Abstract
Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.
Highlights
Major advances in twentieth century antimicrobial therapies significantly decreased mortality associated with microbial infections; a recent global surge of antimicrobial resistance (AMR) has made the treatment of common bacterial and fungal infections increasingly challenging
Antimicrobial peptide discovery, characterization, and the path to clinical relevance is hindered by extreme diversity in sequence, size (2 to >50 residues), charge, post-translational modification, and natural product complexity
Despite similarities in the activity profiles of PP and PS plate-based assays, V. inconspicua library bioactivity was highly variable among plate-based and microfluidic platforms. These results show that significant optimization must be pursued prior to the incorporation of microfluidic technologies into established antimicrobial susceptibility pipelines
Summary
Major advances in twentieth century antimicrobial therapies significantly decreased mortality associated with microbial infections; a recent global surge of antimicrobial resistance (AMR) has made the treatment of common bacterial and fungal infections increasingly challenging. The future of global health relies on an infusion of new antimicrobial therapeutics in the drug discovery pipeline. Natural product drug discovery has often favored the identification and isolation of small molecule constituents; antimicrobial peptides (AMPs) offer a complementary activity profile with the potential for increased selectivity and efficacy against pathogens (Fosgerau and Hoffmann, 2015; Lázár et al, 2018). Despite the characterization of >3,000 AMPs since (Wang et al, 2016), only seven peptide antimicrobial drugs are currently approved by the FDA (Divyashree et al, 2020). Antimicrobial peptide discovery, characterization, and the path to clinical relevance is hindered by extreme diversity in sequence, size (2 to >50 residues), charge, post-translational modification (e.g., cyclic, disulfide-bound), and natural product complexity
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