibitors overcome intrinsic macrolide resistance in multidrug-resistant Klebsiella pneumoniae isolates

Multidrug-resistant (MDR), ESBL- and carbapenemase-producing Enterobacterales represent a major global health concern requiring novel therapeutic strategies. One promising approach is the development of efflux pump inhibitors (EPIs) targeting the predominant AcrAB-TolC system to sensitize these pathogens to antibiotics predominantly used for Gram-positive infections. PyridylPiperazine (PyrPip)-based EPIs are novel allosteric AcrB inhibitors that block antibiotic efflux in Enterobacterales. Medicinal chemistry optimization identified BDM100004 ( 3 ), an alkyne-modified PyrPip analog with improved drug-like properties. In vitro susceptibility profiling of 48 clinical MDR Klebsiella pneumoniae isolates showed that BDM100004 ( 3 ) potentiated macrolide activity in most MDR isolates, with particularly strong effects observed for azithromycin. A small fraction of MDR isolates was found to be highly resistant to macrolides, and poorly potentiated by BDM100004 ( 3 ). Genome analysis revealed these isolates to carry specific macrolide resistance genes, and together with reverse genetics, it was validated that BDM100004 ( 3 ) could not reverse Mph(A) and Mph(E) mediated azithromycin resistance. Interestingly, however, BDM100004 ( 3 ) was found to overcome azithromycin resistance mediated by Erm(B), Msr(E) and Mrx. In vivo, BDM100004 ( 3 ) enhanced azithromycin efficacy both in a Galleria mellonella infection model and in a murine lung infection model of K. pneumoniae . Overall, these findings demonstrate the potential of a PyrPip EPI to repurpose azithromycin against ESBL- and carbapenemase-producing K. pneumoniae with limitations in mph(A) and mph(E) -expressing strains.