New Insights\ninto the Mechanism of Action of L‑681,217,\na Medicinally Promising Polyketide Inhibitor of Bacterial Protein\nTranslation

An attractive strategy for combating\nantibacterial resistance\ninvolves\nthe development of new antibiotics whose mechanisms differ from those\nof existing ones in the clinic. Elfamycin antibiotics, whose prototypes\ninclude kirromycin and aurodox, are illustrative examples based on\ntheir ability to target EF-Tu, an essential component for protein\ntranslation in bacteria. Our efforts to revisit this antibiotic class\nwere enabled by two developments. First, we produced L-681,217, an\nunderstudied member of this polyketide family harboring a terminal\ncarboxylic acid in place of a hydroxypyridone ring, and synthesized\na biotinylated derivative with comparable activity to the natural\nproduct. Second, we established a sensitive cell-free protein synthesis\n(CFPS) assay in which superfolder green fluorescent protein (sfGFP)\nproduction was inhibited by L-681,217. Biotinyl-L-681,217 was used\nto drain the CFPS system of endogenous EF-Tu, allowing replenishment\nwith orthologs to interrogate pathogen selectivity and propensity\ntoward resistance. Comparative <i>in vitro</i> analysis\nof kirromycin and L-681,217 showed that, while both antibiotics are\nequipotent in CFPS assays, they interact distinctly with purified\nEF-Tu, a feature that presumably correlates with prior observations\nthat kirromycin enhances GTP hydrolysis by EF-Tu whereas L-681,217\ndoes not. Analysis of L-681,217 and kirromycin accumulation in selected\nmutant E. coli strains also revealed\nthat antibiotic import and efflux contributed to resistance. The promise\nof L-681,217 as a medicinal lead was underscored by the observation\nthat, unlike aurodox, this polyketide does not inhibit adenylosuccinate\nsynthase.