High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

• Published in: Peptides (New York, N.Y. : 1980) N.Y. : 1980), 2011-08, Vol.32 (8), p.1764-1773
• Main Authors: Walsh, Edwin G., Maher, Sam, Devocelle, Marc, O’Brien, Peter J., Baird, Alan W., Brayden, David J.
• Format: Article
• Language: English
• Subjects: anti-infective properties; antibiotics; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - pharmacology; Antimicrobial peptide; antimicrobial peptides; antimicrobials; bacteria; Biological and medical sciences; Caco-2 Cells; computer software; cytotoxicity; Cytotoxicity screening; developmental stages; drugs; erythrocytes; fish; fluorescence microscopy; food additives; Fundamental and applied biological sciences. Psychology; High-content-analysis (HCA); Humans; insects; Melitten - chemistry; Melitten - pharmacology; Melittin; MTT assay; pharmacokinetics; prediction; screening; toxicity testing; Vertebrates: endocrinology
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2011.06.006

High content analysis for cytoxicity of peptides on epithelial cells. Images of dye-loaded cells are acquired via multi-well automation by epifluorescence microscopy and are then subjected to segmentation analysis. [Display omitted] ► We describe a multi-parameter high-content analysis protocol for assessing peptide toxicity on epithelial cells. ► Selected deletion of amino acids of the bee venom, melittin, reduces its cytotoxicity. ► Loss of cytotoxicity was correlated with loss of epithelial permeation enhancement but not a reduction in antimicrobial activity. Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.