The effect of lysine substitutions in the biological activities of the scorpion venom peptide VmCT1

European Journal of Pharmaceutical Sciences 136 (2019) 104952

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The effect of lysine substitutions in the biological activities of the scorpion venom peptide VmCT1

T

Cibele Nicolaski Pedrona, Cyntia Silva de Oliveirab, Adriana Farias da Silvaa,b, Gislaine Patricia Andradea, Maria Aparecida da Silva Pinhalb, Giselle Cerchiaroa, ⁎ Pedro Ismael da Silva Juniorc, Fernanda Dias da Silvaa, Marcelo Der Torossian Torresd,e, , ⁎⁎ Vani Xavier Oliveiraa,b, a

Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Santo André, SP 09210580, Brazil Universidade Federal de São Paulo, São Paulo, SP 04044020, Brazil Instituto Butantan, São Paulo, SP 05503900, Brazil d Department of Psychiatry and Microbiology, Perelman School of Medicine of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, United States of America e Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, United States of America b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Antimicrobial peptides VmCT1 Structure-activity relationship Scorpion venom peptides

Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysinesubstituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1–6.3 μmol L−1) than VmCT1 (0.8–50 μmol L−1) and higher activity against mammary cancer cells MCF-7 (6.3–12.5 μmol L−1) than VmCT1 (12.5 μmol L−1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8 μmol L−1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.

1. Introduction The multi-drug resistance against bacteria is an important public health problem (van Duin and Paterson, 2016). The treatment of microbial infectious diseases has become increasingly difficult because of the ability of bacteria to develop mechanisms of resistance to antimicrobial agents (Tenover and Georgia, 2006). The development of new strategies to combat microbial infection have become urgent and peptide have emerged as alternative broad-spectrum antimicrobials (de la Fuente-Nunez et al., 2017). AMPs act as essential components of the host defense system against infections and they are versatile biomolecules, which act through a variety of mechanisms against pathogenic

microorganisms such as bacteria (De Breij et al., 2018), fungi (Durnaœ et al., 2016), and parasites (Vinhote et al., 2017). In addition, their activity may extend to enveloped viruses (Monteiro et al., 2018) and cancer cells (Pedron et al., 2018; Torres et al., 2018b). Most AMPs are amphipathic sequences that tend to adopt helical or β-like structures when they reach the interface of biological membranes. These structural tendencies are favored because they stabilize amino acid conformers within peptides' sequence that are exposed to protic solvent, negatively charged phospholipids and the hydrophobic portion of the membranes (Schmidt and Wong, 2013). AMPs partition through the membrane leads to the disruption of cytoplasmic membrane (Torres et al., 2019). They can be found in most living organisms such as

⁎

Correspondence to: M.D.T. Torres, Department of Psychiatry and Microbiology, Perelman School of Medicine of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, United States of America. ⁎⁎ Correspondence to: V. X. Oliveira Junior, Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Santo André, SP 09210580, Brazil. E-mail addresses: [email protected] (M.D.T. Torres), [email protected] (V.X. Oliveira). https://doi.org/10.1016/j.ejps.2019.06.006 Received 13 April 2019; Received in revised form 27 May 2019; Accepted 3 June 2019 Available online 07 June 2019 0928-0987/ © 2019 Published by Elsevier B.V.

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Fig. 1. Helical wheel projections of VmCT1 and its analogs. Yellow circles refer to aromatic and aliphatic hydrophobic residues; gray circles indicate residues with hydrophobicity close to zero; blue circles indicate basic positively charged residues; purple circles represent polar uncharged residues; red circles refer to acidic negatively charged amino acid residues; green circles represent the restricted pseudo amino acid proline; and pink circles represent polar uncharged amino acid residues. Black arrows indicate the direction and intensity of the hydrophobic moment (Gautier et al., 2008). P indicates hydrophilic face and N refers to the hydrophobic face of the amphipathic projection. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Lohner, 2017; Malanovic and Lohner, 2016). Previously, Pedron and collaborators proposed new VmCT1 analogs with higher net positive charge values (+2 to +3), which led to increased antimicrobial activity and higher helical content (Pedron et al., 2017). The positively charged residues Lys and Arg are among the most common amino acids residues found in AMPs (Yount et al., 2019). Arg and Lys side chains contribute substantially for first coulombic interactions between peptide and phospholipids in the membrane. The Lys side chain group (primary amine) presents a more localized positive charge density on its terminal amine leading to fewer atoms interacting with water molecules. Consequently, the localized positive charge leads to lesser interactions with water molecules and the biological membrane phospholipid head groups (Armstrong et al., 2016; Li et al., 2013). Differently, Arg presents a delocalized charge density in its side chain terminal guanidinium group. It contributes to more substantial interactions with hydrophilic/hydrophobic interfaces, water and phospholipid head groups, enabling destabilization of completely hydrophobic environments (e.g., bacterial membrane interior). Thus, the more flexible and less polarizable the amino group side chain of the Lys residue has lesser interactions with water and the membrane compared to the guanidium group from the Arg residue. Solvation, hydrophobicity and partition coefficient are the characteristics of amino acids side chain groups that contribute to cytotoxicity (Rice and Wereszczynski, 2017). For this reason, Arg has a higher tendency to be cytotoxic when compared to Lys. Therefore, in order to analyze the influence of changes in the net positive charge on the antimicrobial activity, we designed Lys-substituted VmCT1 analogs. Based on previous studies, the positions of the peptide sequence that would favor positive charge insertion are Gly3, Asn7 and Ser11, leading to net charge variations from +2 to +5 for the analogs designed. Besides, we showed the importance of structurefunction-guided design to generate non-cytotoxic AMPs with higher net positive charge leading to increased biological activities.

unicellular microorganisms, insects, arachnids, plants, amphibians, and mammals (Jenssen et al., 2006). The biological activities of AMPs isolated from venom have been extensively studied (Ifrah et al., 2005; Mendes et al., 2004; Yan et al., 2011). α-Helical peptides are the most weel-known classes of AMPs, cecropin and melitin are examples of AMPs from insect (Hancock and Chapple, 1999). Melittin was isolated from bee venom of Apis mellifera and exhibited antimicrobial and hemolytic activity (Asthana et al., 2004; Habermann, 1972). Cecropin is also an insect-derived AMP, which was isolated from the hemolymph of the giant silk moth Hyalophora cecropi. Its antimicrobial activity was described by Hultmark et al. (1980). Later several other natural cecropin-like AMPs were described (Andreu et al., 1992; Boman et al., 1989). Arachnids, such as scorpions, have a series of bioactive molecules in their venom, including amphipathic peptides (Fratini et al., 2017; Ortiz et al., 2015; Perumal Samy et al., 2017). However, scorpion venomderived AMPs are frequently hemolytic and cytotoxicity, thus the rational design of this class of AMPs may provide novel antibiotics with broad-spectrum and low cytotoxicity (Torres et al., 2019). In general, AMPs isolated from scorpion venom are classified into two main groups: constrained by disulfide-bridges and linear AMPs (Almaaytah and Albalas, 2014; Fratini et al., 2017). VmCT1 is a linear helical AMP, isolated from the venom of the scorpion Vaejovis mexicanus. It contains 13 amino acid residues and an amidated carboxyl-terminus (Phe-Leu-Gly-Ala-Leu-Trp-Asn-Val-AlaLys-Ser-Val-Phe-NH2). The peptide showed antimicrobial activity ranging from 5 to 25 μmol L−1 against Gram-positive and Gram-negative bacteria. However, VmCT1 is hemolytic at this concentration range (Ramírez-Carreto et al., 2012). There are several biological and structural activity descriptors (Jenssen, 2011; Torres et al., 2019), which define the main properties that are responsible for antimicrobial activity. Cationicity is among the most well-known descriptors reported in literature as crucial for peptide-membrane interactions (Nguyen et al., 2011; Stutz et al., 2017). It is related to the initial electrostatic interactions of AMPs with negatively charged phospholipids from the membranes of microorganisms 2

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2. Methodology

2.5. Cytotoxic activity

2.1. Solid-Phase Peptide Synthesis (SPPS), purification and analysis

MCF-10A human breast epithelial cells were used to assess the cytotoxic activity of the peptides designed in this study as previously described by Pedron et al. (2018). Briefly, the cells were incubated for 24 h at 37 °C and 5% CO2. The next day, cells were treated with solutions of peptide ranging from 12.5 to 100 μmol L−1 for 4 h at 37 °C and the cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Torres et al., 2018a). Experiments were performed in three independent replicates.

Peptides shown in Fig. 1, were synthesized on a peptide synthesizer (PS3 - Sync Technologies) using solid-phase peptide syntheses methodology and fluoromethyloxycarbonyl (Fmoc) strategy and then cleaved as previously described by Torres et al. (2017). The crude lyophilized peptides were purified by semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC) on a Delta Prep 600 (Waters Associates). Selected fractions containing the purified peptides were pooled and lyophilized. The peptides were characterized by liquidchromatography electrospray-ionization mass spectrometry (LC/ESIMS) using a Model 6130 Infinity mass spectrometer coupled to a Model 1260 HPLC system (Agilent) described in details by Torres et al. (2017).

2.6. Activity against Plasmodium gallinaceum sporozoites The anti-plasmodial activity of the peptides on sporozoites was determined by fluorescence microscopy as previously described by Silva et al. (2017). Briefly, the Plasmodium gallinaceum mature sporozoites (9000) were pulled from the salivary glands of Aedes aegypti and they were counted in the Neubauer chamber using a fluorescence microscope with a 40× objective lens. After, sporozoites were incubated with peptides or control, at 37 °C for 1 h of serial dilutions of peptides (0.1 to 1.6 μmol L−1) and 9000 sporozoites. PBS solution was used as a negative control and the surfactant digitonin as a positive control (Fani et al., 2016; Krishan, 1975). Experiments were performed in three independent replicates for three different mosquito batches (n = 9).

2.2. Antimicrobial activity The peptides were tested against the following strains: Micrococcus luteus A270, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Bacillus megaterium ATCC 10778, Escherichia coli SBS 363, Serratia marcescens ATCC 4112, Enterobacter cloacae β-12, Candida albicans MDM8 and Candida tropicalis IOC 4560. The microorganisms were obtained from the American Type Culture Collection (ATCC) and Instituto Butantan, São Paulo, Brazil. The antimicrobial activity was determined by liquid growth inhibition assays, as previously described by Pedron et al. (2017). Peptone Broth and Potato Dextrose Broth (Invitrogen) were used for antibacterial and antifungal assays, respectively. Microorganisms were incubated with two-fold serial dilution of peptide (0.09–50 μmol L−1) at 37 °C for 18 h (bacteria) or 24 h (fungi). The microbial growth was assessed by measurements of the absorbance at 595 nm. The MIC (Minimal Inhibitory Concentration) value of each peptide against the strains studied was defined as the lowest concentration of the peptide at which the growth was not observed. Experiments were performed in three independent replicates.

2.7. Circular dichroism (CD) spectroscopy Circular dichroism assays were performed in a Spectropolarimeter Jasco Mod. J-815 (JascoCorp.) to verify the tendency to fold in secondary structures of the designed peptides. CD spectra were recorded in Far-UV (195–260 nm), and analyzed in the following media: water, PBS (pH 7.4), SDS (20 mmol L−1), TFE/water (3:2, v:v), POPC (10 mmol L−1), POPC:POPG (3:1, mol:mol, 10 mmol L−1) and POPC:DOPE (3:1, mol:mol, 10 mmol L−1) at a fixed peptide concentration (50 μmol L−1). 2.8. Stability assays The resistance to degradation assays were performed in GIBCO fetal bovine serum diluted to 25% in water as detailed by Torres et al. (2017). Briefly, the peptide solutions were added to the serum and kept at 37 °C for 6 h. The experiments were made in three independent replicates and aliquots were taken at 0, 0.5, 1, 2, 4 and 6 h. The degradation kinetics was monitored by RP-HPLC and the percentage of the remaining peptide was reported by integrating the peak area related to the peptide.

2.3. Hemolytic activity assays In the hemolytic activity assays we used freshly collected human erythrocytes that were performed according to Pedron et al. (2017). Briefly, peptide solutions at concentrations ranging from 0.1 to 100 μmol L−1 were incubated at 37 °C with a suspension of erythrocytes. The surfactant SDS was used as positive control (Love, 2005; Shalel et al., 2002), and PBS buffer was used as the negative control. The samples were incubated at room temperature for 1 h. The samples were centrifuged and the absorbance of the supernatant was measured at 405 nm. MHC (Maximal non-Hemolytic Concentration) value for each peptide against red blood cells was defined as the lowest concentration of the peptide at which red blood cells membrane lysis was not observed. Experiments were performed in three independent replicates.

3. Results and discussion 3.1. Design of the peptides Previous results obtained by Pedron et al. (2017) for cationic VmCT1 analogs demonstrated the importance of the increase in the original net positive charge value of VmCT1 (+2). The authors proposed substitutions at positions 3, 7 and 11 in the hydrophilic face of VmCT1 amphipathic structure. These substitutions led to increased antimicrobial and anticancer activities. Additionally, the authors reported higher helical content values of analogs with increased net positive charge in zwitterionic (POPC 10 mmol L−1) and negatively charged (POPC:POPG, 3:1, mol:mol, 10 mmol L−1) lipid vesicles and TFE/water solution by CD measurements. Here, we evaluated the effect of single and multiple Lys-substitutions in the original sequence of VmCT1 on biological activities, varying the net positive charge from +2 (original values for VmCT1-NH2) to +5 (Table 1). Analogs were designed according to the changes in their most important physicochemical properties and helical wheel

2.4. Anticancer activity MCF-7 mammary cancer cells were used to assess anticancer activity of the Lys-substituted VmCT1 in vitro as previously described by Pedron et al. (2018). Briefly, the cells were incubated for 24 h at 37 °C and 5% CO2. On the next day, cells were treated with peptides (0.8 to 100 μmol L−1) for 4 and 24 h at 37 °C and the cell viability was measured through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Torres et al., 2018a). Experiments were performed in three independent replicates. 3

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Table 1 Peptides sequence, molecular characterization and physiochemical properties. Peptide

VmCT1-NH2 [Lys]1-VmCT1-NH2 [Lys]9-VmCT1-NH2 [Lys]1[Lys]12-VmCT1-NH2 [Lys]3[Lys]7-VmCT1-NH2 [Lys]3[Lys]11-VmCT1-NH2 [Lys]7[Lys]11-VmCT1-NH2 [Lys]3[Lys]7[Lys]11-VmCT1-NH2

Sequence

Molecular weight (Da) Theoretical

Observed

1450.7 1431.7 1507.8 1460.8 1534.9 1561.9 1504.9 1576.0

1450.9 1432.3 1508.0 1461.0 1535.9 1563.9 1505.9 1577.0

Phe-Leu-Gly-Ala-Leu-Tep-Asn-Val-Ala-Lys-Ser-Val-Phe-NH 2 Lys-Leu-Gly-Ala-Leu-Trp-Asn-Val-Ala-Lys-Ser-Val-Phe-NH 2 Phe-Leu-Gly-Ala-Leu-Trp-Asn-Val-Lys-Lys-Ser-Val-Phe-NH2 Lys-Leu-Gly-Ala-Leu-Trp-Asn-Val-Ala-Lys-Ser-Lys-Phe-NH2 Phe-Leu-Lys-Ala-Leu-Trp-Lys-Val-Ala-Lys-Ser-Val-Phe-NH2 Phe-Leu-Lys-Ala-Leu-Trp-Asn-Val-Ala-Lys-Lys-Val-Phe-NH2 Phe-Leu-Gly-Ala-Leu-Trp-Lys-Val-Ala-Lys-Lys-Val-Phe-NH2 Phe-Leu-Lys-Ala-Leu-Trp-Lys-Val-Ala-Lys-Lys-Val-Phe-NH2

H

μH

z

P/N

0.82 0.61 0.72 0.44 0.71 0.67 0.72 0.64

0.58 0.50 0.49 0.44 0.68 0.69 0.66 0.72

+2 +3 +3 +4 +4 +4 +4 +5

0.50 0.62 0.62 0.86 0.50 0.50 0.50 0.50

H (hydrophobicity), μH (hydrophobic moment), z (net charge), P/N (ratio of polar/non-polar residues in the sequence) according to Heliquest helical wheel projection (Gautier et al., 2008).

charged residue in the N-terminal extremity of the peptide on the biological activities. The substitution generated an analog with decreased mean hydrophobicity and mean hydrophobic moment values compared to VmCT1-NH2. The Lys-substitution at the center of the hydrophobic face (position 9) led to decreased hydrophobicity-related features. Tripathi and collaborators substituted the Ile residue at position 9 (center of the hydrophobic face) of IsCT1 by a Lys residue, causing decreased hemolytic activity while the antimicrobial activity was maintained (Tripathi et al., 2015). However, some authors observed the reduction in the antimicrobial activity and cytotoxicity by adding a positively charged residue at the center of the hydrophobic face. For instance, by substituting the Ile residue at position 17 by Lys in the hydrophobic face of the peptide AR-23, Zhang and collaborators reported lower antimicrobial activity, but also lower hemolytic activity and cytotoxicity against mammalian cells (Zhang et al., 2016). Another example is the substitution by positively charged amino acids made in any of the positions of the hydrophobic face of Polybia-CP by Torres and collaborators, which led to lower antimicrobial activity when the peptides presented lower helical tendency (Torres et al., 2018b). In addition, we also proposed a double Lys-substitution in the hydrophobic face at positions 1 and 12 (Phe1 and Val12) leading to a peptide with decreased mean hydrophobicity and mean hydrophobic moment values. These substitutions led to a peptide with lower antimicrobial activity and cytotoxicity against human erythrocytes.

projections analyses of VmCT1-NH2 and derivatives caused by Lyssubstitutions, and based on previous results obtained by Pedron et al. (2017). Positions 3, 7 and 11 at the hydrophilic face were estimated as the most prone to multiple Lys-substitutions according to the HeliQuest server (Gautier et al., 2008) (Fig. 1 and Table 1). Thus, all the doublesubstitution combinations for the three chosen positions ([Lys]3[Lys]7VmCT1-NH2, [Lys]3[Lys]11-VmCT1-NH2 and [Lys]7[Lys]11-VmCT1NH2 analogs) were designed, synthesized and tested. Modifications led to peptides with a net positive charge value of +4. The effect of a triple Lys-substitution at the same positions ([Lys]3[Lys]7[Lys]11-VmCT1NH2) was an analog with a net positive charge value of +5. These substitutions led to decreased mean hydrophobicity and increased mean hydrophobic moment values compared to VmCT1-NH2. The hydrophobic moment can be defined as the vectorial sum of individual amino acid hydrophobicity, normalized to an ideal α-helix and it is an important feature to characterize the amphipathicity of peptides (Eisenberg et al., 1982). Lys substitutions were also performed in the hydrophobic face of the amphipathic structure of VmCT1-NH2 to verify the importance of amphipathicity balance on biological activities (Fig. 1). Substitutions by Lys residues in the hydrophobic portion of AMPs are extensively explored in previous studies (Torres et al., 2018c; Tripathi et al., 2015; Wade et al., 2000; Zhao et al., 2016). Singh and collaborators described a Lys-substituted α-MSH derivative. The authors performed the Lys-substitution at position 1 (Ser) and did not alter the conformation compared to the wild-type peptide (WT). The Lys-substituted α-MSH analog presented higher antimicrobial activity than α-MSH with ~18-fold higher binding to vesicles (Singh et al., 2016). Similarly, Wade and collaborators proposed the substitution of the Phe residue at position 1 by a Lys residue in Temporin A. The authors observed reduced antimicrobial activities. The results showed that the amphipathic balance is important to the antimicrobial activity when the Lys residue was added in the first position of the sequence, leading to the destabilization of the helical structure (Wade et al., 2000). Thus, we proposed the replacement of the Phe residue at position 1 by a Lys residue to verify the effect of adding a positively

3.2. Structural analyses Lys-substitutions in the hydrophilic face generated peptides with lower helical content in most of the media tested (Table 2) compared to VmCT1-NH2 and the Lys-substituted analogs described previously (Pedron et al., 2017). In the presence of zwitterionic and negatively charged vesicles, the analogs proposed here showed lower helical content (fH: 0–0.05, Fig. 2 and Table 2) compared to VmCT1-NH2 (fH: 0.42 in POPC and 0.55 in POPC:DOPE - Fig. 2 and Table 2). In helical inductor medium (TFE/water), the peptides with a net positive charge

Table 2 Helical fraction of the WT peptide and analogs in four different media calculated used Lifson-Roig helix-coil theory (Lifson and Roig, 1961). Peptide

VmCT1-NH2 [Lys]1-VmCT1-NH2 [Lys]9-VmCT1-NH2 [Lys]1[Lys]12-VmCT1-NH2 [Lys]3[Lys]7-VmCT1-NH2 [Lys]3[Lys]11-VmCT1-NH2 [Lys]7[Lys]11-VmCT1-NH2 [Lys]3[Lys]7[Lys]11-VmCT1-NH2

Helical fraction (fH) Water

PBS

SDS

TFE/Water

POPC

POPC:DOPE

POPC:POPG

0.05 0.03 0.02 0.02 0 0 0 0

0.03 0.02 0.03 0 0.05 0.04 0.03 0.03

0.17 0.30 0.21 0.19 0.37 0.28 0.27 0.25

0.30 0.32 0.21 0.13 0.52 0.33 0.39 0.23

0.42 0.04 0.03 0 0 0.02 0.01 0.02

0.55 0.07 0.04 0 0.05 0.05 0 0.03

0.25 0.19 0.27 0.11 0.19 0.29 0.18 0.19

4

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Fig. 2. Circular dichroism spectra of the peptides in water, PBS (pH 7.4), SDS (20 mmol L−1), POPC (10 mmol L−1), POPC:POPG (3:1, mol:mol - 10 mmol L−1), POPC:DOPE (3:1, mol:mol - 10 mmol L−1) and TFE/Water (3:2, v:v).CD were recorded after four accumulations at 20 °C, using a 1 mm path length quartz cell, between 195 and 260 nm at 100 nm min−1, with a band width of 0.5 nm. Peptide concentration at 50 μmol L−1.

value of +4 ([Lys]3[Lys]7-VmCT1-NH2, [Lys]3[Lys]11-VmCT1-NH2 and [Lys]7[Lys]11-VmCT1-NH2) exhibited higher helical content (fH: 0,52, 0,33 and 0,39, respectively) than VmCT1-NH2 (fH: 0,29). In the presence of SDS micelles, the peptides with multiple Lys-substitutions ([Lys]3[Lys]7-VmCT1-NH2, [Lys]3[Lys]11-VmCT1-NH2, [Lys]7[Lys]11VmCT1-NH2, and [Lys]3[Lys]7[Lys]11-VmCT1-NH2,) presented higher helical content (fH: 0,37, 0,32, 0,27 e 0,25, respectively) than VmCT1NH2 (fH: 0,17). The [Lys]1-VmCT1-NH2 analog presented lower helical content in the presence of zwitterionic and negatively charged vesicles. Contrarily, when [Lys]1-VmCT1-NH2 helical tendency was analyzed in the presence of SDS micelles (fH: 0.30) and helical inductor medium, its helical content was higher (fH: 0,32) than the helical content of VmCT1-NH2 (Fig. 2 and Table 2). While the peptide [Lys]1[Lys]12-VmCT1-NH2 presented lower helical tendency (Fig. 2 and Table 2) in the media analyzed.

cyclization (Rozek et al., 2003), and utilization of unusual amino acids (Knappe et al., 2010). Chen and collaborators, verified that the substitution of Gly by D-amino acids in cyclic peptide favored proteolytic resistance (Chen et al., 2013). Arias and collaborators reported that the introduction of Arg or Lys residues with modified side chains contributed to increasing resistance to proteases (Arias et al., 2018). From these results, as alternatives to increasing the degradation resistance of this family of peptides we could incorporate D-amino acids or Arg or Lys with modifications in the side chain in the next generations of positively charged VmCT1 analogs. 3.4. Biological activities of VmCT1 analogs with Lys substitutions in the hydrophilic face The peptides designed with Lys substitutions in the hydrophilic face of helical structure presented antimicrobial activity with MIC (Minimal Inhibitory Concentration) values ranging from 0.1 μmol L−1 to 6.3 μmol L−1 (Fig. 3 and Supplementary Information Table 1). [Lys]3[Lys]7-VmCT1-NH2 and [Lys]3[Lys]7[Lys]11-VmCT1-NH2 showed higher activity than VmCT1-NH2 against M. luteus (0.4 μmol L−1), S. marcescens (0.1 μmol L−1), E. cloacae (3.1 μmol L−1), B. megaterium (0.8 μmol L−1 and 0.4 μmol L−1, respectively) and B. megaterium (0.4 μmol L−1). While the activity of the peptides [Lys]3[Lys]11-

3.3. Resistance to degradation The peptides designed in this study were not stable after longer periods of exposure to serum proteases (Fig. 8). The most well-known alternatives to prevent the degradation of peptides by peptidases are replacement of L-amino acids for D-amino acids (Zhao et al., 2016), 5

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VmCT1-NH2 and [Lys]7[Lys]11-VmCT1-NH2 was higher against S. marcescens (0.2 μmol L−1) and E. cloacae (3.1 μmol L−1 and 6.3 μmol L−1, respectively). Candida is one of the most frequent cause fungal infections, which range mucocutaneous infection to invasive candidiasis (Dismukes et al., 2004; Yapar, 2014). The WT peptide presented MIC value of 12.5 μmol L−1 against fungi C. albicans and C. tropicalis. The MIC value of the analogs with double or triple Lys-substitutions at the hydrophilic face (1.6 μmol L−1) was 8-fold lower

against C. albicans, while against C. tropicalis the peptides showed activity at 0.8 μmol L−1, reduced by 16-fold compared to the activity exhibited by the WT peptide. We envision that the increase in the net positive charge favored the interaction of the peptides with the fungal membranes, which besides of phospholipids contain significant amounts of sterols and are also more negative such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylserine (PS) (Rautenbach et al., 2016).

Fig. 3. Antimicrobial activities of VmCT1 and its analogs against fungi, Gram-positive and Gram-negative bacteria. Peptone Broth or Potato Dextrose Broth was used as the control for microbial growth. Absorbance values in purple represent maximal inhibition of microbial growth, while in green, absorbance values related to microbial growth. Experiments were made in three independent replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6

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against MCF-7 cells (Pedron et al., 2018). These results corroborate to earlier reports that demonstrated the introduction of positively charged amino acids was important to promote the interaction with cancer cell membranes. Du and collaborators described peptides with increased net positive charge, designed from the original sequence of AcrAP1 and AcrAP2, the analogs that were generated presented activity against cancer cell lines (Du et al., 2014). Additionally, we evaluated the cytotoxicity of the VmCT1-NH2 analogs designed in this study against MCF-10A human breast epithelial cells (Fig. 6). The Lys-substituted analogs were active against MCF-7 cancer cells at a concentrations range (6.3–12.5 μmol L−1) lower than the concentration where VmCT1-NH2 analogs were cytotoxic against MCF-10A healthy epithelial cells (25 μmol L−1). Besides the anticancer properties of AMPs, these versatile molecules are also well-known for their antiparasitic activity. Among the most important diseases caused by parasites, malaria is one of the most prevalent diseases with a high mortality rate in Africa, Asia and the Americas, which affects mainly children under the age of 5 and pregnant women (Biamonte et al., 2013). According to the World Health Organization, it is estimated that 219 million cases of malaria occurred worldwide in 2018 with > 435,000 deaths (WHO, 2018). Peptides have been used as alternative antiplasmodial agents (Lacerda et al., 2016; Vale et al., 2014). The use of naturally occurring peptides are advantageous compared to small molecule drugs due to their short half-life, presents a diversity of mechanisms actions which makes it difficult to acquire resistance mechanisms, besides the degradation products are natural amino acids (Mahlapuu et al., 2016). Gao and collaborators described the meucin-24, which was isolated from the venom of the scorpion Mesobuthus eupeus and showed antiplasmodial activity (Gao et al., 2010). Silva and collaborators studied angiotensin II analogs and demonstrated that linear and cyclic peptides presented antiplasmodial activity against Plasmodium gallinaceum and Plasmodium falciparum (Silva et al., 2017, 2015). Decoralin, an AMP isolated from wasp venom, did not exhibit antiplasmodial activity; however, through a structure-function-design study the authors finetuned the physicochemical and structural properties of the peptide with single and double substitutions in the sequence transforming it in an active anti-plasmodial agent against Plasmodium gallinaceum. The authors demonstrated that the increase in net positive charge favors the interaction with the membrane of the parasite (Torres et al., 2018c). The antiplasmodial activity of the AMPs designed in this study was assessed against mature Plasmodium gallinaceum sporozoites, an alternative model to Plasmodium falciparum (Chamlian et al., 2013). The antiplasmodial activity of the peptides was assessed through the exposure of the mature sporozoites to peptides serial dilutions for 1 h. The cells exposed to VmCT1 and its analogs were analyzed by fluorescence microscopy to evaluate sporozoites integrity, according to the methodology described by Silva et al. (2017). The WT peptide showed antiplasmodial activity at 0.8 μmol L−1 (93% of fluorescent sporozoites - Fig. 7 and Supplementary Information Table 1), indicating that the peptide kills 93% of sporozoites at submicromolar concentration, characterizing a highly active anti-plasmodial peptide when compared to others in literature (Der Torossian Torres et al., 2014; Marcelo Der Torossian et al., 2015; Silva et al., 2015, 2014). Contrarily, the analogs with multiple Lys-substitutions did not present activity at sub-micromolar concentrations. The increase of the net positive charge at the hydrophilic face did not contribute significantly to increasing antiplasmodial activity, differently from what was observed in antimicrobial and anticancer activities.

Fig. 4. Hemolytic activities of VmCT1 and analogs against human red blood cells in different peptide concentrations (0.1–100 μmol L−1), in PBS at room temperature for 1 h. Experiments were performed in three independent replicates. Surfactant (1% SDS in PBS) was used to ensure complete hemolysis and PBS was used as control preserving erythrocyte integrity. Absorbance values in red represent the hemolytic activity, while in blue, absorbance values related to no hemolytic activity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The introduction of positive charge in the hydrophilic face of VmCT1-NH2 was important to favor antimicrobial activities. However, these modifications did not favor the interaction with microbial membranes. The interactions with mammalian cell membranes were favored instead. [Lys]3[Lys]11-VmCT1-NH2, [Lys]7[Lys]11-VmCT1-NH2 and [Lys]3[Lys]7[Lys]11-VmCT1-NH2 showed MHC values of 3.1 μmol L−1 (Fig. 4 and Supplementary Information Table 1), while the analog [Lys]3[Lys]7-VmCT1-NH2 presented an MHC value of 1.6 μmol L−1 (Fig. 4 and Supplementary Information Table 1). Compared to normal mammalian cell membranes, the cancer cells show a higher net negative charge caused by the abnormal expression of anionic molecules, such as phosphatidylserines, negative glycoproteins, and glycosaminoglycans, which make the cancer cell membranes similar in composition to the bacterial membranes (Gaspar et al., 2013; Liu, 2015). The initial electrostatic interactions between the cationic peptide with the negatively charged cancer cell membranes are important to destabilize the membrane (Hoskin and Ramamoorthy, 2008). The biological activity of the VmCT1 peptides was verified against MCF-7 human breast cancer cells after 4 and 24 h of exposure (Fig. 5 and Supplementary Information Table 1). Multiple Lys-substitutions on the hydrophilic face of the helical structure of VmCT1-NH2 (e.g., peptides [Lys]3[Lys]7-VmCT1-NH2, [Lys]3[Lys]11-VmCT1-NH2, [Lys]7[Lys]11-VmCT1-NH2, and [Lys]3[Lys]7[Lys]11-VmCT1-NH2,) were important to increase the anticancer activity against MCF-7. Previously, Pedron and collaborators described VmCT1 analogs with single substitutions and the peptides with Lys-substitution at the positions 3 or 7 presented activity against MCF-7 cancer cells at 25 μmol L−1. The results obtained by multiple Lys substitutions demonstrate that the introduction of positive charge residues at the hydrophilic face of the WT peptide promotes higher anticancer activity

3.5. Biological activities of VmCT1 analogs with Lys substitutions in the hydrophobic face The Lys-substitutions were also performed in the hydrophobic face of the helical structure to verify whether the destabilization of amphipathic balance affected the biological activities (Fig. 1). Substitutions in 7

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Fig. 5. MTT assay results for VmCT1 and analogs in 4 and 24 h on MCF-7 cell lines. Dulbecco's Modified Eagle Medium was used as the control for cell maximum growth. Absorbance values in yellow represent the decrease of viable MCF-7 cells, while in pink, absorbance values related to maintenance of cells. Experiments were made in three independent replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Antiplasmodial activity against P. gallinaceum expressed as the percent of fluorescent mature sporozoites. (n = 9). Absorbance values in orange represent the activity against Plasmodium gallinaceum (fluorescent sporozoites), while in blue, absorbance values related to not have antiplasmodial activity. Digitonin/PBS solution was used as the positive control and PBS solution was used as the negative control. Experiments were made in three independent replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

GNU7) were designed by Kim and collaborators (Kim et al., 2013). These peptides exhibited high antimicrobial activity and resistance to degradation by proteases, besides of lower cytotoxicity. The substitution of the Phe residue at position 1 by a Lys residue led to decreased hemolytic activity (12.5 μmol L−1) compared to the WT peptide (> 6.3 μmol L−1 - Fig. 4 and Supplementary Information Table 1). The antimicrobial activity of the [Lys]1-VmCT1-NH2 peptide was lower than the WT peptide for most of the microorganisms tested; however, the peptide showed higher antimicrobial activity against C. albicans and C. tropicalis (3.1 μmol L−1 and 6.3 μmol L−1, respectively Fig. 3 and Supplementary Information Table 1) than the WT peptide (12.5 μmol L−1 - Fig. 3 and Supplementary Information Table 1). The

Fig. 6. MTT assay results for VmCT1 and analogs in 4 and 24 h on healthy human breast epithelial cells MCF-10A lines. Dulbecco's Modified Eagle Medium was used as the control for cell maximum growth Absorbance values in pink represent maintenance of healthy MCF-10A cells, while in yellow, absorbance values related to decrease of viable cells. Experiments were made in three independent replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the hydrophobic face were evaluated in previous studies by other research groups (Kim et al., 2013; Tripathi et al., 2015). Peptides with a charged amino acid at the center of hydrophobic face (GNU6 and 8

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Fig. 8. Resistance to degradation of the WT peptide and designer analogs in fetal bovine serum for 6 h. Experiments were performed in three independent replicates.

Lys-substitutions in the hydrophobic face promoted decreased antimicrobial, anticancer and hemolytic activities, as well as lower or similar helical tendency compared to the WT peptide. Additionally, [Lys]1-VmCT1-NH2 and [Lys]9-VmCT1-NH2 showed increased helical content compared to the WT peptide in helical inductor medium and negatively charged vesicles, respectively. The [Lys]9-VmCT1-NH2 analog showed significant antiplasmodial activity and low hemolytic activity, unlike the other peptides with positively charged amino acid substitutions in the hydrophilic face of the helical structure proposed in this study, showing that the antiplasmodial activity was not dependent on the amphipathic balance and helicity of the VmCT1-NH2 analogs. These results pointed to a new direction of the development of synthetic peptides with multiple biological activities. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejps.2019.06.006.

substitution of Ala residue at position 9 of the hydrophobic face by a Lys residue, led to lower hemolytic (MHC > 100 μmol L−1 - Fig. 4 and Supplementary Information Table 1) and antimicrobial (MIC > 100 μmol L−1 - Fig. 3 and Supplementary Information Table 1) activities values. The antimicrobial and hemolytic activities obtained for the double Lys-substitutions at positions 1 and 12 were similar to the other Lys-substituted analogs where Lys was replaced by a residue in the hydrophobic face. The [Lys]1-[Lys]12-VmCT1-NH2 analog presented lower hemolytic activity (> 100 μmol L−1 - Fig. 4 and Supplementary Information Table 1); however, it was the only analog with a Lys-substitution in the hydrophobic face to present antimicrobial activity against M. luteus (25 μmol L−1), C. albicans (12.5 μmol L−1), C. tropicalis (25 μmol L−1) and B. megaterium (6.3 μmol L−1) (Fig. 3 and Supplementary Information Table 1). Lys-substituted analogs at the hydrophobic face did not present activity against MCF-7 cells (Fig. 5 and Supplementary Information Table 1). However, when the substitution was made at the position 9 of the hydrophobic face ([Lys]9-VmCT1-NH2 peptide), the peptide that had not shown antimicrobial nor anticancer activity, exhibited antiplasmodial activity at 0.8 μmol L−1 with 64% fluorescent sporozoites (Fig. 7 and Supplementary Information Table 1). It shows that the amphipathic balance was not a crucial factor for antiplasmodial activity. Our results demonstrate that the substitution of residues from the original sequence by Lys residues at the hydrophilic portion of the helical structure leads to increased antimicrobial and anticancer activities. Furthermore, simultaneous Lys-substitutions, at positions 3, 7 and 11 promotes higher antimicrobial and anticancer activities compared to the WT peptide. Lys-substitutions made at the hydrophobic face lead to analogs with higher antiplasmodial activity and lower hemolytic activity.

Credit author statement Cibele Nicolaski Pedron – design, synthesis and characterization of peptides, tests of antimicrobial activity, circular dichroism assays and paper writing. Cyntia Silva Oliveira - synthesis and characterization of peptides and stability assays Adriana Farias da Silva – assays of the activity against Plasmodium gallinaceum sporozoites and data analysis Gislaine Patricia Andrade - anticancer activity and cytotoxic activity Maria Aparecida Silva Pinhal - anticancer activity, cytotoxic activity and data analysis Giselle Cerchiaro - anticancer activity, cytotoxic activity and data analysis Pedro Ismael da Silva Junior - Hemolytic activity assays and discussion Fernanda Dias da Silva - tests of antimicrobial activity and discussion Marcelo Der Torossian Torres - tests of antimicrobial activity, paper writing and financial support Vani Xavier Oliveira Junior – peptides design, paper writing and financial support

4. Conclusions We rationally designed VmCT1 analogs with Lys-substitutions to verify the effect of the increased net positive charge in their biological activities. The increase in net positive charge played an important role in the antimicrobial activity of this family of peptides. Multiple Lyssubstitutions at positions 3, 7 and 11 in the hydrophilic face resulted in increased antimicrobial and anticancer activities compared to the WT peptide. However, hemolytic activity was also slightly increased. These results show the importance of the electrostatic interactions between peptides and negatively charged phospholipids from the membranes of microorganisms and cancer cells. The peptides with Lys-substitutions at the hydrophilic face showed activity against C. albicans and C. tropicalis fungi, with low micromolar and sub-micromolar MIC values (1.6–0.4 μmol L−1) compared to the WT peptide (12.5 μmol L−1). These analogs also presented lower cytotoxicity at these concentrations, demonstrating that this design strategy can be applied to development of novel topical antifungal peptides.

Declaration of Competing Interest The authors declare that they do not have conflict of interest. Acknowledgments We thank Prof. Dr. Katia Regina Perez for the donation of the POPC, POPC:POPG and POPC:DOPE vesicles, Prof. Dr. Margareth Lara Capurro for the donation of the sporozoites, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (Finance Code 001 -CAPES) and Fundação de Amparo à Pesquisa do Estado de São 9

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Paulo (FAPESP) for funding (VXO #2017/03046-2 and MDTT #2014/ 04507-5) and Multiuser Central Facilities (UFABC) for the experimental support.

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