Armeniaspirols inhibit the AAA+ proteases ClpXP and ClpYQ leading to cell division arrest in Gram-positive bacteria.

Multi-drug-resistant bacteria present an urgent threat to modern medicine, creating a desperate need for antibiotics with new modes of action. As natural products remain an unsurpassed source for clinically viable antibiotic compounds, we investigate the mechanism of action of armeniaspirol. The armeniaspirols are a structurally unique class of Gram-positive antibiotic discovered from Streptomyces armeniacus for which resistance cannot be readily obtained. We show that armeniaspirol inhibits the ATP-dependent proteases ClpXP and ClpYQ in vitro and in the model Gram-positive Bacillus subtilis. This inhibition dysregulates the divisome and elongasome supported by an upregulation of key proteins FtsZ, DivIVA, and MreB inducing cell division arrest. The inhibition of ClpXP and ClpYQ to dysregulate cell division represents a unique antibiotic mechanism of action and armeniaspirol is the only known natural product inhibitor of the coveted anti-virulence target ClpP. Thus, armeniaspirol possesses a promising lead scaffold for antibiotic development with unique pharmacology.