scholarly journals Technologies for High-Throughput Identification of Antibiotic Mechanism of Action

Antibiotics ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 565
Author(s):  
Bernardo Ribeiro da Cunha ◽  
Paulo Zoio ◽  
Luís P. Fonseca ◽  
Cecília R. C. Calado
Keyword(s):  
Mechanism Of Action ◽  
Chemical Genetics ◽  
Phenotypic Screening ◽  
Antimicrobial Compounds ◽  
Raman Scattering Spectroscopy ◽  
Major Bottleneck ◽  
Bacterial Cytological Profiling ◽  
New Generation ◽  
Antibiotic Discovery ◽  
Cytological Profiling

There are two main strategies for antibiotic discovery: target-based and phenotypic screening. The latter has been much more successful in delivering first-in-class antibiotics, despite the major bottleneck of delayed Mechanism-of-Action (MOA) identification. Although finding new antimicrobial compounds is a very challenging task, identifying their MOA has proven equally challenging. MOA identification is important because it is a great facilitator of lead optimization and improves the chances of commercialization. Moreover, the ability to rapidly detect MOA could enable a shift from an activity-based discovery paradigm towards a mechanism-based approach. This would allow to probe the grey chemical matter, an underexplored source of structural novelty. In this study we review techniques with throughput suitable to screen large libraries and sufficient sensitivity to distinguish MOA. In particular, the techniques used in chemical genetics (e.g., based on overexpression and knockout/knockdown collections), promoter-reporter libraries, transcriptomics (e.g., using microarrays and RNA sequencing), proteomics (e.g., either gel-based or gel-free techniques), metabolomics (e.g., resourcing to nuclear magnetic resonance or mass spectrometry techniques), bacterial cytological profiling, and vibrational spectroscopy (e.g., Fourier-transform infrared or Raman scattering spectroscopy) were discussed. Ultimately, new and reinvigorated phenotypic assays bring renewed hope in the discovery of a new generation of antibiotics.

scholarly journals Antibiotic Discovery with Synthetic Fermentation: Library Assembly, Phenotypic Screening, and Mechanism of Action of Beta-Peptides Targeting Penicillin-Binding Proteins

2019 ◽  
Author(s):  
Iain A. Stepek ◽  
Trung Cao ◽  
Anika Koetemann ◽  
Satomi Shimura ◽  
Bernd Wollscheid ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Target Identification ◽  
Building Blocks ◽  
Hek293 Cells ◽  
Peptide Antibiotic ◽  
Phenotypic Screening ◽  
Bioactive Natural Products ◽  
Random Fashion ◽  
New Strategy ◽  
Antibiotic Discovery

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing for the resulting cultures to be directly screened for biological activity. In this work, a novel beta-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing for simple identification of active cultures. Subsequent deconvolution, focused screening and structure-activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human Hek293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant (Kd) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds.

scholarly journals Antibiotic Discovery with Synthetic Fermentation: Library Assembly, Phenotypic Screening, and Mechanism of Action of Beta-Peptides Targeting Penicillin-Binding Proteins

2019 ◽  
Author(s):  
Iain A. Stepek ◽  
Trung Cao ◽  
Anika Koetemann ◽  
Satomi Shimura ◽  
Bernd Wollscheid ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Target Identification ◽  
Building Blocks ◽  
Hek293 Cells ◽  
Peptide Antibiotic ◽  
Phenotypic Screening ◽  
Bioactive Natural Products ◽  
Random Fashion ◽  
New Strategy ◽  
Antibiotic Discovery

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing for the resulting cultures to be directly screened for biological activity. In this work, a novel beta-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing for simple identification of active cultures. Subsequent deconvolution, focused screening and structure-activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human Hek293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant (Kd) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds.

scholarly journals Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes

2018 ◽  
Vol 15 (8) ◽  
pp. 3404-3416 ◽  
Author(s):  
Yang Sun ◽  
David K. Heidary ◽  
Zhihui Zhang ◽  
Christopher I. Richards ◽  
Edith C. Glazer
Keyword(s):  
Metal Complexes ◽  
Mechanism Of Action ◽  
Bacterial Cytological Profiling ◽  
Cytological Profiling

scholarly journals Metabolic Fingerprinting with Fourier-Transform Infrared (FTIR) Spectroscopy: Towards a High-Throughput Screening Assay for Antibiotic Discovery and Mechanism-of-Action Elucidation

Metabolites ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 145
Author(s):  
Bernardo Ribeiro da Cunha ◽  
Luís P. Fonseca ◽  
Cecília R.C. Calado
Keyword(s):  
Fourier Transform ◽  
Ftir Spectroscopy ◽  
High Throughput ◽  
Mechanism Of Action ◽  
High Throughput Screening ◽  
Fourier Transform Infrared ◽  
Metabolic Fingerprinting ◽  
High Throughput Screening Assay ◽  
Metabolic Fingerprint ◽  
Antibiotic Discovery

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.

scholarly journals The Mechanism of Action of Cyclophosphamide and Its Consequences for the Development of a New Generation of Oxazaphosphorine Cytostatics

2020 ◽  
Vol 88 (4) ◽  
pp. 42
Author(s):  
Georg Voelcker
Keyword(s):  
Mechanism Of Action ◽  
Active Metabolite ◽  
Dna Alkylation ◽  
In Vivo Metabolism ◽  
Pharmacologically Active ◽  
New Generation ◽  
Anti Tumor Activity ◽  
Oxazaphosphorine Cytostatics

Although cyclophosphamide (CP) has been used successfully in the clinic for over 50 years, it has so far not been possible to elucidate the mechanism of action and to use it for improvement. This was not possible because the basis of the mechanism of action of CP, which was found by lucky coincidence, is apoptosis, the discovery of which was honored with the Nobel Prize only in 2002. Another reason was that results from cell culture experiments were used to elucidate the mechanism of action, ignoring the fact that in vivo metabolism differs from in vitro conditions. In vitro, toxic acrolein is formed during the formation of the cytotoxic metabolite phosphoreamidemustard (PAM), whereas in vivo proapoptotic hydroxypropanal (HPA) is formed. The CP metabolites formed in sequence 4-hydroxycyclophosphamide (OHCP) are the main cause of toxicity, aldophosphamide (ALDO) is the pharmacologically active metabolite and HPA amplifies the cytotoxic apoptosis initiated by DNA alkylation by PAM. It is shown that toxicity is drastically reduced but anti-tumor activity strongly increased by the formation of ALDO bypassing OHCP. Furthermore, it is shown that the anti-tumor activity against advanced solid P388 tumors that grow on CD2F1 mice is increased by orders of magnitude if DNA damage caused by a modified PAM is poorly repairable.

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