International Fundation forBiotechnology Research &Early Stimulation in theCulture of Health, Nutrition,Sport, Art, Science,Technology & Society, A.C.

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Fabián Fernández–Luqueño

Fernando López–Valdez

Susana Lozano-Muñiz

Editors:

Web VersionISBN: 978-607-9023-25-6

Appropriate form to cite the articles of this compendious (example): Híjar-Soto LE, González-Horta C, Chávez-Flores D, Torres-Muñoz JV, Sánchez-Ramírez B (2014) Antineoplastic potential of bioactive fractions of Rhus trilobata on colon cancer cells CaCo-2. In: Fernández-Luqueño F, López-Valdez F, Lozano-Muñiz S. (Eds.). Biotechnology Summit 2014. Cinvestav, México. pp. 2-6. This compendious of Biotechnology Summit 2014 is property and responsibility of Authors. All or any part of this publication may be reproduced or transmitted, by any means, electronic or mechanical (Including photocopying, recording or any recovery system and storage), and must be included with the corresponding citation of this compendious and their authors. International Foundation for Biotechnology Research & Early Stimulation in the Culture of Health, Nutrition, Sport, Art, Science, Technology & Society A.C., Non-profit Organization (IFBRESCHNSASTSAC). D.R. © 1st Edition Centro de Investigación y de Estudios Avanzados del I.P.N. Cinvestav, 2014 Publisher Cinvestav, AV. I.P.N. 2508 07360, MÉXICO, D.F. Web version ISBN: 978-607-9023-25-6

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Design of short alpha-helical peptides and their activity against pathogenic bacteria Mycobacterium tuberculosis

Alexis J. Rodríguez-Solís1,2, Elba C. Villegas-Villarreal1, Alejandra Montoya-Rosales3,

Bruno Rivas-Santiago3, Gerardo A. Corzo-Burguete1

1Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad

Nacional Autónoma de México, Cuernavaca Morelos, México, 2Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México,

3Medical Research Unit-Zacatecas, Mexican Institute of Social Security, UIMZ-IMSS, Zacatecas, Mexico. corzo@ibt.unam.mx

Abstract: The contention of multidrug-resistant bacteria requires the use of new antibiotics. Pandinin 2 (Pin2) is a highly hemolytic antimicrobial peptide that has a central proline residue. Proline forms a structural ‘‘kink’’ linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked by Glycine residues according to the low hemolytic activities of antimicrobial peptides as Magainins and Ponericins, respectively. Both Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was 30% less hemolytic. To avoid the drawback associated to the cost of synthesis of large peptides, two short peptides were designed and synthesized based on Pin2 [G] and Pin2 [GPG]. Both Pin2 [14] and Pin2 [17] short variants, showed antibiotic activities against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis at 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides showed better activity at molar concentrations against multidrug resistant M. tuberculosis respect to conventional antibiotics. Pin2 [14] and Pin2 [17] have the potential to be used as alternative antibiotics with reduced hemolytic effects. Keywords: antimicrobial peptides ● Mycobacterium tuberculosis ● hemolytic activities Introduction: Cationic Antimicrobial Peptides (CAMPs) are components of the biological defense system of microorganisms, plants, animals and humans (Bulet et al., 2004). CAMPs with alpha-helical conformation share some common characteristics such as broad-spectrum antimicrobial activities at low micromolar concentrations and alpha-helix conformation in hydrophobic environments (Nguyen et al., 2011). They have potent antibacterial activities that made them promissory candidates to develop novel antibiotics towards multidrug resistant pathogenic bacteria, as well as towards clinically important yeasts such as Candida albicans (Park et al., 2011; Yeung et al., 2011). Pin2 is a 24-residue alpha-helical antimicrobial peptide characterized from the venom of the African scorpion Pandinus imperator, this peptide has broad spectrum antimicrobial activities in the micromolar range; however, it shows hemolytic activity at similar concentrations (Corzo et al., 2001). Pin2 shows an alpha-helical with a central proline “kink” structure. The proline ‘‘kink’’ is a structural characteristic of some CAMPs, such as Alamethicin, Melittin and Pardaxin, that confer them high pore-forming abilities but also a high hemolytic activities (Dathe et al., 1998; Dempsey et al., 1991). On the other hand, CAMPs such as Magainin 2, Oxypinin 2b and Ponericin G1, showed antimicrobial activities with low cytotoxic effects towards erythrocytes (Corzo et al., 2002; Orivel

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et al., 2001; Zasloff, 1987). These peptides have different amino acid motifs in the central region of their primary structures. For example, Magainin 2 has a single glycine in the middle of its structure, Oxypinin 2b (Oxki2b) has a GlyValGly motif, and Ponericin G has glycine residues flanking the central proline, a GlyProGly motif. Likewise, the substitution of the proline residue (P14) in Pin2 for the residues Val, GlyVal, ValGly or GlyValGly reduced the hemolytic activity of Pin2 without any significant changes in its antimicrobial activity (Rodriguez et al., 2011). In this work, based on the low hemolytic activities shown by the antimicrobial peptides Magainin 2 and Ponericin G1, two synthetic variants of Pin2, Pin2 [G] and Pin2 [GPG] were chemically synthesized with the aim to reduce the hemolytic activity and preserve the antibiotic activities of Pin2. In addition, two short variants of Pin2, with 14 and 17 residues, respectively, were designed and chemically synthesized with the aim to continue reducing their hemolytic but keeping their antimicrobial activities as well as to reduce the number of residues to have a low cost CAMP. Here these antimicrobial peptide variants are proposed as potential antibiotics for the clinical treatment of pathogenic bacteria including Mycobacterium tuberculosis. Materials and Methods: Bacterial strains and peptides. The bacterial strains used in this report were Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Mycobacterium tuberculosis H37Rv (ATCC 27294) and M. tuberculosis multidrug resistant strain (MDR). Pin2 and its variants were chemically synthesized by solid phase method using the Fmoc methodology. Each crude synthetic peptide was then purified by reverse phase HPLC (RP-HPLC) on an analytical C18 column. The final purity of the peptides was higher than 95%. Antimicrobial assays. Minimal inhibitory concentrations (MIC) and bacterial growth inhibition curves were obtained using pure peptides, at different concentrations, in the presence of bacteria using two different methods, agar diffusion susceptibility assays and broth microdilution assays in accordance to the procedures from the Clinical and Laboratory Standards Institute (CLSI, http://www.clsi.org). Mycobacterium tuberculosis assays. The Resazurin microtitre assay plate (REMA) method was conducted to determine the M. tuberculosis susceptibility to the action of Pin2 and the variants studied in this work. Resazurin is an oxidation–reduction indicator and has been used to assess viability, bacterial contamination and to test for antimicrobial activity. The M. tuberculosis H37Rv (ATCC 27294) and a clinically isolated multidrug resistant (MDR) strain were used in these experiments. Hemolytic assays. Hemolytic activity was determined by incubating suspensions of human red blood cells with serial dilutions of each selected peptides. Red blood cells were counted by a hemocytometer and adjusted to 7.7x106 cells mL-1. Red blood cells were then incubated at room temperature for 1 h in 10% Triton X-100 (positive control), in PBS (blank), or with amphipathic peptides at concentrations of 0.4, 0.8, 1.6, 3.1, 6.2, 12.5 and 25 µM, only for Pin2 [14] and Pin2 [17] the 50 and 100 µM concentrations were evaluated. Circular Dichroism (CD) measurements. The CD experiments were recorded on a Jasco model J-720 spectropolarimeter (Tokyo, Japan). The different spectra were measured from 260 to 190 nm on samples in water, 20, 40 and 60% trifluoroethanol (TFE), at room temperature, with a 1-

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mm pathlength cell. Data were collected at 1 nm with a scan rate of 100 nm min-1 and a time constant of 0.5 s. The concentration of each peptide was 150 µg mL-1 Data were the average of five separate recordings and were analyzed on line by the software K2d. Results and Discussion: Solid phase antimicrobial activities. The antimicrobial activity of Pin2 and its variants were tested in solid phase against S. aureus and E. coli. Pin2 [G] variant displayed the highest antimicrobial activity with a MIC value of 12.5 µM, towards both S. aureus and E. coli. Both parental Pin2 and Pin2 [GPG] showed MIC values of 37.5 and 25 µM for S. aureus, and 18.8 and 25 µM for E. coli, respectively. However, the short variants Pin2 [14] and Pin2 [17] had MIC values >300 and 80 µM towards S. aureus, respectively, but they both Pin2 [14] and Pin2 [17] were more active towards E. coli with MIC values of 25 µM. Broth culture antimicrobial activities. The bacterial growth of both S. aureus and E. coli was observed in the presence of serial concentrations of all Pin2 variants from 25 to 0.4 µM. Most of the synthetic peptides showed bactericidal or bacteriostatic antimicrobial activity against the two strains with MIC values of 12.5 and 25 µM. Pin2 [G] and Pin2 [GPG] showed MIC values of 12.5 µM for both E. coli and S. aureus. The short variant Pin2 [14] had a MIC value of 25 µM against E. coli but a bacteriostatic effect at 25 µM against S. aureus. Finally, the short variant Pin2 [17] only showed bacteriostatic effect on E. coli at 25 µM, and no antimicrobial activity towards S. aureus was observed (Table 1).

Table 1. Antimicrobial and hemolytic activities of Pin2 and the Pin2 variants.

M. tuberculosis activity. The antibiotic capacities of Pin2 variants were tested in two strains of M. tuberculosis using the REMA methodology. All synthetic peptide variants showed antimicrobial activities over both M. tuberculosis strains (Table 2). The MIC values observed were from 11 to 30 µM for the M. tuberculosis H37Rv and from 6 to 33 µM for M. tuberculosis MDR. It was interesting to observe that the two short variants Pin2 [14] and Pin2 [17] had MIC values of 11.9

Minimum Inhibitory Concentrations (µM) Peptide name Assay S. aureus E. coli Hemolysis (IC50)‡ Pin2 MHA 37.5 18.8 3.3 [1.9-5.7] MHB 12.5 12.5 Pin2 [G] MHA 12.5 12.5 1.4 [0.4-4.3] MHB 6.25 12.5 Pin2 [GPG] MHA 25 25 46.6 [34-64]* MHB 25 25 Pin2 [14] MHA >300 25 418.4 [291-602]* MHB 50 25 Pin2 [17] MHA 80 25 ND§ MHB NA >25† MHA, Mueller-Hinton Agar; MHB: Mueller Hinton Broth; ‡ Mean and 95% confidence intervals, values expressed in µM; *The numeric IC50 value was obtained from the Boltzmann sigmoid equation fit; † Bacteriostatic effect; NA Non Activity observed; § Any hemolytic effect at 100 µM was observed because of that the value of IC50 did not calculate.

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and 11.6 µM towards M. tuberculosis H37Rv and MIC values of 6 and 14.8 µM against M. tuberculosis MDR, respectively.

Table 2. Antimicrobial activity of Pin2 and the Pin2 variants on two M. tuberculosis strains. Mycobacterium tuberculosis

H37Rv Mycobacterium tuberculosis

MDR* Peptide name

MW (Da) MIC (µg mL-1) MIC (µM) MIC (µg mL-1) MIC (µM)

Pin2 2612.1 57.7±22.3 22.1±8.6 86.5 33.1 Pin2 [G] 2572.0 48.1 18.7 48.1 18.7 Pin2 [GPG] 2762.2 80.1±24.8 29±9 48.1 17.4 Pin2 [14] 1680.1 20 ± 6.2 11.92±3.7 10± 3.1 6±1.8 Pin2 [17] 1891.3 22 ± 4.9 11.65±2.59 28.04 ± 9.814 14.8±5.2 Ethambutol§ 204.3 0.5 2.5 20 97 Isoniazid 137.1 24 ±8.8 175.1±63.9 6 ±2.2 43.8±16 Rifampicin§ 823.0 0.4 0.5 32 38.9 The MIC values were calculated using the Resazurin dye reduction method, 500,000 bacteria per well were evaluated; §, MIC values reported by Rastogi, et al., (1996); *, Clinically isolated strain characterized with resistance to rifampicin and isoniazid in UIMZ-IMSS, Zacatecas, Mexico.

Hemolytic activity of Pin2 variants. The hemolytic assays of Pin2 and the Pin2 variants on human erythrocytes (Figure 1) showed that the parental peptides Pin2 as the Pin2 [G] variant at 25 µM had the highest hemolytic activities of all five peptides evaluated, with a 91% and a 100% of hemolysis, respectively. However at the same concentration the Pin2 [GPG] variant showed only a 30% of hemolysis, indicating that the insertion of the GPG motif is relevant for hemolytic activity reduction. The two short variants had not hemolytic effects at 25 µM. That is, they were assayed up to 100 µM observing that the peptide Pin2 [14] showed only 25% of hemolysis, while Pin2 [17] did not show any hemolytic effect at such concentration.

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Figure 1. Hemolytic activity in human red blood cells. Data are the average of at least four

independent experiments. Error bars represent the standard deviations. Circular dichroism secondary structure analysis. The CD spectrum of Pin2 was compared to the CD spectra of the Pin2 [G] and Pin2 [GPG] variants. The secondary structure analysis was performed using CD spectral data from 190 to 260 nm, in the presence of 60% aqueous TFE, the CD spectra of Pin2 [G] and Pin2 [GPG] showed a clear ordered structure with two minimum ellipticity values at 208 and 222 nm (Figure 2A), indicating an alpha-helix conformation. In order to obtain more information concerning the propensity of these peptides to adopt alpha-helical structures, their CD spectra at different TFE proportions (0, 20, 40 and 60%) was acquired. The parental peptide Pin2 (Figure 2B) and the Pin2 [GPG] (Figure 2D) variant showed a clear ‘‘random coil’’ CD profile in the absence of TFE, but interestingly, the Pin2 [G] variant showed a 20% of alpha-helical structure in the absence of TFE (Figure 3C), suggesting a more structured Pin2 [G]. Pin2 [G] and Pin2 [GPG] adopted a maximum of 60% of alpha-helical structure at 40% TFE. Similarly, the CD spectra of the short variants Pin2 [14] and Pin2 [17] were conducted at 0, 20, 40 and 60% TFE (Figures 2E and 2F). In the absence of TFE both Pin2 [14] and Pin2 [17] had clear positive ellipticities around 220 nm, and the increment in the TFE proportion induced a reduction in their ellipticity values. Furthermore, in the absence of TFE Pin2 [14] showed a strong negative ellipticity at 205 nm, and the increase of TFE enhanced its CD ellipticity value. Similar behavior was observed for Pin2 [17]. The CD deconvolution for Pin2 [14] and Pin2 [17] resulted in unstructured peptides even at the highest TFE concentration of 60%.

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Figure 2. Circular dichroism Pin2 and its variants. A. Circular dichroism spectra of Pin2 and its variants in 60% TFE, B. Pin2 TFE alpha-helix induction kinetics, C. Pin2 [G] TFE alpha-helix

induction kinetics, D. Pin2 [GPG] TFE alpha-helix induction kinetics, Pin2 [14] TFE alpha-helix induction kinetics and Pin2 [17] TFE alpha-helix induction kinetics.

The differences among the antimicrobial activities of the different peptides reported here are first related to the length of to peptides, as was observed by Deslouches et al. (2005), in which peptides with short sequences display lower antimicrobial activities, observing that for their study a 24 amino acid length is optimal for antimicrobial activity. Respect to the selectivity, in the same study

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short peptides showed better activity towards Gram negative bacteria, indicating an effect of the thickness of the cell wall of Gram positive bacteria. As was observed here, short peptides displayed lower antimicrobial activities towards Gram positive S. aureus. In other study, Liu et al. (2007) observed that the antimicrobial activity of a peptide series was increasing according to length of the peptides observing the lower MIC values with peptides with larger peptides. Here, the same profile is observed in both solid phase and broth culture assays. The lower MIC values observed in the solid phase respect to the broth culture assay could be related to a limited diffusion through the agar media primarily because of the hydrophobic nature of the peptides. Similar results were observed for Nisin an antimicrobial peptide produced by Lactococcus lactis (Chandrapati and O’Sullivan, 1998). Respect to the antimicrobial activity towards M. tuberculosis, better molar antimicrobial activities were observed with the short variants, respect to the other CAMPs and conventional antibiotics evaluated here, especially with the MDR strain. These short peptides could be evaluated in combination with commercial antibiotics as Rifampicin to increase the affectivity of these antibiotics over clinically important Mycobacterium strains, in especial those with antibiotic resistance (Khara et al., 2014). The antimicrobial potency and hemolytic profile of CAMPs are closely related to the alpha-helical structure (Jiang et al., 2014; Ramón-García et al., 2013). So that, the peptides with higher helical profiles have lower MIC values but also they become more hemolytic. The hemolysis reduction observed in the Pin2 [GPG], Pin2 [14] and Pin2 [17], could be related to the DC profiles observed here. Pin 2 [G] has a higher helical profile respect to the other peptides and it is more hemolytic. While the short peptides, they do not show an alpha-helical profile, displaying low or none hemolytic effects. Conclusions: Here we based the design of low hemolytic and short antimicrobial peptides on observed patterns in nature and on theoretical calculations. We found a strong correlation in hydrophobicity and alpha-structured molecules with high hemolytic activity; however, the antimicrobial capacity could be sustained with low eukaryotic lytic activities in short hydrophilic antimicrobial peptides such that Pin2 [14] and Pin2 [17] with a plus of maintaining a wider antimicrobial spectrum; that is affecting Gram-positive and Gram-negative bacteria as well as multi-drug resistant Mycobacterium tuberculosis. References: Bulet P, Stocklin R, Menin L 2004. Anti-microbial peptides: from invertebrates to vertebrates.

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