title: Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.
abstract: Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.
authors.affiliation: †Department of Chemistry, Infection Innovative Medicines Unit, AstraZeneca-US, 53 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: †Department of Chemistry, Infection Innovative Medicines Unit, AstraZeneca-US, 53 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
authors.forename: Camil
authors.initials: C
authors.lastname: Joubran
authors.affiliation: ‡Department of Biosciences, Infection Innovative Medicines Unit, AstraZeneca-US, Waltham, Massachusetts 02451, United States.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Miller-Vedam
authors.affiliation: ‡Department of Biosciences, Infection Innovative Medicines Unit, AstraZeneca-US, Waltham, Massachusetts 02451, United States.
authors.forename: Vincent
authors.initials: V
authors.lastname: Isabella
authors.affiliation: ‡Department of Biosciences, Infection Innovative Medicines Unit, AstraZeneca-US, Waltham, Massachusetts 02451, United States.
authors.forename: Asha
authors.initials: A
authors.lastname: Nayar
authors.affiliation: †Department of Chemistry, Infection Innovative Medicines Unit, AstraZeneca-US, 53 Gatehouse Drive, Waltham, Massachusetts 02451, United States.
authors.forename: Sharon
authors.initials: S
authors.lastname: Tentarelli
authors.affiliation: ‡Department of Biosciences, Infection Innovative Medicines Unit, AstraZeneca-US, Waltham, Massachusetts 02451, United States.
authors.forename: Alita
authors.initials: A
authors.lastname: Miller
mesh_terms: D000081:Acetamides / Q000032:analysis / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D016905:Gram-Negative Bacterial Infections / Q000188:drug therapy / Q000382:microbiology*; D006801:Humans; D000069349:Linezolid; D013058:Mass Spectrometry; D023303:Oxazolidinones / Q000032:analysis / Q000378:metabolism*; D010539:Permeability; D011550:Pseudomonas aeruginosa / Q000378:metabolism
chemical_list: D000081:Acetamides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D023303:Oxazolidinones; D000069349:Linezolid