%0 Journal Article %A Puneet Labana %A Mark H. Dornan %A Matthew Lafrenière %A Tomasz L. Czarny %A Eric D. Brown %A John P. Pezacki %A Christopher N. Boddy %T Armeniaspirols inhibit the ATP-dependent proteases ClpYQ and ClpXP in Gram-positive bacteria, dysregulating the divisome %D 2019 %R 10.1101/685669 %J bioRxiv %P 685669 %X The emergence of multi-drug resistant organisms clinically presents major challenges to managing human health and threaten the great progress that has been made in preventing morbidity in the age of antibiotics. In order to combat these pathogens, new antibiotics with diverse mechanisms of action will be required. Armeniaspirols represent a novel class of natural product-based antibiotic molecules with unknown mechanisms of action. Herein, we synthesized analogs of armeniaspirol and studied their antibiotic properties and mechanism of action. Using a combination of chemoproteomics, quantitative proteomics, and a battery of functional assays we discovered that armeniaspirols inhibit the bacterial divisome through direct inhibition of the ClpYQ and ClpXP proteases. This was validated by comparisons with genetic knockouts. Sub-lethal challenges suggested that resistance to armeniaspirol inhibition of ClpYQ and ClpXP proteases was difficult to achieve without catastrophic consequences for the bacteria. Thus, the armeniaspirols represent an important new tool to combat multi-drug resistance, through a potent and highly novel mechanism of action. %U https://www.biorxiv.org/content/biorxiv/early/2019/06/28/685669.full.pdf