Butelase-Mediated Ligation as an Efficient Bioconjugation Method for

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Butelase-mediated ligation as an efficient bioconjugation method for the synthesis of peptide dendrimers yuan cao, Giang K. T. Nguyen, samuel Chuah, James P. Tam, and Chuan-Fa Liu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00538 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 15, 2016

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Butelase-mediated ligation as an efficient bioconjugation method for the synthesis of peptide dendrimers Yuan Cao, Giang K T Nguyen, Samuel Chuah, James P Tam* and Chuan-Fa Liu* School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 Supporting information ABSTRACT: Herein we report a novel enzymatic bioconjugation method to prepare peptide dendrimers. Under the catalysis of a newly discovered peptide ligase, butelase 1, peptide dendrimers of di-, tetra- and octabranches were successfully synthesized using thiodepsipeptides as acyl donors for ligation with lysyl dendrimeric scaffolds. The efficient assembly of the highly clustered dendrimeric structure highlighted the versatility of butelase 1. We also showed that our synthetic antibacterial peptide dendrimers containing an RLYR motif are highly potent and broadly active against antibiotic-resistant strains.

Bioconjugation provides essential tools to manipulate biomolecules such as peptides/proteins, nucleic acids and carbohydrates under mild conditions. Current bioconjugation toolkits consist of various chemical and enzymatic strategies that allow for sitespecific modification of biomolecules with a biophysical probe, a bioactive cargo, a synthetic polymer or other moieties for numerous applications in biology, medicine and materials science1-3. For example, chemical strategies for site-selective protein conjugation4, 5 include targeting a natural or non-natural amino acid side chain with orthogonal reactivity,6-10 N-terminus-directed tagging11-14 and other emerging methods15-16.These strategies can overcome the limitations associated with classic chemistries that modify internal Lys, Asp/Glu or other residues which often exist in multiple copies in the substrate protein3. Enzymatic methods offer attractive bioconjugation solutions due to their high reaction specificities and mild operating conditions17,18. In particular, peptide ligases, enzymes that catalyse the formation of peptide bonds, provide exciting opportunities for peptide and protein conjugation. The ligases are ideal to prepare peptide/protein bioconjugates through N- or C-terminal attachment19. Previously known peptide ligases include sortase A20 and subtiligase21. Recently, a new peptide ligase, namely butelase 1, was isolated from a cyclotideproducing plant Clitoria ternatea22. Butelase 1 is the enzyme responsible for the macrocyclization of cliotides, cyclotides from C. ternatea, during their biosynthesis and recognizes a linear precursor with a C-terminal tripeptide motif Asn/Asp(Asx)-His-Val. It cleaves the bond between Asx and His to accept an N-terminal residue Xaa as the incoming nucleophile, resulting in a new AsxXaa bond in the cyclized peptide. Butelase 1 exhibits not only the highest catalytic kinetics among all the peptide ligases found so far, but also a broad substrate specificity for the N-terminal amino acid, Xaa, which can be any amino acid except Pro, making it an attractive tool for bioconjugation and peptide ligation23-25. Peptide dendrimers are a type of synthetic bioconjugates26. Multiple antigen peptide system (MAP), a strategy to present multiple functional peptides in a clustered dendrimeric format, was first developed by Tam to amplify the immunogenicity of small antigenic peptides27, 28. This chemical platform has also found wide applications in peptide-based therapeutics and biomaterials26. A common MAP design contains a scaffold, a branching oligolysine dendron core, to which various copies of a functional peptide are attached. Often, stepwise solid phase peptide synthesis (SPPS) or a convergent synthetic strategy is used to prepare such peptide dendrimers29. For many applications, stepwise SPPS suffices; however, as the size of a MAP increases, the likelihood of synthetic errors such as residue deletion also increases, making purification a challenge to arrive at a homogeneous product from a synthetic mixture. Although this problem could be partially mitigated by an optimized SPPS methodology, increased attention has

been focused on employing a convergent strategy in which the dendron core and the antigenic peptides are separately synthesized and purified to homogeneity and then brought together using chemoselective conjugation reactions. Conjugation chemistries developed thus far include disulfide30, thioether31, thiazolidine32, oxime33, hydrazone34, maleimide35 and native chemical ligation36. Compared with the stepwise SPPS method, the convergent strategy often gives higher yield and greater purity of the final MAP products.

A) Butelase-mediated ligation using native peptide as acyl donor X1

Xn Asn

Y1 Y2

O

R

HN

+

Val

O

Y1 Y2

K

Butelase 1 X1

Xn Asn Y1 Y2

X1

Xn

R

Asn Y1 Y2

K

H2N

+

Val O

(substrate of butelase 1) B) Butelase-mediated ligation using thiodepsipeptide as acyl donor X1

Xn Asn

O

Y1 Y2

R

S O

+

Val

Y1 Y2

K

Butelase 1

X1 X1

Xn Asn Y1 Y2 Xn Asn Y1 Y2

K

+

R HS

Val O

(non-substrate of butelase 1)

Scheme 1. Butelase-mediated peptide dendrimer synthesis (bivalent format as example), using A) native peptides and B) thiodepsipeptides as acyl donor substrates.

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We envisioned that, with its exquisite site-specificity and site selectivity, butelase 1 would be useful for peptide dendrimer synthesis whereby a peptide as an acyl donor could be ligated to a lysyl dendron core containing appropriate N-terminal acceptor amino acids to form a multi-valent dendrimeric product (Scheme 1 and Figure 1). An enzymatic synthesis of a highly clustered peptide dendrimer (such as the octavalent one) would present a stringent test of butelase-mediated ligation (BML) for bioconjugation and help to advance the development of peptide-based vaccines and therapeutics. B)

A)

Peptide

Peptide

Peptide Lys

Peptide

Tyr Peptide

Lys Lys

Lys

Tyr

Peptide

C) Peptide Lys

Peptide Peptide Peptide

Lys Lys

Peptide

Lys Lys

Lys

Tyr

Peptide Lys

Peptide Peptide

Peptide

βAla = Ac-Arg-Tyr-Arg-Leu-Asn-Arg-Ile-β

Figure 1. Schematic presentation of selected peptide dendrimer structures assembled by butelase-mediated bioconjugation. A) bivalent peptide dendrimer 3; B) tetravalent peptide dendrimer 8; C) octavalent peptide dendrimer 9.

Table 1. Sequences of the peptides in this study. Numbering in this table is used throughout the paper.

Number 1 2 3 4 5 6 7 8 9 10 11 12 13

Sequence Ac-RYRLN-thioglc-V (RIβA)2KY (Ac-RYRLNRIβA)2KY RYRLNHV (RYRLNRIβA)2KY (RIβA)4K2KY (RIβA)8 K4K2KY (Ac-RYRLNRIβA)4K2KY (Ac-RYRLNRIβA)8K4K2KY Ac-RLYRN-thioglc-V RLYR (Ac-RLYRNRIβA)2KY (Ac-RLYRNRIβA)4K2KY

Figure 2. Analytical HPLC monitoring of butelase-mediated peptide dendrimer bioconjugation using A) thiodepsipeptide – peak a: peptide 1; peak b: desired bivalent product 3 with an observed mass of 2478.5 (calc. 2478.4 Da) or B) normal peptide – peak a’: peptide 4; peak b’: dendron core 2; peak c’: the monovalent product; peak d’: the desired bivalent product 5.

We started with a trial reaction in which a conventional peptide substrate containing the C-terminal -NHV motif (peptide 4, Table 1) was used to bioconjugate to a bivalent lysyl dendron core (entry 2, Table 1), we observed a sluggish reaction even with an excess of the monomeric peptide 4 and in a prolonged period. We attributed this poor outcome to the reversibility of the BML reaction because the released dipeptide His-Val acts as a competing nucleophile (Scheme 1A). Prompted by this observation, we sought to use thiodepsipeptides as acyl donors for peptide dendrimer synthesis to overcome the reversibility issue. We have previously shown that thiodepsipeptides are superior substrates for butelase-mediated Nterminal labeling of proteins24. The use of thiodepsipeptide has two advantages. First, it contains a thioester linkage as the scissile bond which is more susceptible to enzymatic cleavage compared to its amide bond counterpart. Second, after the reaction, it releases a thiol byproduct which, unlike its native dipeptide counterpart, is a poor acyl-acceptor substrate of butelase 1, essentially rendering the BML reaction irreversible (Scheme 1B). To test our hypothesis, we first conducted a bioconjugation between an Nacetylated thiodepsipeptide 1 (Table 1) and the bivalent lysyl

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scaffold 2. The N-terminus of the thiodepsipeptide was acetylated because we found self-ligation of N-terminus-free thiodepsipeptide as a minor side reaction in our preliminary study (Figure S1) possibly due to high reactivity of the thioester. Thiodepsipeptide 1 was conveniently prepared by Boc chemistry as previously reported24. Using HPLC, we evaluated the ligation reaction of 1 with 2 in forming the bivalent product 3 (Figure 1A). The reaction was performed in the presence of 50 µM scaffold 2, 150 µM thiodepsipeptide 1 (1.5 equiv. per branch), 100 nM butelase 1 (0.001 equiv. per branch), 1 mM TCEP, pH 6.5 phosphate buffer at 42 o C. The dendron core was consumed in 30 min and the desired product 3 was formed in > 95% yield based on HPLC analysis (Figure 2A). This is in strong contrast to the sluggish ligation between the native peptide 4 which contains the –NHV motif and the scaffold 2. Under similar conditions, >50% of the starting materials were not consumed after 2h (Figure 2B). HPLC analysis showed that the monovalent product was the major product (peak c’ in Figure 2B) whereas the desired bivalent product was a minor product (peak d’ in Figure 2B). Side reactions due to self-ligation of the unacetylated peptide 4 were not observed.

Figure 3. Analytical HPLC monitoring of butelase-mediated preparation of (A) tetravalent dendrimer – peak a: linear peptide 1; peak b: the desired tetravalent product 8 with an observed mass of 4903.5 Da (calc. 4903.8 Da) and (B) octavalent dendrimer – peak a: linear peptide 1; peak b’: the desired octavalent product 9 with an observed mass of 9753.4 Da (calc. 9754.7 Da).

To further test enzymatic multimerization of peptides at a higher density level, tetra- and octavalent lysly dendron cores 6 and 7 with four and eight branches, respectively, were then prepared by SPPS using a low-loading resin. Under similar reaction conditions, clean and efficient ligations of thiodepsipeptide 1 with these two dendron cores were observed in HPLC profiles and dendron cores 6 and 7 were consumed after 45 min and 180 min (Figure 3), respectively, to give the corresponding tetra- and octavalent products 8 and 9 (peak b and b’ in Figure 3A and B) in excellent

yields (> 85%), MS analysis on isolated products confirmed their identities (inserts in Figure 3A and B). It should be noted that upon completion of the reaction, few intermediate low-order ligation products were found, a desirable scenario for purification purpose to achieve high product homogeneity. The synthesis of these MAPs with four and eight peptide branches showed the powerful nature of the butelase-mediated ligation reaction. To the best of our knowledge, this is the first time that a peptide ligase has been used for the synthesis of an octabranched dendrimeric peptide. Moreover, our bioconjugation method forms native peptide bonds between the monomeric peptides and lysyl dendron cores, as opposed to most chemical conjugation methods which form non-peptidic linkages. There is a growing interest developing peptide-based antibiotics25-28 because, compared with conventional antibiotics, they are less likely to develop drug resistance and have fewer side effects29-32. To apply our enzymatic method in preparing dendrimeric antimicrobials, we synthesized two antimicrobials 12 (bivalent) and 13 (tetravalent) (MS shown in supporting information) which harbored a tetrapeptide RLYR as the antibacterial sequence33 by ligating N-acetylated thiodepsipeptide 10 Ac-RLYRN-thioglc-V with dendron core 2 and 6 respectively. The RLYR tetrapeptide contains a BHHB motif (B = basic, H = hydrophobic) which is commonly found in certain potent and broad-spectrum β-stranded antimicrobial peptides34 such as PG (protegrins)35 and RTD1(rhesus monkey theta defensin)36. This consensus motif with positive charged and hydrophobic residues was successfully used as a dendrimeric antimicrobial in 200233. Such short peptidebased dendrimeric antimicrobials are broad-spectrum antibiotics and kill bacterial cells through electrostatic and hydrophobic interactions with the negatively charged microbial cytoplasmic membranes37. Using a radial diffusion assay, we tested the antimicrobial activities against E. coli and S. aureus of these two dendrimeric peptides together with prototypical monomer 11 RLYR, mono-, tri-lysine dendron core 2 and 6 for comparison. All assays were performed under a high-salt condition (100 mM NaCl) to simulate physiological conditions. The monomeric peptide 11 showed no appreciable activity against either E. coli or S. aureus (Table 2). However, significant antimicrobial activities were observed with the bivalent product 12 which had a MIC of 18.3 µM against E. coli and 3.4 µM against S. aureus. The tetravalent dendrimeric structure 13 had further improved antimicrobial activities with MICs < 3 µM against both strains. When testing 13 against several drug-resistant strains under high-salt condition, the tetramer construct was broadly active against all six tested strains with MICs ranging from 0.87 to 4.8 µM (Table 3). Our results suggest that MAP-based dendrimeric peptides, which can be easily prepared using BML, are an effective platform for the design of antimicrobial agents. A possible reason for this success is that the dendrimeric structure increases the effective molarity of the functional monomeric units and reduces the entropy cost associated with peptide self-assembly on bacterial plasma membranes33. Table 2. Antimicrobial activity of different peptides against E. coli and S. aureus. MIC (µM) Peptide E. coli S. aureus Monomer 11

> 300

> 300

Bivalent dendron core 2

> 150

> 150

Tetravalent dendron core 6

> 150

> 150

Dimer 12

18.3

3.4

Tetramer 13

2.4

1.4

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REFERENCES Table 3. Antimicrobial activity of tetramer 13 against different drug-resistant strains. Organism E.cloacae DM 09800

MIC (µg/mL) 4.41

MIC (µM) 0.9

K. species DR 13779

7.36

1.5

E. coli DM 04604

22.07

4.5

S. aureus DB 14329

23.54

4.8

P. aeruginosa DM 14158

11.28

2.3

E. coli DU 09777

10.79

2.2

In conclusion, we have developed a novel method to prepare peptide dendrimers using butelase 1 as the catalyst for bioconjugation of thiodepsipeptides with a lysyl dendron core. Using a small excessive of a monomeric peptide substrate (1.5 equiv. per branch to the dendron core) and a low catalytic amount of the enzyme butelase 1 (0.001 equiv.), we obtained very efficient synthesis of the bi-, tetra- and octavalent dendrimers. No other peptide ligases have been used for the synthesis of such dendrimeric peptides. We further used this bioconjugation method to evaluate the polyvalent feature of peptide dendrimers as antimicrobials. We found that a tetravalent dendrimer containing a tetrapeptide sequence with the BHHB motif is a potent broad-spectrum antimicrobial (MICs < 5 µM), including several drug-resistant bacterial strains. Our butelase-mediated bioconjugation method works under mild conditions and is user-friendly as it requires no complicated chemistry operations. We anticipate this method to be a useful tool in preparing peptide dendrimers.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (J. P. Tam), [email protected] (C.-F. Liu).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the NTU iFood Research Grant M4081467.080 and Singapore National Research Foundation NRF-CRP8-2011-05.

1. King, M., and Wagner, A. (2014) Developments in the field of bioorthogonal bond forming reactions-past and present trends, Bioconjugate Chem. 25, 825-839. 2. Kalia, J., and Raines, R. T. (2010) Advances in Bioconjugation, Curr. Org. Chem. 14, 138-147. 3. Hermanson, G. T. (2008) Bioconjugate Techniques, Elsevier, San Diego, CA. 4. Boutureira, O., and Bernardes, G. J. (2015) Advances in chemical protein modification, Chem. Rev. 115, 2174-2195. 5. Francis, M., Kapoor, T. M., Schreiber, S., and Wess, G. (2007) New Methods for Protein Bioconjugation Chemical Biology - From Small Molecules to Systems Biology and Drug Design, Vol. 2, Academic Press, London. 6. Xie, J., and Schultz, P. G. (2006) A chemical toolkit for proteins-an expanded genetic code, Nat. Rev. Mol. Cell Biol. 7, 775-782. 7. Cornish, V. W., Hahn, K. M., and Schultz, P. G. (1996) Sitespecific protein modification using a ketone handle. J. Am. Chem. Soc. 118, 8150-8151. 8. Deiters, A., Cropp, T. A., Mukherji, M., Chin, J. W., Anderson, J. C., and Schultz, P. G. (2003) Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J. Am. Chem. Soc. 125, 11782-11783. 9. Szobota, S., Gorostiza, P., Del Bene, F., Wyart, C., Fortin, D. L., Kolstad, K. D., Tulyathan, O., Volgraf, M., Numano, R., Aaron, H. L., et al. (2007) Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 54, 535-545. 10. van Hest, J. C. M., Kiick, K. L., and Tirrell, D. A. (2000) Efficient incorporation of unsaturated methionine analogues into proteins in vivo. J. Am. Chem. Soc. 122, 1282-1288. 11. Baker, D. P., Lin, E. Y., Lin, K., Pellegrini, M., Petter, R. C., Chen, L. L., Arduini, R. M., Brickelmaier, M., Wen, D. Y., Hess, D. M., et al. (2006) N-terminally PEGylated human interferon-beta-1a with improved pharmacokinetic properties and in vivo efficacy in a melanoma angiogenesis model. Bioconjug. Chem. 17, 179-188. 12. Chan, A. O. Y., Ho, C. M., Chong, H. C., Leung, Y. C., Huang, J. S., Wong, M. K., and Che, C. M. (2012) Modification of N-Terminal alpha-Amino Groups of Peptides and Proteins Using Ketenes. J. Am. Chem. Soc. 134, 2589-2598. 13. Geoghegan, K. F., and Stroh, J. G. (1992) Site-Directed Conjugation of Nonpeptide Groups to Peptides and Proteins Via PeriodateOxidation of a 2-Amino Alcohol - Application to Modification at NTerminal Serine. Bioconjugate Chem. 3, 138-146. 14. Singudas, R., Adusumalli, S. R., Joshi, P. N., and Rai, V. (2015) A phthalimidation protocol that follows protein defined parameters. Chem. Commun. 51, 473-476. 15. Lac, D., Feng, C., Bhardwaj, G., Le, H., Tran, J., Xing, L., Fung, G., Liu, R., Cheng, H., and Lam, K. S. (2016) Covalent Chemical Ligation Strategy for Mono- and Polyclonal Immunoglobulins at Their Nucleotide Binding Sites. Bioconjugate Chem. 27, 159-169,

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16. Takaoka, Y., Ojida, A., and Hamachi, I. (2013) Protein organic chemistry and applications for labeling and engineering in live-cell systems. Angew. Chem. Int. Ed. Engl. 52, 4088-4106. 17. Uttamapinant, C., White, K. A., Baruah, H., Thompson, S., Fernandez-Suarez, M., Puthenveetil, S., and Ting, A. Y. (2010) A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl. Acad. Sci. U. S. A. 107, 10914-10919. 18. McFarland, J. M., and Rabuka, D. (2015) Recent advances in chemoenzymatic bioconjugation methods. Org. Chem. Insights 5, 714. 19. Rashidian, M., Dozier, J. K., and Distefano, M. D. (2013) Enzymatic labeling of proteins: techniques and approaches, Bioconjugate Chem. 24, 1277-1294. 20. Ritzefeld, M. (2014) Sortagging: a robust and efficient chemoenzymatic ligation strategy, Chemistry. 20, 8516-8529. 21. Chang, T. K., Jackson, D. Y., Burnier, J. P., and Wells, J. A. (1994) Subtiligase: a tool for semisynthesis of proteins, Proc. Natl. Acad. Sci. U. S. A. 91, 12544-12548. 22. Nguyen, G. K. T., Wang, S. J., Qiu, Y. B., Hemu, X., Lian, Y. L., and Tam, J. P. (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis, Nat. Chem. Biol. 10, 732738. 23. Cao, Y., Nguyen, G. K., Tam, J. P., and Liu, C. F. (2015) Butelase-mediated synthesis of protein thioesters and its application for tandem chemoenzymatic ligation, Chem. Commun. (Camb). 51, 17289-17292. 24. Nguyen, G. K., Cao, Y., Wang, W., Liu, C. F., and Tam, J. P. (2015) Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide, Angew. Chem. Int. Ed. Engl.54, 15694-15698. 25. Nguyen, G. K., Kam, A., Loo, S., Jansson, A. E., Pan, L. X., and Tam, J. P. (2015) Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization, J. Am. Chem. Soc. 137, 15398-15401. 26. Sadler, K., and Tam, J. P. (2002) Peptide dendrimers: applications and synthesis, J. Biotechnol. 90, 195-229 . 27. Posnett, D. N., McGrath, H., and Tam, J. P. (1988) A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain, J. Biol. Chem. 263, 1719-1725. 28. Tam, J. P. (1988) Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system, Proc. Natl. Acad. Sci. U. S. A. 85, 5409-5413. 29. Lu, Y. A., Clavijo, P., Galantino, M., Shen, Z. Y., Liu, W., and Tam, J. P. (1991) Chemically Unambiguous Peptide Immunogen Preparation, Orientation and Antigenicity of Purified Peptide Conjugated to the Multiple Antigen Peptide System, Mol. Immunol. 28, 623-630. 30. Drijfhout, J. W., and Bloemhoff, W. (1991) A new synthetic functionalized antigen carrier, Int. J. Pept. Protein Res. 37, 27-32. 31. Defoort, J. P., Nardelli, B., Huang, W., and Tam, J. P. (1992) A rational design of synthetic peptide vaccine with a built-in adjuvant. A modular approach for unambiguity, Int. J. Pept. Protein Res. 40, 214221.

32. Rao, C., and Tam, J. P. (1994) Synthesis of Peptide Dendrimer, J. Am. Chem. Soc. 116, 6975-6976. 33. Rose, K., Zeng, W. G., Brown, L. E., and Jackson, D. C. (1995) A synthetic peptide-based polyoxime vaccine construct of high purity and activity, Mol. Immunol. 32, 1031-1037. 34. Spetzler, J. C., and Tam, J. P. (1995) Unprotected Peptides as Building-Blocks for Branched Peptides and Peptide Dendrimers, Int. J. Pept. Prot. Res. 45, 78-85. 35. Monso, M., de la Torre, B. G., Blanco, E., Moreno, N., and Andreu, D. (2013) Influence of conjugation chemistry and B epitope orientation on the immune response of branched peptide antigens, Bioconjugate Chem. 24, 578-585. 36. Fujita, Y., Moyle, P. M., Hieu, S., Simerska, P., and Toth, I. (2008) Investigation toward multi-epitope vaccine candidates using native chemical ligation, Biopolymers. 90, 624-632. 37. Boman, H. G. (1995) Peptide Antibiotics and Their Role in Innate Immunity, Annu. Rev. Immunol. 13, 61-92. 38. Boman, H. G. (2003) Antibacterial peptides: basic facts and emerging concepts, J. Intern. Med. 254, 197-215. 39. Giuliani, A., Pirri, G., and Nicoletto, S. F. (2007) Antimicrobial peptides: an overview of a promising class of therapeutics, Cent. Eur. J. Biol. 2, 1-33. 40. Liu, S. P., Zhou, L., Lakshminarayanan, R., and Beuerman, R. W. (2010) Multivalent Antimicrobial Peptides as Therapeutics: Design Principles and Structural Diversities, Int. J. Pept. Res. Ther. 16, 199213. 41. Koh, J. J., Lin, H. F., Caroline, V., Chew, Y. S., Pang, L. M., Aung, T. T., Li, J. G., Lakshminarayanan, R., Tan, D. T. H., Verma, C., Tan, A. L., Beuerman, R. W., and Liu, S. P. (2015) N-Lipidated Peptide Dimers: Effective Antibacterial Agents against GramNegative Pathogens through Lipopolysaccharide Permeabilization, J. Med. Chem. 58, 6533-6548 . 42. Tam, J. P., Lu, Y. A., and Yang, J. L. (2000) Design of saltinsensitive glycine-rich antimicrobial peptides with cyclic tricystine structures, Biochemistry-Us 39, 7159-7169. 43. Tam, J. P., Wu, C., and Yang, J. L. (2000) Membranolytic selectivity of cystine-stabilized cyclic protegrins, Eur. J. Biochem. 267, 3289-3300. 44. Thakur, N., Qureshi, A., and Kumar, M. (2012) AVPpred: collection and prediction of highly effective antiviral peptides, Nucleic Acids Res. 40, 199-204. 45. Tam, J. P., Lu, Y. A., and Yang, J. L. (2002) Antimicrobial dendrimeric peptides, Eur. J. Biochem. 269, 923-932. 46. Gwyer Findlay, E., Currie, S. M., and Davidson, D. J. (2013) Cationic host defence peptides: potential as antiviral therapeutics, BioDrugs 27, 479-493. 47. Kokryakov, V. N., Harwig, S. S. L., Panyutich, E. A., Shevchenko, A. A., Aleshina, G. M., Shamova, O. V., Korneva, H. A., and Lehrer, R. I. (1993) Protegrins - Leukocyte Antimicrobial Peptides That Combine Features of Corticostatic Defensins and Tachyplesins, Febs Lett. 327, 231-236. 48. Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., and Selsted, M. E. (1999) A cyclic antimicrobi-

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al peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins, Science 286, 498-502. 49. Dewan, P. C., Anantharaman, A., Chauhan, V. S., and Sahal, D. (2009) Antimicrobial Action of Prototypic Amphipathic Cationic Decapeptides and Their Branched Dimers, Biochemistry-US 48, 56425657.

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H

Xaa

H Xaa H Xaa H Xaa

Lys Lys

Asn

O S

Lys

O

Val

Butelase 1 Asn

Xaa

Asn Xaa Asn

Xaa

Lys Lys

Lys

Asn Xaa

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