Optimizing Intracellular Transport of Antimicrobial Metallohelices Delivers Selective Nanomolar Potency in <i>E. coli</i>

To investigate large discrepancies in antimicrobial potency between cationic amphipathic metallohelix architectures, 22 new optically pure candidates were synthesized via self-assembly. A total of 34 compounds were tested against <i>S. aureus</i>, <i>E. coli</i>, and, for the most active, against a panel of ESKAPE pathogens. While addition of substituents reduced activity in a 3-fold symmetric "flexicate" series, a potent compound (∼500 nM) with promising selectivity against a challenging <i>E. coli</i> microbe emerged in the hitherto inactive "triplex" series. This and other key compounds were studied by using techniques focused on transport and localization in Gram-positive and Gram-negative bacteria. Zeta-potential measurements at model membranes revealed affinities that mirror the antimicrobial activity. Extensive temperature- and concentration-dependent intracellular accumulation studies via isotopic labeling revealed that antimicrobial activity (within each architecture) is strongly dependent on the ability to enter the cell via passive diffusion. Mechanistic differences across metallohelix classes are confirmed by checkerboard activity assays and confocal microscopy studies via Click-labeled alkyne derivatives. The most active (and bactericidal) enantiomer achieves a growth-inhibiting concentration across the microbial population (apparently not restricted to dividing cells) at ca. 250 nM applied dose. Extraordinarily, given this very high potency, the mirror image of this compound is essentially inactive.