To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

Marco Cavaco; Clara Pérez-Peinado; Javier Valle; Rúben D M Silva; João D G Correia; David Andreu; Miguel A R B Castanho; Vera Neves

doi:10.3389/fbioe.2020.552035

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

Front Bioeng Biotechnol. 2020 Sep 9:8:552035. doi: 10.3389/fbioe.2020.552035. eCollection 2020.

Authors

Marco Cavaco^{1

2}, Clara Pérez-Peinado², Javier Valle², Rúben D M Silva³, João D G Correia³, David Andreu², Miguel A R B Castanho¹, Vera Neves¹

Affiliations

¹ Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
² Proteomics and Protein Chemistry Unit, Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona, Spain.
³ Centro de Ciências e Tecnologias Nucleares and Departamento de Engenharia e Ciências Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.

Abstract

The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood-brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.

Keywords: BBB peptide shuttles; anticancer peptides; fluorescence; fluorophore; labeling.