# Click-based determination of accumulation of molecules in Escherichia coli

## TL;DR

The paper introduces a new method called CHAMP to quickly measure how well small molecules enter the cytosol of E. coli, aiding in the development of antibiotics against Gram-negative bacteria.

## Contribution

The CHAMP assay is a novel, high-throughput method for measuring cytosolic accumulation of azide-tagged molecules in Gram-negative bacteria.

## Key findings

- CHAMP enables rapid and robust accumulation measurements of over 1000 azide-tagged molecules in E. coli within hours.
- The assay was validated in multiple contexts, including hyperporinated and TolC-impaired E. coli strains.
- CHAMP provides comprehensive accumulation profiles at a larger scale than previous methods.

## Abstract

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.

In this work, the authors present their Chloroalkane Azide Membrane Permeability (CHAMP) assay that measures cytosolic accumulation of small molecules in Escherichia coli, enabling rapid profiling of compounds, with the goal to simplify and accelerate antimicrobial drug discovery.