Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr Targeting Trypanosoma evansi with disulphide-rich peptides derived from a phage display library Julio Cesar Moraes, Lina Maria Salazar Echeverri, Maria de Lourdes Borba Magalhães, Gustavo Felippe Da Silva * , Luiz Claudio Miletti ** Biochemistry Laboratory, Agroveterinary Science Center, State University of Santa Catarina, Lages, Santa Catarina, Brazil ARTICLE INFO Keywords: Defensins Kunitz BTK-2 ABSTRACT A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of “Surra”, a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections. 1. Introduction The discovery of new antimicrobials, as well as novel infectious disease biomarkers, is a continuing need for the biomedical field. Historically, major sources of bioactive compounds originated from nature, such as molecules found in plants, fungi, and bacteria and various animals. Among these compounds, several bioactive peptides called attention by acting over a wide range of biological receptors, causing a myriad of physiological effects. Among these, there are var- ious protein families such as the knottins (Moore and Cochran, 2012), the Kunitz (Ding et al., 2015), defensins (Li et al., 2017) and disulfide- rich peptides (Chaudhuri et al., 2019), which all hold potent biological effects. The above-mentioned proteins present a constrained tertiary structure due to the presence of intramolecular disulfide bonds, which also contribute to their marked stability (Kolmar, 2008). Among the secondary structures directly involved in biological action, constrained loops are frequently observed and often involved in target binding (Zoller et al., 2011). Interestingly, Cochran and collaborators demon- strated that a Knottin function can be altered via loop transplant from a natural protein, keeping the initial loop specificity within the new scaffold, highlighting the importance of loop-target interactions (Moore et al., 2013). Alternatively, toxins can also exert biological function via alpha helix or beta-sheets interactions. Recent studies demonstrated the biological relevance of alpha-helices in protein-protein interactions (PPIs) and their involvement in key mechanisms, such as gene regula- tion (Azzarito et al., 2013). Therefore, due to the significance of alpha helices and constrained loops in PPIs, many groups have explored and expanded their features to target and/or modulate biological interac- tions of biomedical importance. The improved stability of disulfide-rich peptide scaffolds in toler- ating mutations to produce novel proteins has been the focus of many studies. The diversification on loops, alpha helix or beta sheets, while keeping intact the disulfide bridges, keeps the scaffold preserved, pro- ducing variants that can be explored by display techniques allowing the discovery of new specific ligands against relevant biological targets (Zoller et al., 2011). Among the most widely used biotechnology methods for protein diversification and selection, is the phage display technique. In this https://doi.org/10.1016/j.exppara.2020.107885 Received 1 November 2019; Received in revised form 21 February 2020; Accepted 20 March 2020 * Corresponding author. State University of Santa Catarina; 2090 Luiz de Camões, 88520-000, Lages, SC, Brazil. ** Corresponding author. State University of Santa Catarina; 2090 Luiz de Camões, 88520-000, Lages, SC, Brazil. E-mail addresses: gustavo.silva@udesc.br (G.F. Da Silva), luiz.miletti@udesc.br (L.C. Miletti). Experimental Parasitology 212 (2020) 107885 Available online 28 March 2020 0014-4894/ © 2020 Published by Elsevier Inc. T