Click-based determination of accumulation of molecules in Escherichia coli

Gram-negative bacterial pathogens pose a significant challenge in drug development because their outer membranes hinder the permeation of small molecules. The lack of widely adoptable methods for measuring the cytosolic accumulation of compounds in bacterial cells further hinders drug discovery efforts. To address this challenge, we report the development of the Chloroalkane Azide Membrane Permeability (CHAMP) assay, which we designed specifically to assess molecule accumulation in the cytosol of Gram-negative bacteria. The CHAMP analysis utilizes bioorthogonal epitopes anchored within HaloTag-expressing bacteria and measures the cytosolic arrival of azide-bearing test molecules through strain-promoted azide-alkyne cycloaddition. This workflow enables robust and rapid accumulation measurements of thousands of azide-tagged small molecules. Our approach consistently produces comprehensive accumulation profiles that surpass the scale of previous measurements in Escherichia coli (E. coli). We validated the CHAMP assay across various chemical and biological contexts, including hyperporinated cells, membrane-permeabilized cells, and E. coli strains with impaired TolC function, a key component of the efflux pump. The CHAMP platform provides a simple, high-throughput, and accessible method that enables the analysis of over 1000 molecules within hours. This technique addresses a critical gap in antimicrobial research and has the potential to accelerate the development of effective agents against Gram-negative pathogens.