Computer-Aided Design of Mastoparan-like Peptides Enables the

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Computer-aided design of mastoparan-like peptides enables the generation of non-toxic variants with extended antibacterial properties Karen G. N. Oshiro, Elizabete de Souza Cândido, LAI YUE CHAN, Marcelo Der Torossian Torres, Bruna E. D. Monges, Silvia Gomes Rodrigues, William F Porto, Suzana Meira Ribeiro, Sónia Troeira Henriques, Timothy K. Lu, Cesar de la Fuente-Nunez, David J Craik, Octávio Luis Franco, and Marlon H. Cardoso J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00915 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Journal of Medicinal Chemistry

Computer-aided design of mastoparan-like peptides enables the generation of non-toxic variants with extended antibacterial properties

Karen G. N. Oshiroa,b, Elizabete S. Cândidob,c, Lai Y. Chand, Marcelo D. T. Torrese,f,g,h,i, Bruna E. D. Mongesb, Silvia G. Rodriguesb, William F. Portob,j, Suzana M. Ribeirok, Sónia T. Henriquesl, Timothy K. Lue,f, Cesar de la Fuente-Nuneze,f,h,i,, David J. Craikd, Octávio L. Francoa,b,c* and Marlon H. Cardosoa,b,c,d*

aPrograma

de Pós-Graduação em Patologia Molecular, Faculdade de Medicina, Universidade de

Brasília, 70910900, Brazil; bS-inova

Biotech, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom

Bosco, 79117900, Brazil; cCentro

de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e

Biotecnologia, Universidade Católica de Brasília, 70790160, Brazil; dInstitute

for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072,

Australia; eSynthetic

Biology Group, MIT Synthetic Biology Center; The Center for Microbiome Informatics

and Therapeutics; Research Laboratory of Electronics, Department of Biological Engineering, and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States of America; fBroad

Institute of MIT and Harvard, Cambridge, MA, 02139, United States of America;

gCentro

de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP,

09210170, Brazil; hDepartment

of Psychiatry, and Department of Microbiology, Perelman School of Medicine,

University of Pennsylvania, Philadelphia, PA, 19104, United States of America; 1

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

iDepartment

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of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19104, United States

of America; jPorto

Reports, Brasília, DF, Brazil;

kPrograma

de Pós-Graduação em Ciências da Saúde, Universidade Federal da Grande Dourados,

Dourados, MS, 79825070, Brazil; lFaculty

of Health, School of Biomedical Sciences, Institute of Health & Biomedical Innovation,

Queensland University of Technology, Translational Research Institute, Brisbane, QLD 4102, Australia.

*Corresponding

authors:

Dr. Octávio L. Franco, S-Inova Biotech, Universidade Católica Dom Bosco, Programa de PósGraduação em Biotecnologia. Email: [email protected]; Dr. Marlon H. Cardoso, S-Inova Biotech, Universidade Católica Dom Bosco, Programa de PósGraduação em Biotecnologia. Email: [email protected].

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ABSTRACT Diverse peptides have been evaluated for their activity against pathogenic microorganisms. Here, five mastoparan variants were designed based on mastoparan-L, among which two (R1 and R4) were selected for in-depth analysis. Mastoparan-L (parent/control), R1 and R4 inhibited susceptible/resistant bacteria at concentrations ranging from 2 to 32 μM, whereas only R1 and R4 eradicated P. aeruginosa biofilms at 16 μM. Moreover, the toxic effects of mastoparan-L toward mammalian cells were drastically reduced in both variants. In skin infections, R1 at 64 M was the most effective variant, reducing P. aeruginosa bacterial counts 1,000-times at day four postinfection. Structurally, all the peptides showed varying levels of helicity and structural stability in aqueous and membrane-like conditions, which may affect the different bioactivities observed here. By computationally modifying the physicochemical properties of R1 and R4 we reduced the cytotoxicity and optimized the therapeutic potential of these mastoparan-like peptides both in vitro and in vivo.

Keywords: mastoparan, antibacterial, antibiofilm, anti-infective, structural biology.

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Introduction Persistent bacterial infections are among the greatest threats to human health and are directly responsible for high levels of morbidity and mortality.1 Bacteria from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.)2, 3 are often associated with resistance events and hospital infections.2 Moreover, in the case of constant exposure to antimicrobial agents, nutrient limitation or temperature variation, bacterial cells can form multicellular communities, known as biofilms.4 Biofilms are uni- or poly-microbial structured consortia, presenting an extracellular matrix that may act as physical and chemical barriers in response to antibiotic administration.5 In addition to the challenge of bacterial resistance and biofilm formation, the misuse of antibiotics may cause severe side-effects6 and exacerbated immune response.7 Antimicrobial peptides (AMPs) have been considered as alternatives for the treatment of bacterial and biofilm infections that are no longer responsive to the conventional antibiotic medicines.8 The net positive charge and hydrophobicity of these peptides are related to their antimicrobial, hemolytic and cytotoxic activity, as they may influence cell selectivity and membrane insertion, as well as affecting their interaction with intracellular targets.9 However, toxicity toward mammalian cells is still one of the major challenges in working with AMPs, and an increasing number of studies have focused on the reduction of this undesired biological property.10, 11

Among the naturally occurring AMPs, the mastoparan peptides have attracted great attention due to their multifunctional properties.12-16 Mastoparans include mainly cationic, amphipathic AMPs usually organized in α-helical structure (environment-dependent) that have been isolated from the venom of several wasp species.17 Currently, the mastoparan peptides are known for their potential against susceptible and resistant bacterial strains and, most recently, antibiofilm activities have also been reported against methicillin-resistant S. aureus (MRSA).18 However, some 4


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members of this peptide family still lack therapeutic effectiveness due to their hemolytic and cytotoxic properties toward mammalian cells, thus directly impacting the therapeutic index of these peptides and, therefore, encouraging novel strategies for the generation of improved variants.19 Diverse rational design methods for obtaining optimized AMP sequences have been described in the literature.20 Among them, an increasing number of studies have focused on amino acids frequency and their positions as a base for the de novo design of novel AMPs with limited homology with known AMPs.21 This strategy has highlighted the modular nature of AMPs, thus allowing the development of novel bioinformatics tools for designing these molecules. Considering this modularity among AMPs, Loose et al.21 presented a linguistic model, in which AMP sequences were treated as a formal language, with grammatical rules (amino acid patterns and motifs). Consequently, novel AMP sequences were generated without using structure-function information, but a combination of patterns.21 Years later, this concept was further explored by Porto et al.,22 which developed a novel algorithm for designing optimized AMPs, called Joker, through the insertion of antimicrobial patterns into peptide sequences (AMPs or not) in a sliding window fashion. Here, we describe the computer-aided design of novel mastoparan-like peptides (Figure 1) through the Joker algorithm.22 For this, we used the sequence of the cytolytic peptide mastoparan-L as input for Joker aiming to optimize the antimicrobial activities of the designed variants and reduce their toxic effects on mammalian cells when compared to the parent peptide. A total of five variants were generated, of which two (R1 and R4) were selected for detailed analysis of their antibacterial, antibiofilm, cytotoxic, hemolytic and anti-infective properties. Moreover, the structure profile of these variants was investigated in different biological conditions.

Results Design, molecular modeling and initial screening for antimicrobial activities 5



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The Joker algorithm22 designs peptide variants by inserting amino acid patterns in a template sequence, in a non-cumulative sliding window system. Using mastoparan-L as the template and the pattern “K[ILV][AL]x[RKD][ILV]xxKI”, five variants were designed. All the sequences generated were aligned with other mastoparan peptide sequences deposited in public databases, including the Antimicrobial Peptide Database (APD).23 The fourth sequence (INLKILARLAKKIL) designed in this study had a complete sequence match with a mastoparan previously described, named [I5R8].24 We discarded this sequence from further analysis, and the four remaining variants were named mastoparan-R1 to -R4. Since the pattern used is derived from α-helical AMPs, it is expected that variants showed α-helix formation (Table 1). This was confirmed by molecular modeling, showing that all variants (R1 to R4) adopt -helix conformation, along with well-defined amphipathic segments according to the Adaptive Poisson-Boltzmann Solver (APBS) calculations25 (Figure S1). The statistics for the theoretical models are summarized in Table S1. The calculated theoretical physicochemical properties show that among all variants, R1 and R4 present the highest positive net charge (+6). In addition, when compared to the parent sequence, as well as to R2 and R3, the variants R1 and R4 revealed reduced hydrophobicity (which is known to reduce toxic effects against mammalian cells, including hemolysis)26 and increased hydrophobic moment (Table 1). The antimicrobial activity of all variants was assessed against a bioluminescent strain of P. aeruginosa (H1001) (Table 1), revealing minimal inhibitory concentrations (MIC) ranging from 4 to 30 M, with the highest activities observed for mastoparan-R1 and R4 (MIC = 4 M in both cases). Therefore, we selected the variants R1 and R4 for further functional and structural characterization. Subsequently, mastoparan-L (control for all experiments), R1 and R4 were chemically synthesized by solid-phase (Fmoc) at >95% purity, which was confirmed by ultra-high performance liquid chromatography (UHPLC) and matrix-assisted laser desorption/ionization, time

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of flight (MALDI-TOF). They presented monoisotopic masses of 1479.84 Da, 1636.14 Da and 1636.39 Da for mastoparan-L (Figure S2), R1 (Figure S3) and R4 (Figure S4), respectively.

Antibacterial and antibiofilm properties The peptides were evaluated against Gram-positive and Gram-negative, susceptible and resistant bacterial strains. MIC results are summarized in Table 2. R1 and R4 inhibited E. coli (ATCC 25922) at 4 μM and E. coli (KpC+ 001812446) at 8 μM; whereas mastoparan-L inhibited only the E. coli ATCC strain at 32 μM. Furthermore, all peptides (control and variants) inhibited E. coli (BL21) at 2 μM. When evaluated against K. pneumoniae (ATCC 13883) only R1 was active at 8 μM. In addition, all the peptides were unable to inhibit the growth of K. pneumoniae (KpC+ 001825971), A. baumannii (clinical isolate 003326263) and E. cloacae (clinical isolate 1383251) at the maximal concentration tested (32 μM). Gram-positive strains, including S. aureus (ATCC 12600) were inhibited by mastoparan-L at 8 M and by R1 and R4 at 4 M. Moreover, only R4 inhibited the growth of S. aureus (ATCC 25923) and methicillin-resistant S. aureus (MRSA; clinical isolate 713623) at 8 μM. Finally, all the peptides were active against P. aeruginosa strains (PAO1 and PA14) at 8 μM, for mastoparan-L, and 4 μM, for R1 and R4. Based on the MIC values obtained against P. aeruginosa (PAO1), and since this strain represents a model organism for biofilm growth and is standardized for antibiofilm assays,27 flowcell studies were carried out to further evaluate the antibiofilm potential of these peptides. We observed that both R1 and R4 were capable of eradicating two-day-old preformed P. aeruginosa biofilms at the tested concentration of 16 μM (Figure 2c and d). However, the control peptide (mastoparan-L) did not show the same antibiofilm potential, leading only to a slight dispersion of the biofilm cells (Figure 2b) compared to the untreated control (Figure 2a).

In vitro cytotoxic and hemolytic studies 7



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One of the challenges in working with mastoparan-like peptides is their undesired hemolytic and cytotoxic effect toward healthy mammalian cells. We therefore performed cytotoxicity assays using mouse adipocytes (3T3-L1) and human umbilical vein endothelial cells (HUVEC). We observed that mastoparan-L exhibits cytotoxic activity (IC50: 50% cell viability) at 12.5 μM and 50 μM against 3T3-L1 and HUVEC, respectively. In contrast, variants R1 and R4 were not cytotoxic at the range of concentrations tested (100 μM – Table 2). Moreover, hemolytic assays were also carried out against human erythrocytes. We observed that mastoparan-L, at 100 μM, led to ~45% hemolysis, whereas R1 and R4 caused less than 10% hemolysis at the same concentration (Table 2). Previous studies have reported additional biological properties for AMPs, including anticancer activities. Studies with mastoparan-like peptides have demonstrated their pro-necrotic activity on human glioblastoma multiforme cells,28 as well as anticancer potential toward leukemia, myeloma and breast cancer cell lines.14 We therefore investigated the anticancer potential of mastoparan-L and its variants against human prostate cancer (PC-3), human breast cancer (MCF-7) and human colon adenocarcinoma (HT-29) cell lines. As a result, we observed that mastoparan-L was the only peptide capable of compromising the viability (IC50) of cancerous cells from 12.5 M (PC-3) to 25 M (MCF-7 and HT-29) (Table 2).

In vivo studies Skin infections caused by P. aeruginosa, in their severe stage, may evolve to invasive infections and, eventually, result in sepsis.29 The mastoparan variants studied here were active against P. aeruginosa planktonic cells and pre-formed biofilms (Table 2 and Figure 2); however, they did not cause adverse effects on mammalian cells. Therefore, we investigated the anti-infective potential of these peptides (single dose at 64 M) in skin infections caused by P. aeruginosa (PA14) in mice. We observed that the anti-infective potential of the peptides was time-dependent, as all the 8


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peptides (both parent and synthetic variants) were capable of reducing bacterial counts at days two and four post-infection. As observed in Figure 3, all peptides reduced P. aeruginosa counts by approximately 100-times two days post-infection (Figure 3). On day four, the parent peptide mastoparan-L and the variant R4 had a slight loss of effectiveness (Figure 3). Otherwise, the variant R1 showed improved anti-infective potential by reducing P. aeruginosa bacterial counts 1,000times at day four post-infection (Figure 3). Structural analysis AMPs are known for their wide structural diversity, which may directly impact their mechanisms of action against microorganisms.30 In the present work, we used experimental biophysical techniques, including CD and NMR spectroscopies, to characterize the secondary structure of mastoparan-like peptides. CD spectra were recorded in different environments, as described in the methodology section. When analyzed in ultrapure water, the variants R1 and R4 showed CD signatures characteristic of random coiled structures (Figure 4b and c). In addition, spectra recorded in buffer revealed that a higher salt concentration does not influence the helicity of the designed peptides (Figures 4b and c; Table 3). In contrast, the CD spectra of the parent peptide mastoparan-L in water has a minimum at 200 nm, typical random coil, but also a minimum at 222 nm, suggesting a small contribution of -helix; in buffer the contribution of -helix is further increased (Figure 4a; Table 3). In 30% 2,2,2-trifluoroethanol (TFE) in water (v/v) and 25 mM dodecyl sodium sulfate (SDS) (anionic micelles), all the peptides showed strong signatures of αhelix conformation from 46.7% to 100% of helicity (Figures 4a, b and c; Table 3). In this study, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles were used to mimic mammalian healthy cells and a mixture (1:1) of POPC and 1-palmitoyl-2-oleoyl-snglycero-3-phospho-(1′–sn-glycerol) (POPG) was used for bacterial cells. Interestingly, the spectra of the variants R1 and R4 in the presence of POPC model membranes are highly similar to those obtained in buffer, suggesting that these two peptides have low affinity for zwitterionic membranes 9



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(Figure 4 b and c; Table 3). Similar CD signatures were also observed for mastoparan-L in POPC and buffer and, therefore, indicate a contribution of -helix due to a defined minimum at 222 nm (Figure 4a; Table 3). Mastoparan-L has been described as a membranolytic peptide that adopts helical arrangements in contact with biological membranes to trigger its membrane-associated activities.31 Therefore, our findings suggest that the transient state from random coil to -helix in mastoparan-L when in contact with zwitterionic vesicles might play a crucial role in the hemolytic and cytotoxic effects of this peptide on mammalian cells when compared to its variants R1 and R4. Finally, when evaluated in a bacterial membrane-like condition (SDS micelles and POPC/POPG vesicles), mastoparan-L and the variant R1 showed higher -helical contents compared to the variant R4 (Figures 4a, b and c; Table 3). Nevertheless, although R4 presented lower helicity compared to R1, we concluded that the -helical contents of these peptides are directly correlated with their interaction with micelles and vesicles mimicking bacterial surfaces. In addition to the CD studies, NMR spectroscopy experiments were carried out to evaluate the H secondary chemical shifts for each peptide in 30% TFE-d3 (v/v), thus determining which residues participate in the formation of secondary structure. All the H chemical shifts for mastoparan-L, R1 and R4 in the presence of 30% TFE-d3 (v/v) are summarized in Table S2, Table S3 and Table S4, respectively. In agreement with the CD data, NMR spectroscopy revealed that all the peptides adopt -helical structure in 30% TFE, with helical segments from residues Leu3 to Ile13 for mastoparan-L; residues Lys4 to Ile13 for R1; and residues Lys4 to Lys12 for R4 (Figure 5a). In addition, we conducted temperature coefficient experiments from 285 K to 310 K, at intervals of 5 K. We observed that residues Asn2 to Ala8, Lys11 and Lys12 in mastoparan-L are protected from the solvent as their amide protons showed temperature gradients more positive than -4.6 ppb.K-1, thus indicating intrapeptide hydrogen bonding.32 The protected residues in R1 were Leu3, Leu6, Ala8, Lys9 and Lys12, whereas the residues Asn2, Lys4, Lys5, Ala8, Arg9, Lys12 and Lys13 were protected in R4. Considering that the higher the number of residues protected from the solvent the higher the 10


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number of intrapeptide hydrogen bonds involved in structure stabilization, we conclude that the levels of structural stability of the peptides follow the order: mastoparan-L>R4>R1. These findings also confirm our CD data, where mastoparan-L had higher helicity in all cases.

Discussion and Conclusions The indiscriminate use of antibiotics has become a major concern in our society, leading to multidrug-resistance,33 especially in hospitals and communities where antibiotics are no longer effective at treating infections caused by resistant bacteria.34 Antimicrobial resistance is estimated to cause ~700,000 deaths worldwide annually.35 In this context, efforts have been made by the scientific community to develop viable alternatives to restrain infections caused by these pathogenic strains. AMPs appear as promising candidates, representing a class of multifunctional molecules produced by most organisms in nature.36 Currently, over 3,000 AMPs have been deposited in public databases,23 providing a rich source of information that can be used in drug AMP design strategies. In the present study we successfully designed four novel mastoparan-like AMPs (mastoparan-R1 to R4) according to the -helical pattern "K[ILV][AL]x[RKD][ILV]xxKI" and using the mastoparan-L sequence as input for the Joker algorithm.22 The initial screening for antibacterial activities against P. aeruginosa showed that R1 and R4 had the highest efficacy. The residues Ile1, Asn2, Ala5, Leu9 and Ala10 in mastoparan-L were replaced by Lys1, Ile2, Arg5, Lys9 and Ile10 in R1, whereas the residues Ala5, Leu9, Ala10, Ile13 and Leu14 in mastoparan-L were replaced by Lys5, Arg9, Ile10, Lys13 and Ile14, in R4 (Figure 1). The modifications in R1 and R4 sequences provided these variants with a net-positive charge that was twice that of mastoparan-L, increasing from +3 to +6, which favor their interaction with negatively charged bacterial membranes to exert their actions. Moreover, detailed studies on the influence of lysine residue positioning along mastoparan sequences have shown that variants with lysine residues at position 4/5 and/or from 11 to 13 are more likely to present antibacterial properties,37 as reported for R1 and 11



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R4 in the present study. We also observed reduced hydrophobicity and increased hydrophobic moment in the variants, leading to increased amphipathicity, as shown in Figure S1. It has been reported that the positive net charge of peptides, as well as their amphipathicity, are a crucial aspect in their antimicrobial action and selectivity.38 In this context, we observed that the physicochemical modifications in variants R1 and R4 can be directly related to the loss of the hemolytic and cytotoxic activities when compared to their parent peptide. Similar findings were also reported by Irazazabal et al.24, who applied techniques of rational design to modify the sequence of mastoparan-L, generating a variant denominated [I5R8] (INLKILARLAKKIL-NH2). In that study, single substitutions at positions 5 and 8 were performed and resulted in increased positive net charge from +3 in mastoparan-L to +4 in [I5R8], which was described by the authors as the cause for its enhanced antimicrobial activity and lower toxicity against mammalian cells. Moreover, single amino acid substitutions aiming to modulate the hydrophobicity of mastoparanlike peptides have also been shown to interfere in their membrane selectivity and therapeutic potential.39 Diverse mastoparan-like peptides have been reported to have antimicrobial activity, mostly acting against bacteria.37, 40 The variants R1 and R4 described here were mostly active against E. coli, P. aeruginosa and S. aureus planktonic strains (Table 2). Similar findings were also reported by Lin et al.41, who developed six mastoparan-like peptides that potentially inhibited E. coli and S. aureus. Interestingly, other biological activities, including cell degranulation and different degrees of hemolytic properties, were also observed and correlated with the physicochemical parameters of these peptides.41 It was concluded that the higher the mean hydrophobicity of these mastoparan-like peptides the higher their hemolytic effects on erythrocytes from different sources.41 These findings support our data. Although mastoparan-L presented antibacterial properties against E. coli, P. aeruginosa and S. aureus, the higher hydrophobicity and lower hydrophobic moment of this peptide could be related to its hemolytic and cytotoxic effects on mammalian cells. Here, we confirmed that 12


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mastoparan-L exerts hemolytic activity at 100 μM and dose-dependent cytotoxicity against 3T3-L1 and HUVEC cells (IC50 = 12.5 and 50 M). Differently, R1 and R4 variants are non-toxic and caused less than 10% hemolysis at the range of concentrations tested, reinforcing the therapeutic advantages of these variants compared to their parent peptide. Considering the non-selective nature of mastoparan-L in contact with bacterial and healthy human cells, we conducted further studies to evaluate whether this peptide could also present anticancer properties compared to its variants. As observed in our previous biological analysis, the higher hydrophobic character of mastoparan-L, along with its reduced charge and hydrophobic moment, favored its anticancer properties against all cell lines tested from 12.5 to 25 µM; whereas variants R1 and R4 were not effective. These results corroborate those reported by da Silva et al.28, who observed lower IC50 values of glioblastoma viability for the most hydrophobic mastoparan-like peptide tested (i.e. HR1). Moreover, studies have shown that mastoparan peptides present toxic effects on breast, leukemia and myeloma cells.14 Cancerous cells, in comparison to healthy cells, lose their membrane asymmetry, leading to the exposure of negatively charged phosphatidylserine phospholipids on the cell surface.42 This event favors the anchoring of cationic peptides on cancerous cells and may trigger higher toxic effects when compared to those on healthy zwitterionic cells, as observed for mastoparan-L in the present work (Table 2). Mastoparan-L has been described as a membranolytic peptide that adopts -helical arrangements in contact with biological membranes to trigger its membrane-associated activities.31 Therefore, the structure-function relationship of the mastoparan peptides here studied was also investigated. In parallel with previous literature on mastoparan peptides,43 we observed that the helical contents in mastoparan-L, R1 and R4 are correlated with their biological activities (antibacterial, hemolytic and cytotoxic for mastoparan-L; antibacterial for the variants R1 and R4). Based on that, we concluded that not only the higher mastoparan-L hydrophobicity is involved in its poor cell selectivity, but also the adoption of α-helical arrangements in hydrophobic (TFE) and 13



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membrane-like environments (SDS micelles and POPC, POPC/POPG vesicles). On the contrary, both the R1 and R4 variants presented varying levels of helicity only in bacterial membrane-like conditions that, among other factors (e.g., number of peptides that bind and insert into bacteria), may explain their selective and extended activities against bacteria. Here, in addition to the antibacterial properties of mastoparan-L, R1 and R4 against planktonic bacteria, their antibiofilm potential was also evaluated against P. aeruginosa. This bacterium is a Gram-negative, opportunistic pathogen responsible for numerous infections. Moreover, it is known that persistent infections caused by P. aeruginosa are commonly associated with biofilm formation.44,

45

The results obtained by flow cell assays show that both R1 and R4

eradicated two-day-old biofilms at 16 μM. However, mastoparan-L did not present the same potential of eradication, causing a slight dispersion of biofilm cells. The results obtained for mastoparan-L may be explained by its higher hydrophobicity, which has been hypothesized to compromise the translocation of mastoparan peptides through the biofilm matrix and, therefore, reduces their concentration around biofilm-constituting cells.46 Nevertheless, further investigations are needed to support this hypothesis. Flexibility in AMPs has been indicated as a structural determinant for improved and/or extended antibacterial and antibiofilm activities, as well as cell selectivity.47 Here, NMR experiments were performed to investigate the H secondary shifts for all the peptides studied in 30% TFE-d3. Because of its ability to displace water molecules around the peptide, TFE has been used as a co-solvent in the determination of structures and stability of these molecules, favoring the formation of intrapeptide hydrogen bonds.48 The two-dimensional NMR experiments showed that mastoparan-L has -helix signatures from residue 3 to 13; the variant R1 from residue 4 to 13; and R4 from residue 4 to 12. These data corroborate the temperature coefficient experiments, in which mastoparan-L presented a higher number of residues protected from the solvent (higher intrapeptide hydrogen bonding), followed by R4 and R1, respectively. These results suggest different levels of 14


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structural stability/flexibility. Similar studies were reported for mastoparan-X in TFE/water mixtures.49 Some works have suggested that the antimicrobial potency of AMPs may be facilitated by greater conformational flexibility when binding to bacterial membranes.50, 51 Amos et al.50 tested two cationic peptides, pleurocidin (isolated from Pleuronectes americanus) and magainin 2 (isolated from Xenopus laevis), which share approximate absolute values for a number of physicochemical properties. The authors observed that pleurocidin has greater conformational flexibility compared to magainin 2, which was further correlated to the antibacterial potency of that peptide by penetrating into the hydrophobic core of the lipid bilayer. Similarly, works with the AMP carein 1.1 have revealed that its flexible hinge around Pro15 allows the optimal orientation of this peptide into bacterial membranes.52 More recently, Cardoso et al.53 also showed that an helical peptide with flexible termini, as is the case with R1 and R4, presented dual antibacterial and antibiofilm properties. Here, we observed that variants R1 and R4 are more flexible in terms of structure, which may be associated with their higher antibacterial potential against planktonic cells and biofilm. In addition to the in vitro antibacterial data, we also evaluated the anti-infective potential of mastoparan-L, R1 and R4 at 64 µM in a mouse model, where 20 L were added to a cutaneous infection caused by P. aeruginosa. A time-dependent anti-infective potential was observed, as all the peptides tested were capable of reducing the bacterial counts at the site of infection. Brunetti et al.54 used a similar skin scarification model, where mice were infected with P. aeruginosa (P1242), demonstrating a significant reduction in the bacterial counts two days post-infection when treating the mice with the peptide SET-M33L at 10 mg.mL-1 (50 μL based lotion). In addition, previous studies have been performed with the same mouse model and bacteria (P. aeruginosa PA14) as used in our present study. Pane et al.55, for instance, showed that three recombinant peptides, at 50 M, were able to reduce P. aeruginosa counts from 100- to 10,000-times at day four post-infection. Furthermore, Cardoso et al.53 reported that a single dose of the computationally designed peptide 15



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EcDBS1R5, at 64 M, reduced bacterial count in skin infections by 100-times at day two postinfection. Similarly, we observed that a single dose of the most flexible variant (R1), at 64 µM, reduced bacterial counts by 1,000-times at day four post-infection. In this work, we have successfully generated improved mastoparan-like peptides with extended antibacterial properties by using the sequence of mastoparan-L as input for the Joker algorithm. In summary, we characterized the variants R1 and R4 as -helical, flexible peptides with improved antibacterial, antibiofilm and anti-infective potential, when compared to their parent peptide (mastoparan-L). The cytotoxic and hemolytic effects of mastoparan-L were drastically reduced in the designed variants, which is an important feature in drug development strategies.8 These differences in terms of biological activities are related to the changes in the physicochemical properties (increased net charge and hydrophobic moment, and reduced hydrophobicity) in R1 and R4 compared to their parent peptide, along with the greater structural flexibility observed for these two variants. Moreover, considering that -helical structures are usually required for mastoparan peptides to exert their biological activities, we also concluded that the selective antibacterial potential of R1 and R4 might be directly related to their high helical content in bacterial membranelike environments. Taken together, our findings clearly indicate the advantages of using the Joker algorithm to generate improved mastoparan-like peptides, with selective activities toward planktonic bacteria in vitro and in vivo, as well as toward biofilms. Overall, the variants studied here represent a promising starting point for further studies on mastoparan-like peptides and provide new scaffolds for antibacterial drug design strategies.

Experimental section Design of mastoparan variants and molecular modeling We have used the Joker algorithm22 to design the mastoparan variants. This algorithm uses as input a template peptide sequence (a known AMP or not) and an antimicrobial pattern, which 16


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guides the amino acid replacements in the template in a non-cumulative sliding window system. Here we used mastoparan-L (INLKALAALAKKIL-NH2) as the template together with the antimicrobial pattern “K[ILV][AL]x[RKD][ILV]xxKI”. This is the same pattern used in the initial Joker study,22 where amino acid residues between brackets indicate that this position could be filled by one of them and “x” indicates a wild card position, that can be filled by any of the 20 natural amino acid residues. This pattern was previously assembled from a set of patterns retrieved from αhelical antimicrobial peptides,22 where 248 α-helical AMP sequences were initially retrieved from the Antimicrobial Peptides Database (APD),23 and then were submitted to the PRATT pattern discovery tool.56 The pattern “K[ILV][AL]x[RKD][ILV]xxKI” was generated by the visual alignments of patterns B to F,22 whereas pattern A was used to fish for some sequences in the original Joker study.22 This approach generated a number of AMPs, including the recently described PaDBS1R1,57 PaDBS1R658 and EcDBS1R5.53 Thus, combining the mastoparan sequence with this antimicrobial pattern, five mastoparan variants were generated (Table 1). Nonetheless, the fourth variant (INLKILARLAKKIL) presented a complete sequence match with a mastoparan peptide already described in the literature.24 We therefore discarded this variant for further analysis and named the remaining ones mastoparan-R1 to -R4. After the computer-aided design, all variants were submitted to comparative modeling studies using the solution NMR structure of mastoparan-L (parent peptide - PDB ID: 1D7N)31 as a template. All target sequences were individually aligned to the template and further submitted to comparative modeling on MODELLER v.9.20.59 A total of 100 theoretical structures were generated for each variant and ranked according to their free energy scores (DOPE score). The lowest free energy models were validated regarding their stereochemistry and fold quality on PROCHECK60 and ProSA-web,61 respectively. The helical diagram and physicochemical properties were calculated on the HeliQuest server.62

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Solvation potential energy calculation The solvation potential energy calculation was measured for the lowest energy tridimensional theoretical structures generated by molecular modeling. The conversion of .pdb files into .pqr files was performed on the PDB2PQR server using the AMBER force field.63 The grid dimensions for APBS calculation were also determined by PDB2PQR. Solvation potential energy was calculated on APBS.25 Surface visualization was performed using the APBS plugin for PyMOL (Figure S1).

High-throughput peptide synthesis on cellulose arrays A peptide array composed of the mastoparan variants (mastoparan-R1 to -R4) was designed and synthesized by Kinexus Bioinformatics Corporation (Vancouver, BC). Eighty μg of peptides were synthesized by SPOT technology in cellulose support.64 Briefly, cellulose membranes were incubated in an activated glycine solution to achieve a base for peptide synthesis. Peptide synthesis was performed by an F-moc strategy using an F-moc-protected N-terminus and a Cterminal Opfp-activating group, with double coupling at each cycle aiming at higher coupling efficiency at each amino acid position. The cleavage of the side-chain protecting group for 30 min using 90% trifluoroacetic acid, 3% tri-isobutylsilane, 2% water, 1% phenol in dichloromethane. Subsequently, it was incubated for 120-min with 50% trifluoroacetic acid, 3% tri-isobutylsilane, 2% water and 1% phenol in dichloromethane. The peptides were cleaved from the membranes in an ammonia atmosphere overnight, resulting in free peptides. All peptides had amidated C-terminus. Crude peptides released from the membranes were screened for antimicrobial activities using an engineered luminescent P. aeruginosa (H1001) strain, as described bellow.

Screening for antimicrobial activity by bioluminescence assay The antimicrobial activity of variants R1 to R4 was evaluated against an engineered 18


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luminescent P. aeruginosa (H1001) strain in 96-well microplates, as previously described.65 Briefly, all peptides were released from the cellulose spots in aqueous solution and diluted two-fold in 96-well microplates using Basal Medium 2 (BM2) [62 mM potassium phosphate buffer pH 7; 2 mM MgSO4; 10 μM FeSO4; 0.4% (wt/vol) glucose]. Subsequently, 50 μL of overnight culture of P. aeruginosa (H1001) (fliC::luxCDABE) were subcultured in 5 mL of fresh LB media and grown until they reached an OD600 of 0.4. The bacterial culture was diluted 4:100 (v/v) into fresh BM2 media, and 25 μL was transferred to the microplate wells containing 25 μL of peptide solution (concentrations ranging from 128 to 2 μM). The plates were incubated for 4 h at 37 °C with constant shaking at 50 rpm. Luminescence was measured on a TecanSPECTRAFluor Plus Microplate Reader (Tecan US, Morrisville, NC). The antimicrobial activity was evaluated by the ability of the peptides to reduce the luminescence of P. aeruginosa-lux strain compared to untreated cells. The carbapenem meropenem was used as positive control and distilled water was used as a negative control.

Chemical synthesis, UHPLC and mass spectrometry of the parent (mastoparan-L) and the selected variants R1 and R4 After the initial screening for antibacterial activities, variants R1 and R4 were selected and submitted to further studies. In addition, the parent peptide (mastoparan-L) was used as control in all experiments. The peptides mastoparan-L, R1 and R4 were purchased from Peptide 2.0 Incorporated (USA) and Aminotech (Brazil), which synthesized these peptides by F-moc (9fluorenylmethyloxycarbonyl) solid phase methodology with >95% purity. Purity was analyzed by a Nexera UHPLC (LC-30AD, Shimadzu), flow rate of 0.4 mL.min-1 on a 0.8 mL.min-1 Agilent column using a 4% gradient of 0-60% solvent B (90% MeCN in 0.045% aq. TFA). The molecular masses were determined by MALDI-TOF analysis (Ultraflex III type mass spectrometer, Bruker

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Daltonics) in the reflector mode, using the Peptide Calibration Standard II (Bruker Daltonics) as an external calibration (ranging up to 4,000 Da).

Minimal inhibitory concentration assay The MICs for mastoparan-L, R1 and R4 were evaluated against E. coli (ATCC 25922, BL21 and KpC+ 001812446), S. aureus (ATCC 25923 and ATCC 12600), MRSA (clinical isolate 713623), K. pneumoniae (ATCC 13883 and KpC+ 001825971), P. aeruginosa (PAO1 and PA14), A. baumannii (clinical isolate 003326263) and colistin-resistant Enterobacter cloacae (clinical isolate 1383251) strains. The bacterial strains were plated on Muller-Hinton agar plates (MHA) and incubated at 37 °C for approximately 18 h. After this period, three colonies isolated from each bacterium were inoculated in 5 mL of Muller-Hinton broth (MHB) and incubated at 37 °C, overnight, at 200 rpm. Bacterial growth was measured in a spectrophotometer at 600 nm using 100 μL of each bacterial suspension. MIC assays were performed according to the protocol established by the Clinical & Laboratory Standards Institute (CLSI) using the 96-well microplate dilution method.66 Three independent experiments were performed in the microplates at a final bacterial concentration of 2-5 x 105 CFU.mL-1. The peptides were tested at concentrations ranging from 2 to 32 μM. As positive control, chloramphenicol was used at the same concentrations as the peptides, whereas the bacterial suspension (5 x 105 CFU.mL-1) in MHB was used as negative control. The microplates were incubated at 37 °C for 18 h and the readings were performed on a microplate reader at 600 nm after the incubation time.66

Preformed Biofilm Eradication Assay For the treatment of preformed biofilms, strains of P. aeruginosa (PAO1) were cultured in BM2 [62 mM potassium phosphate buffer, pH 7.0, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 M FeSO4] 20


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supplemented with 0.4% (wt/vol) glucose for 24 h. The system was assembled and sterilized with a 10% hypochlorite solution using a multichannel peristaltic pump. The system was rinsed with sterile water and medium for 5 min each. Subsequently, flow chambers were inoculated with PAO1 by injecting 400 μL of an overnight culture diluted to an optical density at 600 nm of approximately 0.5. After biofilm growth had been established, a continuous flow of mastoparan-L, R1 and R4 at 16 µM in BM2 medium was programmed for 24 h. Biofilms were stained with SYTO-9 (green fluorescence, live cells) and propidium iodide (red fluorescence, dead cells). Biofilm eradication was analyzed by confocal microscopy (Zeiss LSM 700 Laser Scanning Confocal), and the threedimensional constructs were generated by the Imaris (Bitplane, AG) computational package. Two independent experiments were performed for each condition.

Cell culture and cell cytotoxicity assays using non-cancerous and cancerous cell lines Healthy cells, including HUVEC and 3T3-L1, were cultivated using EGM™-2 BulletKit™ supplemented with SingleQuots™ (supplements: growth factors, cytokines, antibiotics; Lonza) and 10% BCS/DMEM (catalog no. 12133C; Sigma) with 1% (v/v) penicillin–streptomycin (5000 U.mL-1; Life Technologies), respectively. Cancerous cells, including MCF-7 and HT-29, were cultivated in 10% (v/v) FBS/DMEM with 1% (v/v) penicillin–streptomycin (5000 U.mL-1; Life Technologies); whereas PC-3 cells were cultivated in 10% (v/v) FBS/RPMI 1640 media with 1% (v/v) penicillin – streptomycin (5000 U.mL-1; Life Technologies). All cells were cultured at 37 ºC in 5% CO2. Cytotoxicity assays were performed according to Chan et al.67 Briefly, passages 2–10 were used for all cell lines mentioned above. Final concentrations of 5 x 103 cells per well (100 μL) were used for both cell lines. Cells were allowed to attach for 24 h after plating, and treated with fresh media prior to the addition of mastoparan-L, R1 and R4 (final concentrations ranging from 0.781 to 100 μM per well). Moreover, 0.1% (v/v) Triton X-100 was used as positive control. To evaluate cell viability, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (5 21



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mg.mL-1 in PBS) was added after 2 h incubation. Subsequently, cells were incubated for an additional 4 h. The supernatants were removed, and 100 μL DMSO was added. Three independent experiments were performed. Absorbance was measured at 600 nm using a microplate reader (BioTekPowerWave XS).

Hemolytic Assay Hemolytic assays were performed with human erythrocytes isolated from healthy volunteers. Fresh venous blood was collected and stored in tubes containing phosphate-saline buffer (PBS). Blood samples were centrifuged at 4,000 rpm for 1 min. Thereafter, the supernatant was discarded and the blood cells were washed three times in 1 mL PBS. Further, solutions of 0.25% erythrocytes were prepared in PBS. Peptides were prepared in serial dilutions from an initial stock concentration of 100 μM (highest concentration per well in 96-well microplates). PBS was used as a negative control, whereas 1% Triton X-100 (100% lysis of erythrocytes) was used as a positive control. Assays were performed on 96-well polypropylene plates at 37 °C for 1 h. After that, the plates were centrifuged at 1,000 rpm for 5 min and supernatants (100 μL) were transferred onto 96well flat bottom plates. Absorbance was measured at 415 nm. Three replicates were performed for this assay.

In vivo assay (Scarification skin infection model) The in vivo assay was performed as previously described.53 To generate skin infection, sixweeks-old female CD-1 IGS mice (Swiss) were anesthetized with isoflurane, and the fur on the back of the mouse was shaved. Superficial linear skin abrasions were made to damage the stratum corneum only and upper-layer of the epidermis, but not the dermis. Five minutes after wounding, an aliquot of 50 μL suspensions containing 5 x 107 CFU of P. aeruginosa in PBS was inoculated over wound with a pipette tip. Two days later the peptides (64 M.20 L-1) were inoculated on the same 22


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area. Animals were euthanized and the infected area was excised and homogenized for CFU quantification. Two independent experiments were performed with four mice per group in each condition. Animals were maintained in accordance with institutional guidelines as defined by Institutional Animal Care and Use Committee for U.S. institutions. All the procedures were approved by MIT’s Institutional Animal Care and Use Committee (IACUC), protocol number 1016-064-19. Statistical significance was assessed using a one-way ANOVA, followed by Dunnett’s multiple comparison tests.

Circular Dichroism spectroscopy The secondary structure of mastoparan-L, R1 and R4 at 50 µM was investigated in ultrapure water, 10 mM potassium phosphate buffer with 150 mM NaF (pH 7.4), 30% TFE in water (v/v) and 25 mM SDS, pure POPC and POPC/POPG (1:1). LUVs - peptide-to-lipid ratio was 1:10 molar. Lipid samples were prepared in 10 mM phosphate buffer with 150 mM NaF (pH 7.4) at 500 M. CD spectra of all peptides were performed on a JASCO (J-810) spectropolarimeter coupled to a Peltier Jasco temperature control system. CD spectra were obtained at 25 °C using 0.1 cm path length quartz cells and acquired from 185 to 260 nm at a scan speed of 50 nm.min-1. The resolution was 0.1 nm with a response time of 1 s and five scan accumulations for each sample. Spectra were corrected by subtracting the appropriate blank (buffer and lipid in buffer) and converted to mean residue molar ellipticity. All spectra were smoothed using the Jasco Fast Fourier algorithm and baseline corrected. Helix content for all peptides was calculated from the mean residue ellipticity at 222 nm ([θ]222), as previously described.68

Nuclear Magnetic Resonance (NMR) Spectroscopy NMR spectra were obtained for mastoparan-L, R1 and R4 in 60% H2O, 30% TFE-d3 and 10% D2O (v/v) (pH 4.3), at a final peptide concentration of 1 mM for each sample, according to 23



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Cardoso et al.53. 4,4-Dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as a chemical shift reference for spectral calibration. One-dimensional 1H and two-dimensional spectra (TOCSY and NOESY) were recorded for peptides at 298 K on a Bruker Avance 600 MHz spectrometer, with mixing times of 200–300 ms for NOESY experiments. All spectra were processed using TopSpin 2.1 (Bruker) and assigned using CCPNMR.69 All amino acid spin systems were specifically assigned based on Wüthrich et al.70. The αH secondary shifts were analyzed by subtracting the random coil 1H NMR chemical shifts reported by Wishart et al.71 from our experimental αH chemical shifts. Temperature coefficient experiments were carried out in 70% (v/v) D2O and 30% (v/v) TFE-d3 (pH 4.3). Spectra were recorded from 285 to 310 K, at intervals of 5 K. Coefficients more positive than -4.6 ppb.K-1 were interpreted as the presence of a hydrogen bond, as previously described.32

Corresponding Author Information Dr.

Octávio

L.

Franco

([email protected]).

Dr.

Marlon

H.

Cardoso

([email protected]).

Author Contributions: L.Y.C., C.F.N., D.J.C., O.L.F. and M.H.C. designed the research. W.F.P. performed the computer-aided design of the mastoparan variants. S.M.R. performed the screening for antimicrobial activity. K.G.N.O. and M.H.C. performed the in silico simulations. K.G.N.O., E.S.C., M.D.T.T., B.E.D.M., S.G.R., C.F.N. and M.H.C. performed the antimicrobial assays. M.D.T.T. and C.F.N. performed the flow-cell analysis and in vivo assays. E.S.C., L.Y.C. and M.H.C. performed the hemolytic and cytotoxicity assays. E.S.C., L.Y.C., S.T.H. and M.H.C. performed and analyzed the CD experiments. L.Y.C. and M.H.C. performed the NMR experiments. K.G.N.O., L.Y.C. and

24


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M.H.C. analyzed the NMR experiments. K.G.N.O. and M.H.C. wrote the paper with input from all authors. T.K.L, C.F.N., D.J.C., O.L.F. and M.H.C. supervised the research.

Acknowledgment This work was supported by grants from Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (to M.H.C. 88881.134423/2016-01 and 88887.351521/2019-00), Conselho Nacional de Desenvolvimento e Tecnológico (CNPq) (to M.H.C. 141518/2015-4) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT), Brazil, Ramon Areces Foundation (to CFN), DTRA (DTRA HDTRA1-15-1-0050) (to TKL), Fundação de Amparo à Pesquisa do Estado de São Paulo (MDTT # 2014/04507-5 and 2016/24413-0). The Translational Research Institute is supported by a grant from the Australian Government. S.T.H. is an Australian Research Council (ARC) Future Fellow (FT150100398). D.J.C. is an ARC Australian Laureate Fellow (FL150100146). L.Y.C. was supported by the Advance Queensland Women’s Academic Fund (WAF-6884942288). We acknowledge Dr. Evan F. Haney and Dr. Robert E. W. Hancock from the Centre for Microbial Diseases and Immunity Research, University of British Columbia, Canada, for their support with the SPOT synthesis of mastoparan-R1 to R4. We thank Robin Kramer for her assistance with the skin scarification mouse model.

Abbreviations used: ESKAPE,

(Enterococcus

faecium, Staphylococcus

aureus, Klebsiella

pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species); WHO, world health organization; AMP, antimicrobial peptide; UHPLC, ultra-high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization - time of flight; QSAR, quantitative structure–activity relationship; CD, circular dichroism; NMR, nuclear magnetic 25



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resonance; PDB, protein data bank; APBS, Adaptive Poisson-Boltzmann Solver; KpC, Klebsiella pneumoniae Carbapenemase; HUVEC, Human umbilical vein endothelial cells; 3T3-L1, mouse adipocytes; TFE, 2,2,2-trifluoroethanol; SDS, sodium dodecyl sulfate; LUVs, large unilamellar vesicles; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoylsn-glycero-3-phospho-(1′–sn-glycerol); DSS, 4,4-Dimethyl-4-silapentane-1-sulfonic acid; MIC, minimal inhibitory concentration.

Competing financial interest statement: The authors declare no competing financial interests. T.K.L is a co-founder of Senti Biosciences, Synlogic, Engine Biosciences, Tango Therapeutics, Corvium, BiomX, and Eligo Biosciences. T.K.L. also holds financial interests in nest.bio, Ampliphi, IndieBio, and MedicusTek.

Atomic coordinates for the mastoparan theoretical models: Authors will release the atomic coordinates and experimental data upon article publication (Supporting information).

Supporting information Table S1, listing the fold quality and stereochemical validations for all theoretical models; Tables S1, S2 and S3, listing the secondary chemical shifts for mastoparan-L, R1 and R5 in the 30% TFE/water; Figure S1, showing the helical wheel diagrams and tridimensional theoretical models for mastoparan-R1 to -R4; Figure S2, S3 and S4, showing the UHPLC/MALDI-TOF analyses for mastoparan-L, R1 and R4.

26


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35



Page 36 of 47

Figure legends Figure 1. Computer-aided design of mastoparan-L variants. Four novel mastoparan variants were

generated

using

the

Joker

algorithm

according

to

the

-helical

pattern

“K[ILV][AL]x[RKD][ILV]xxKI” and using the mastoparan-L sequence as a template. Highlighted residues in yellow correspond to residues modified in variants R1 to R4 in comparison to mastoparan-L. *The designed sequence (INLKILARLAKKIL) presented a complete match with a previously described mastoparan ([I5R8])24 and, for this fact, it was discarded from the present study.

Figure 2. Two-day-old P. aeruginosa (PAO1) biofilms in the absence (control) and presence of mastoparan-L, R1 and R4. Preformed biofilm of P. aeruginosa prior to treatment (control) (a) and treated (16 M) with mastoparan-L (b), R1 (c) and R4 (d). Scale bar = 20 m.

Figure 3. Anti-infective properties of mastoparan-L, R1 and R4 (64 M) on P. aeruginosa (PA14) skin infections. The anti-infective potential was evaluated on six-weeks-old female CD-1 mice two and four days post-infection using 64 M of each peptide in the site of infection. Two independent experiments were performed with four mice per group in each condition. Statistical significance was assessed using one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p>0.001. Error bars represent standard deviation values.

Figure 4. Circular dichroism analysis of the peptides mastoparan-L (a), R1 (b) and R4 (c). CD spectra were recorded in ultrapure water, 10 mM potassium phosphate buffer, 150 mM sodium fluoride (pH 7.4), 30% TFE in water (v/v), 25 mM SDS and 500 M of pure POPC and POPC/POPG (1:1) LUVs.

36


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Figure 5. Secondary chemical shifts and temperature coefficient analysis. Mastoparan-L, R1 and R4 αH secondary chemical shifts were determined from 1H NMR spectra at 298 K in 60% (v/v) H2O, 30% (v/v) TFE-d3 and 10% (v/v) D2O (a). Amide proton chemical shifts in temperature coefficient experiments were obtained from 285 to 310 K in 70% (v/v) D2O and 30% (v/v) TFE-d3 (b). Temperature coefficient values more positive than -4.6 ppb.K-1 (dashed line) were interpreted as the presence of a hydrogen bond.32 Asterisks indicate conserved residues between all sequences.

37



Page 38 of 47

Tables Table 1. Screening for antibacterial activities against bioluminescent P. aeruginosa (H1001) and physicochemical properties for the mastoparan variants. Physicochemical propertiesb Peptide

Sequencea

MIC

Net

Hydrophobicity

Hydrophobic

(µM)

charge

(%)

moment72

mastoparan-L

INLKALAALAKKIL

-

+3

57.6

0.398

mastoparan-R1

KILKRLAAKIKKIL

4

+6

36.9

0.775

mastoparan-R2

IKLLARIALKIKIL

30

+4

76.0

0.365

mastoparan-R3

INKIALRILAKIIL

30

+3

79.5

0.387

[I5R8]c

INLKILARLAKKIL

-

+4

58.9

0.471

mastoparan-R4

INLKKLAARIKKKI

4

+6

20.4

0.472

aResidues

modified in the variants R1 to R4 in comparison to mastoparan-L are in bold type. bThe

physicochemical properties were calculated on the Heliquest server.62 cThe designed sequence (INLKILARLAKKIL) presented a complete match with a previously described mastoparan ([I5R8])24 and, for this fact, it was discarded from the present study.

38


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Table 2. Antibacterial, cytotoxic and hemolytic properties for mastoparan-L and its variants (μM). Bacterial strains (MICa)

mastoparan-L

mastoparan-R1

mastoparan-R4

(µM)

(µM)

(µM)

A. baumannii (clinical isolate 003326263)

>32

>32

>32

E. cloacae (clinical isolate 1383251)

>32

>32

>32

E. coli (ATCC 25922)

32

4

4

E. coli (KpC+ 001812446)

>32

8

8

2

2

2

K. pneumoniae (ATCC 13883)

>32

8

>32

K. pneumoniae (KpC+ 001825971)

>32

>32

>32

S. aureus (ATCC 25923)

>32

>32

8

S. aureus (ATCC 12600)

8

4

4

>32

>32

8

P. aeruginosa PA14

8

4

4

P. aeruginosa PAO1

8

4

4

3T3-L1

12.5

>100

>100

HUVEC

50

>100

>100

Human prostate cancer, PC-3

12.5

>100

>100

Human breast cancer, MCF-7

25

>100

>100

Human colon adenocarcinoma, HT-29

25

>100

>100

45

3

8

E. coli (BL21)

MRSA (clinical isolate 713623)

Cell lines (IC50b)

Cell type (hemolysis (%) at 100 M)* Human red blood cells aMIC:

minimum inhibitory concentration; bIC50: half maximal inhibitory concentration.

*: % hemolysis 39



Page 40 of 47

Table 3. Calculated percentage of helicity for mastoparan-L, R1 and R4 in different mimetic conditions. Water

10 mM KH2PO4, 150 mM NaF, pH 7.4

30% TFE

25 mM SDS

POPC

POPC/POPG (1:1)

Mastoparan-L

28.6

55.2

97.3

100

61.3

87.6

Mastoparan-R1

5.5

3.4

55.5

72.2

7.6

90.4

Mastoparan-R4

4.8

8.2

46.7

65.6

13.3

45.4

Helix content (%) for all peptides was calculated from the mean residue ellipticity at 222 nm ([θ]222), as previously described.68

40


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Figure 1.

41



Figure 2.

42


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Figure 3.

43



Figure 4.

44


Page 44 of 47

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Figure 5.

45



46


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Table of Contents Graphic

47


Computer-Aided Design of Mastoparan-like Peptides Enables the

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