Abstract
The discovery of antibiotics has been slowing to a halt. Phenotypic screening is once again at the forefront of antibiotic discovery, yet Mechanism-Of-Action (MOA) identification is still a major bottleneck. As such, methods capable of MOA elucidation coupled with the high-throughput screening of whole cells are required now more than ever, for which Fourier-Transform Infrared (FTIR) spectroscopy is a promising metabolic fingerprinting technique. A high-throughput whole-cell FTIR spectroscopy-based bioassay was developed to reveal the metabolic fingerprint induced by 15 antibiotics on the Escherichia coli metabolism. Cells were briefly exposed to four times the minimum inhibitory concentration and spectra were quickly acquired in the high-throughput mode. After preprocessing optimization, a partial least squares discriminant analysis and principal component analysis were conducted. The metabolic fingerprints obtained with FTIR spectroscopy were sufficiently specific to allow a clear distinction between different antibiotics, across three independent cultures, with either analysis algorithm. These fingerprints were coherent with the known MOA of all the antibiotics tested, which include examples that target the protein, DNA, RNA, and cell wall biosynthesis. Because FTIR spectroscopy acquires a holistic fingerprint of the effect of antibiotics on the cellular metabolism, it holds great potential to be used for high-throughput screening in antibiotic discovery and possibly towards a better understanding of the MOA of current antibiotics.
Highlights
The discovery of antibiotics has been considered a miracle of modern medicine, but since the golden age of antibiotic discovery, when most classes were introduced, innovation has been slowing to a halt [1].The genomics era inspired target-based screening but hits generally proved ineffective at reaching their target within the cell and not a single new drug reached the market from target-based screening programs [2]
To evaluate Fourier-Transform Infrared (FTIR) spectroscopy as a metabolic fingerprinting technique suitable for distinguishing the MOA of different antibiotics, E. coli was exposed to 15 compounds belonging to different classes and acting on different key biosynthetic pathways (Table 1)
The metabolic fingerprints obtained with FTIR spectroscopy were closely related to the MOA of all the antibiotics tested, which include examples that target protein, DNA, and cell wall biosynthesis
Summary
The discovery of antibiotics has been considered a miracle of modern medicine, but since the golden age of antibiotic discovery, when most classes were introduced, innovation has been slowing to a halt [1].The genomics era inspired target-based screening but hits generally proved ineffective at reaching their target within the cell and not a single new drug reached the market from target-based screening programs [2]. Phenotypic screening is a preferred strategy for antibiotic discovery, mostly because compounds that are effective against whole cells have a higher likelihood of becoming candidate molecules and can target poorly understood metabolic pathways [3]. These assays do not reveal the Mechanism-Of-Action (MOA) of candidate compounds, which requires considerable efforts at a later stage of the discovery process [4]. The second is the specific compound—substrate interactions:the drug target [9] Both concepts are very important in antibiotic discovery, given the exploratory purpose of this study, a less stringent definition of MOA identification was used. We refer to MOA elucidation as an approximation using a mechanism-specific fingerprint, rather than the identification of the specific targets of a given molecule, and the pathways affected, which formally constitute MOA identification
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