Implementation of permeation rules leads to a FabI inhibitor with activity against Gram-negative pathogens

Gram-negative bacterial infections are a significant public health concern, and the lack of new drug classes for these pathogens is linked to the inability of most drug leads to accumulate inside Gram-negative bacteria 1 – 7 . Here, we report the development of a web application—eNTRyway—that predicts compound accumulation (in Escherichia coli ) from its structure. In conjunction with structure–activity relationships and X-ray data, eNTRyway was utilized to re-design Debio-1452—a Gram-positive-only antibiotic 8 —into versions that accumulate in E. coli and possess antibacterial activity against high-priority Gram-negative pathogens. The lead compound Debio-1452-NH3 operates as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of the enoyl-acyl carrier protein reductase FabI, as validated by in vitro enzyme assays and the generation of bacterial isolates with spontaneous target mutations. Debio-1452-NH3 is well tolerated in vivo, reduces bacterial burden in mice and rescues mice from lethal infections with clinical isolates of Acinetobacter baumannii , Klebsiella pneumoniae and E. coli . This work provides tools for the facile discovery and development of high-accumulating compounds in E. coli , and a general blueprint for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.