Turning a biochemical GatCAB inhibitor into a live-cell active antibacterial probe through lipid and trehalose conjugation.

The heterotrimeric Asp-tRNAAsn/Glu-tRNAGln amidotransferase, GatCAB, is vital for protein synthesis in many bacterial pathogens, including mycobacteria, that lack glutaminyl- and/or asparaginyl-tRNA synthetases and depend on the indirect aminoacylation pathway. Despite this potential as an antibacterial target, biochemical GatCAB inhibitors have failed to achieve live-cell activity due to poor cell permeability. Here, we converted a biochemically active chloramphenicol-methionine sulfone, a transition-state mimic of GatCAB transamidation, into live-cell-active antibacterial probes by appending two structurally distinct delivery moieties. The lipid-conjugate enabled passive cellular uptake and demonstrated broad-spectrum antibacterial activity that correlated with genetic dependence on GatCAB, but was also cytotoxic to mammalian HepG2 and Vero cells. The trehalose-conjugate, despite being highly hydrophilic, exploited the mycobacterium-specific transporter LpqY-SugABC to enter Mycobacterium smegmatis and Mycobacterium tuberculosis selectively, and exhibited reduced mammalian cell cytotoxicity. Direct intracellular target engagement by both conjugates was confirmed by a gain-of-function dual-luciferase mistranslation assay, and molecular dynamics simulation supported the target-engagement model at the GatB active site. These results show that trehalose conjugation is an effective Trojan-horse strategy for delivering biochemical inhibitors selectively into mycobacteria, and that the choice of conjugation strategy outlines both the antibacterial spectrum and the safety profile of the resulting probes.