Untargeted Metabolomics To Ascertain Antibiotic Modes of Action.

Isabel M Vincent,Manos Perros,Scott D Mills,Michael P Barrett,David E Ehmann

doi:10.1128/aac.02109-15

Isabel M Vincent, Manos Perros + Show 3 more

Open Access

https://doi.org/10.1128/aac.02109-15

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Abstract

Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5′-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5′-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.

Highlights

Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance
Assays that are able to determine the mode of action of new antibiotics are needed to accompany renewed interest in phenotypic screening of chemical libraries for antimicrobial activity
Where chemicals hit enzymes involved in metabolic pathways, perturbation to those pathways can be detected using metabolomics technology

Summary

Introduction

Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. The hope for rational target-based discovery drove a burst of activity, leading to an era of screening chemicals for inhibitory activity against protein targets This approach uncovered compounds too far from pharmacological utility to succeed in development [3]. The history of antimicrobial development led to a quest for useful drug targets that ended prematurely, and large areas of microbial metabolism have yet to be targeted using small-molecule chemicals. An advantage of phenotypic screening over the target-based screens that became popular in the 1980s to 2000s is that effective compounds are already endowed with key pharmacological attributes, such as selectivity for the microbe of interest, microbial membrane permeability, and chemical stability (or, the ability of prodrug to be metabolized to active compound), when identified. Metabolomics aims to identify all small-molecule metabolites in a given system, and the current technologies make use of nuclear magnetic resonance (NMR) analyses for highly abundant metabolites [11, 12] or high-resolution mass spectrometry coupled to sophisticated chromatography for more global analyses [13]

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