Armeniaspirol analogues with more potent Gram-positive antibiotic activity show enhanced inhibition of the ATP-dependent proteases ClpXP and ClpYQ.

Antibiotics with fundamentally new mechanisms of action such as the armeniaspirols, which target the ATP-dependent proteases ClpXP and ClpYQ, must be developed to combat antimicrobial resistance. While the mechanism of action of armeniaspirol against Gram-positive bacteria is understood, little is known about the structure-activity relationship for its antibiotic activity. Based on the preliminary data showing that modifications of armeniaspirol's N-methyl group increased antibiotic potency, we probed the structure-activity relationship of N-alkyl armeniaspirol derivatives. A series of focused derivatives were synthesized and evaluated for antibiotic activity against clinically relevant pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Replacement of the N-methyl with N-hexyl, various N-benzyl, and N-phenethyl substituents led to substantial increases in antibiotic activity and potency for inhibition of both ClpYQ and ClpXP. Docking studies identified binding models for ClpXP and ClpYQ that were consistent with the inhibition data. This work confirms the role of ClpXP and ClpYQ in the mechanism of action of armeniaspirol and provides important lead compounds for further antibiotic development.

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.

Draft Genome Sequence of the Type Strain Streptomyces armeniacus ATCC 15676.

The Streptomyces genus represents a prolific and significant source for secondary metabolite discovery. Here, we report a de novo assembly and draft genome sequence for the type strain Streptomyces armeniacus ATCC 15676.

An evolutionary model encompassing substrate specificity and reactivity of type I polyketide synthase thioesterases.

Bacterial polyketides are a rich source of chemical diversity and pharmaceutical agents. Understanding the biochemical basis for their biosynthesis and the evolutionary driving force leading to this diversity is essential to take advantage of the enzymes as biocatalysts and to access new chemical diversity for drug discovery. Biochemical characterization of the thioesterase (TE) responsible for 6-deoxyerythronolide macrocyclization shows that a small, evolutionarily accessible change to the substrate can increase the chemical diversity of products, including macrodiolide formation. We propose an evolutionary model in which TEs are by nature non-selective for the type of chemistry they catalyze, producing a range of metabolites. As one metabolite becomes essential for improving fitness in a particular environment, the TE evolves to enrich for that corresponding reactivity. This hypothesis is supported by our phylogenetic analysis, showing convergent evolution of macrodiolide-forming TEs.