Armeniaspirol analogues disrupt the electrical potential (ΔΨ) of the proton motive force

[Display omitted] The armeniaspirol family of natural product antibiotics have been shown to inhibit the ATP-dependent proteases ClpXP and ClpYQ and disrupt membrane potential through shuttling of protons across the membrane. Herein we investigate their ability to disrupt the proton motive force (PMF). We show, using a voltage sensitive, that armeniaspiols disrupt the electrical membrane potential (ΔΨ) component of the PMF and not the transmembrane proton gradient (ΔpH). Using checkerboard assays, we confirm this by showing antagonism, with kanamycin, an antibiotic that required ΔΨ for penetration. By evaluating the antibiotic activity and disruption of the PMF by sixteen armeniaspirol analogs, we find that disruption of the PMF is necessary but not sufficient for antibiotic activity. Analogs that are potent disruptors of the PMF without possessing the ability to inhibit ClpXP and ClpYQ are not potent antibiotics. Thus we propose that the armeniaspirols utilize a dual mechanism of action where they disrupt PMF and inhibit the ATP-dependent proteases ClpXP and ClpYQ. This type of dual mechanism has been observed in other natural product-based antibiotics, most notably chelocardin.