Overcoming the Gram-Negative Barrier: Intracellular Drug Tracking with CHAMPGram-negative bacterial pathogens represent a formidable challenge in modern drug development due to their complex outer membrane structures, which act as a selective barrier preventing the passage of small molecules. In scientific literature, the lack of standardizable methods to measure the accumulation of compounds within the bacterial cytoplasm is considered one of the most significant methodological hurdles in novel antibiotic discovery. While existing LC-MS/MS (liquid chromatography-tandem mass spectrometry; a method that separates chemical components based on their mass) techniques are successful in measuring whole-cell accumulation, they fall short in distinguishing the precise location of molecules between the periplasm and the cytoplasm. This situation prevents the full definition of the physicochemical rules governing molecular structure and intracellular accumulation for bacteria.
In the study conducted by Ongwae and colleagues, the aim was to develop a new assay system called CHAMP (Chloroalkanoic Acid Membrane Permeability), which can directly, unbiasedly, and with high efficiency measure the amount of molecules reaching the cytoplasm of Gram-negative bacteria. The researchers aimed to overcome the limitations of current methods regarding low throughput and subcellular localization (determining which compartment of the cell the molecule is in). The central research question of the study was based on whether bioorthogonal (not interfering with biological processes) chemical reactions could be utilized to map cytosolic accumulation on a scale of thousands of molecules. Accordingly, the systematic determination of the rules for small molecules to pass through the outer membrane and achieve cellular accumulation was targeted.
The CHAMP method utilizes HaloTag (a protein tag that selectively and covalently binds to specific chemical groups) expressing Escherichia coli cells as a fundamental genetic platform. In the experimental workflow, DBCO (a strained alkyne group) molecules are first fixed to the HaloTag proteins located in the cytoplasm via a chloroalkane linker. In the second stage, bacterial cells are exposed to azide-tagged test molecules (the “pulse” step), and these molecules reaching the cytoplasm are bound to the DBCO groups through a SPAAC (strain-promoted azide-alkyne cycloaddition; a reaction that allows two molecules to quickly form a covalent bond) reaction. In the final stage, the remaining empty DBCO sites in the cell are labeled with a fluorescent azide; thus, the level of cytosolic accumulation is quantified via flow cytometry (a technique that measures cell properties using lasers) or plate readers based on the decreasing fluorescent signal.
As a result, the developed CHAMP platform has been proven to have an efficiency capable of analyzing more than 1,000 molecules within hours, with a very high reliability coefficient (Z’ score) of 0.954. The method has been validated in different biological contexts using hyperporination (engineered pore formation in the outer membrane), outer membrane permeabilizers (adjuvants such as PMBN), and strains with impaired TolC (a core component of the efflux pump) function. Analyses have revealed that features such as the number of hydrogen bond acceptors, polar surface area, and amide bonds increase the probability of molecules being recognized by efflux systems. Furthermore, the critical role of primary amines in improving accumulation was observed. The findings obtained indicate that the CHAMP technique serves as a powerful data production tool in the processes of determining structure-accumulation rules and developing next-generation antimicrobial agents in the fight against antibiotic resistance.
Author: Berfin Kayabaşı
Editor: Damla Özdemir
Reference: Ongwae, G.M., Liu, Z., Feng, S. et al. Click-based determination of accumulation of molecules in Escherichia coli. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68717-5
-Bioinfocodes Scientific News Service-
News articles prepared by our team members, reviewing and compiling scientific research
published in journals with and impact factor greater than 20 (click here for the list)