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

2019 ◽  
Vol 14 (5) ◽  
pp. 1030-1040 ◽  
Author(s):  
Iain A. Stepek ◽  
Trung Cao ◽  
Anika Koetemann ◽  
Satomi Shimura ◽  
Bernd Wollscheid ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Binding Proteins ◽  
Phenotypic Screening ◽  
Penicillin Binding Proteins ◽  
Antibiotic Discovery

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.

Biochemical and genetical approaches to the mechanism of action of penicillin

1980 ◽  
Vol 289 (1036) ◽  
pp. 273-283 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Binding Proteins ◽  
Biological Effects ◽  
Sodium Dodecyl ◽  
Multiple Forms ◽  
Considerable Effort ◽  
Lethal Effects ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Synthesis

Since the discovery in 1965 that penicillin inhibits the transpeptidation reaction in peptidoglycan synthesis, a considerable effort has been put into the purification of enzymes that catalyse this reaction. This has resulted in the recognition that bacteria possess multiple forms of these penicillin-sensitive enzymes and has made it difficult to identify the precise target that penicillin inactivates to kill the organism. Recently penicillin-sensitive enzymes have been detected and studied as penicillin-binding proteins on sodium dodecyl sulphate polyacrylamide gels. The availability of this convenient method for identifying penicillin-sensitive enzymes has allowed biochemical and genetical approaches to be used to dissect their roles in the lethal effects of penicillin and other β-lactam antibiotics. Three penicillin-binding proteins (1B, 2 and 3) have been identified as killing targets for penicillin in Escherichia coli , whereas four other binding proteins are not implicated in the mechanism of action of the antibiotic. The complex biological effects that β-lactam antibiotics produce on the growth of E. coli can be explained by their interaction with the three killing targets. Progress in the correlation of penicillin-binding proteins with penicillin-sensitive enzymes and in the development of strains of E. coli that overproduce penicillin-binding proteins is discussed.

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