Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx

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In vitro studies of antimicrobial activity of Gly-His-Lys conjugates as potential and promising candidates for therapeutics in skin and tissue infections Monika Kukowska ⇑, Magdalena Kukowska-Kaszuba, Krystyna Dzierzbicka Department of Organic Chemistry, Chemical Faculty, Gdansk University of Technology, 11/12 G. Narutowicza Street, 80-233 Gdansk, Poland

a r t i c l e

i n f o

Article history: Received 21 October 2014 Revised 9 December 2014 Accepted 9 December 2014 Available online xxxx Keywords: Antimicrobial peptide Lipopeptide Fatty acid Gly-His-Lys Therapeutics Skin and tissue infections

a b s t r a c t In this Letter, we presented in vitro studies of antimicrobial activity of Gly-His-Lys conjugates that are important point in preliminary biological evaluation of their potential application in skin and tissue therapies. The novel compounds include the conjugation of fatty acids with a modification of the amino acid sequence in the primary structure of Gly-His-Lys (6i). All the compounds exhibited strong to moderate activity. Compound 1d had the most potent antimicrobial activity at MIC ranges 31.3–125.0 lg/mL (against Escherichia coli spp. and Staphylococcus aureus spp.), 375.0–500.0 lg/mL (against Pseudomonas aeruginosa spp.). Conjugate 5b expressed activity against Staphylococcus aureus spp. and Escherichia coli spp. at MIC ranges 250.0–500.0 lg/mL and 62.5–125.0 lg/mL, respectively. Both conjugates 1d and 5b possessed rapid bactericidal activity against Gram-positive bacteria at 2MIC or 4MIC. Conjugates 1b–c, 1e, 2a–b and 4b showed noticeable effect against both Gram-positive and Gram-negative bacteria. Compounds 1d, 1e and 2e were the most active against fungus. Ó 2014 Elsevier Ltd. All rights reserved.

The interesting achievement in therapy of skin and tissue infections is the use of endogenous peptidic antibiotics.1–3 The peptidic compounds are essential due to the natural origin and low incidence of bacterial resistance. They possess a broad spectrum of antimicrobial,2,3 immunomodulatory4 and strong repairing activities.2,4 One of the recently marketed drug is daptomycin (a cycle lipopeptide) which possesses really strong activity and low resistance in treating skin and tissue infections caused by Gram-positive cocci (mainly Staphylococcus spp. and Streptococcus spp.).5–7 Many experimental works proved surprisingly high activity of oligopeptides against different microorganisms. Moreover, they are also active against biofilm formation.8,9 The activity is also potentiated by fatty acid chain linked to the N-terminal end of peptide.10 Known as lipids, fatty acids are the second group of compounds, that are involved in both physical and immunologic function barriers of the skin. They have been known for nearly several decades for their antimicrobial activity and played a direct role in innate immune defense against epidermal infections.11–13 Fatty acids also possess antimicrobial activity especially against Gram-positive cocci (Micrococci spp., Staphylococci spp., Streptococci spp.), Propionibacterium acnes, and yeast (Candida albicans), rather than ⇑ Corresponding author. Tel.: +48 58 347 20 54; fax: +48 58 347 26 94. E-mail address: [email protected] (M. Kukowska).

Gram-negative bacteria such as Escherichia coli spp. and Pseudomonas aeruginosa spp.12–17 Therefore research involving the possibility of their application for therapy of skin infections is still widely discussed. One of the major advantages of this group of compounds is the ability to decrease the development of bacterial resistance in comparison with conventional antibiotics used in treatment of skin lesions and vast array of wounds.18–20 Additionally, it was proved that the highest level of biological activity among the saturated fatty acids and their derivatives is represented by lauric acid. It also assuages in in vivo studies the effect of inflammation-related infections1,21,22 and has a great potential in treatment of acne.14,15 In spite of the fact that most fatty acids possess a very strong activity and a low toxicity, especially at higher concentration in comparison with the conventional antibiotics, it makes them very attractive candidates for further modifications.21 It is also well known that the effective topical therapeutics with antimicrobial activity should selectively target microorganisms, killing bacteria and other microbes, with minimal adverse on healthy cells. Many fatty acid–oligopeptide conjugates exhibit improved biological activity with low cytotoxic activity.8,23 However, they represent a relatively new group of compounds and the data on their activity are limited to in vitro and in vivo studies. Their amphipathic structure seems to be required for membrane binding. The length of

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fatty acid chain controls the pathogen specificity of the lipopeptides, overall hydrophobicity and oligomeric state of lipopeptides in solution.24 The unique mechanism of action and relatively safe profile in comparison with native lipo-antibiotics makes them extremely attractive candidates for potential therapeutics in skin and tissue infections. In our studies, we applied Glycyl-L-Histydyl-L-Lysine as a peptidyl molecule with a variety of important regulatory role in skin inflammation and tissue regeneration. Tripeptide is liberated from extracellular matrix protein, especially a-II chain of human collagen or secreted protein called SPARC in response to even soft tissue damage.25–30 The first report on antimicrobial activity of tripeptide was presented by Panlabs and included in vivo strong protection against infection caused by Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes spp.31 Liakopoulou-Kyriakides et al.32 described in vitro inhibitory activity of tripeptide against Escherichia coli spp., which is associated with the inhibition of both protein and DNA synthesis. The activity of tripeptide to overcome bacterial infections is also a result of its ability as a copper complex to interact on metabolism of iron ions. It seems that tripeptide copper complex naturally inhibits the release of iron from ferritin, preventing the microbial growth. Besides peptide induces the gene expression of natural antimicrobial defensins—DEFB1 and DEFB5.33–35 We designed a series of peptide conjugates containing Gly-HisLys analogues modified with N-acyl of lauric, palmitic and stearic acid, with C-amide or free carboxyl group. The modifications of the amino acid sequence are based on numerous studies reports, that comprise the replacement of histidine to other residue. This modification is a very attractive target in the development of novel analogues due to the improvement of stability and biological activity of the compounds.36–39 Encouraged by the results obtained earlier, we also replaced His with Hyp, Met, Gly-Hyp, Hyp-Met residues. Some analogues possess additionally D-Lys instead of Lys (Fig. 1). The modification of Gly-His-Lys analogues with fatty acids increased the affinity to phospholipids membrane in in vitro studies.27,40–43 We hypothesized that it could also increase the bacterial and fungal membrane binding and insertion, that could also be important point in preliminary studies on their potential application in skin and tissue therapy. In this Letter, we presented in vitro antimicrobial activity of lipopeptides. Gly

X

Y

O

R1

H N

CH

C

O

O NH

H

All the peptides investigated in our studies were prepared in our laboratory by manual methodology using Fmoc solid-phase technique44,45 (synthesis details in Supplementary data). Antimicrobial assays for Gly-His-Lys (standard, 6i) and all the conjugates were performed by determination of MIC and MBC/MFC values (Supplementary data). Minimal inhibitory concentration values against representatives of Gram-positive (Staphylococcus aureus spp.), Gram negative (Escherichia coli spp., Pseudomonas aeruginosa sp.) bacteria and fungus (Candida albicans sp.) are presented in Figure 2. In our previous effort in the development of Gly-His-Lys conjugates (data not shown), we reported that the replacement of natural histidine for hydroxyproline in peptide 1i gave significantly lower effect of the peptide on Gram-positive bacteria (Staphylococcus aureus spp.), whereas it possessed similar activity in comparison with peptide 6i against representatives of Gram-negative bacteria (Escherichia coli spp.). The compound 4i showed improved activity against Staphylococcus aureus spp. in comparison with peptide 6i. At the same time, the activity of the compounds 3i and 4i against Escherichia coli spp. was attenuated, in comparison with more hydrophilic compounds 6i and 1i. It could also be associated with the phenotype of genome in Escherichia coli spp., that is involved in metabolism of sulfurcontaining compounds.46,47 The most attractive molecule to further structural modifications was compound 1i. Firstly, it was found that peptide 1b exhibited better bacteriostatic activity against Staphylococcus aureus spp. and Pseudomonas aeruginosa sp. in comparison with peptide 1i (and 6i) and conferred also bactericidal activity (Figs. 2B, C and 3A). Moreover, peptides 5a, 5b with the substitution of D-Lys endowed antibacterial activity against Escherichia coli spp. with 2- and 4-fold reduction of MIC values, respectively, in comparison with compound 1b. In spite of the fact that compounds 1b and 5b possessed similar inhibitory effect, killing experiments exhibited higher activity of compound 5b against Staphylococcus aureus spp. Whereas the strong bactericidal activity of compound 1b was observed (at 8MIC) after 18 h, conjugate 5b presented shorttime killing activity at 2-fold lower concentration (4MIC, after 4 h). Palmitoyl derivative 1c possessed 2-fold lower MIC value against Escherichia coli spp. than peptide 1b. Moreover, it prevented the growth of Staphylococcus aureus spp. in the first time of incubation at 1/2MIC and MIC, whereas at 4MIC it possessed

CH

C

CH2

H N

CH

C

R3

CH2 CH2

N

CH2 NH CH2 NH

R2

1 - X=Hyp, Y=Lys

i: R1=H, R2=H, R3=OH

2 - X=Gly-Hyp, Y=Lys

a: R1=CH3COO, R2=H, R3=OH

3 - X=Met, Y=Lys

b: R1=CH3(CH2)10COO, R2=H, R3=OH

4 - X=Hyp-Met, Y=Lys

c: R1=CH3(CH2)14COO, R2=H, R3=OH

5 - X=Hyp, Y=D-Lys

d: R1=CH3(CH2)14COO, R2=H, R3=NH2

6 - X=His, Y=Lys

e: R1=CH3(CH2)16COO, R2=H, R3=OH

Figure 1. The structure of synthesized conjugates of Gly-His-Lys analogues.

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Figure 2. Minimal inhibitory concentrations of compounds against (A) Escherichia coli spp., (B) Staphylococcus aureus spp., (C) Pseudomonas aeruginosa sp., (D) Candida albicans spp., h = mean.

strong bactericidal activity. In fact, peptide 1c in comparison with 1b, at the same concentration, caused short-time killing activity (4 h vs 18 h). As we expected, conjugate 1d possessed the most potent activity against Gram-positive as well as Gram-negative bacteria (Fig. 2A–C, Table 1). It possessed really strong bactericidal activity against Staphylococcus aureus spp. and Pseudomonas aeruginosa spp. at 4MIC (Fig. 3A, B).

In fact, at concentration above 100 lg/mL, it possessed bactericidal activity against Staphylococcus aureus spp. during the first few hours of incubation. It was worth noticing that at 6MIC the peptide 1d possessed bactericidal activity directly after inoculum engrafted (in microdilution method). The differences between both microand macro-dilution methods could also be explained by the scale of experiment. Nevertheless, it caused strong and rapid bactericidal activity.

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Figure 3. Time-killing curves of lipopeptides against A. Staphylococcus aureus spp., B. Pseudomonas aeruginosa spp., C. Candida albicans spp., mean ± SD.

Table 1 Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of compound 1d Compd 1d

Staphylococcus aureus spp.

Escherichia coli spp.

Pseudomonas aeruginosa sp.

Staphylococcus aureus PCM 2054 (ATCC 25923)

Staphylococcus aureus PCM 2602 (ATCC 6538)

Escherichia coli DSM11250 (PCM2560, NTCC 10538)

Escherichia coli PCM 2561 (ATCC 8739, DSM 1576)

Pseudomonas aeruginosa PCM 2562 (ATCC9027)

MIC (lg/mL)

MIC50 31.3

MIC90 62.5

MIC50 31.3

MIC90 31.3

MIC50 31.3

MIC90 62.5

MIC50 62.5

MIC90 125.0

MIC50 375.0

MIC90 500.0

MBC (lg/mL)

MBC50 93.7

MBC90 125.0

MBC50 93.7

MBC90 125.0

MBC50 n.d.

MBC90 n.d.

MBC50 n.d.

MBC90 n.d.

MBC50 1500.0

MBC90 2000.0

n.d.: not determined; Staphylococcus aureus spp. 3MIC = 93.7 lg/mL, 4MIC = 125.0 lg/mL, 6MIC = 187.5 lg/mL; Pseudomonas aeruginosa sp. 2MIC = 1000.0 lg/mL, 4MIC = 2000.0 lg/mL.

The high activity of peptide 1d could be potentiated not only by the additional, C-terminal hydrophobic motif, but also by the appearance of the positive charge at physiological pH in comparison with peptide 1c. Thus, the increase of the net positive charge of peptide 1d enhanced its binding to lipid membrane of Gram-negative bacteria Escherichia coli spp. and Pseudomonas aeruginosa spp. Conjugate 1e possessed lower activity than 1c, but this biological effect against Gram-negative bacteria was slightly stronger than the effect exhibited by compounds 1b and 5e. Additionally, it presented biological activity profile against Staphylococcus aureus spp. similar to peptide 1b, but also at 2-fold higher concentration (4MIC). It also did not demonstrate short-term bactericidal activity in comparison with peptides 5b and 1c. Whereas the peptide 1c showed quite strong bactericidal activity at 4MIC after 4-h incubation, peptide 1e presented the long-term bactericidal activity against Staphylococcus aureus spp. It proved that N-acyl modification of peptides 3i and 4i improved the activity against Escherichia coli spp. It could also be associated with other mechanism of action where the hydrophobic interaction became important. Moreover, conjugates 4a and 4b also decreased MIC values against Staphylococcus aureus spp. in comparison with 4i (and 6i). This biological effect was not so evident for the modification of peptide 3i. Fatty acid chain is important for express bactericidal activity. Whereas peptide 4a prevented the growth of Staphylococcus aureus spp. at 4MIC during the whole time of incubation, conjugate 4b possessed rapid bactericidal activity at the same concentration (8MIC). Killing kinetics

presented their action in comparison with peptide 5b as well, but at 2-fold higher concentration (Fig 3A). Conjugate 2b possessed an inhibitory activity against Escherichia coli spp. similar to compound 1c, but its activity against Staphylococcus aureus spp. was 4-fold lower. Besides derivative 2a presented improved activity (similar to 5b and 1c) against Escherichia coli spp. and Staphylococcus spp. In spite of the fact that derivative 2a presented improved activity (similar to 5b and 1c) against Escherichia coli spp. and Staphylococcus aureus spp. in comparison with peptide 2b, the action profile indicates for the importance of lauric acid in its biological activity (Fig. 3A). The results of antifungal activity was interesting for conjugates 1d, 1e, 2e, 4e. As we expected, the compounds with the fatty acid chains, comprising more than C16, possessed lower antibacterial but improved antifungal activity. It is well correlated with the studies of other groups of scientists reported that lipopeptides with long chain fatty acids attenuated the activity against bacteria and improved it against Candida albicans spp.42,48 The strong activity was observed at relatively high concentration of peptides in comparison with Nystatin. Conjugate 1e exhibited the most potent activity against Candida albicans spp. The strong activity of peptide 1e was observed above 250 lg/mL (MIC). Compound 2e possessed inhibitory activity at 2–4-fold higher concentration. In fact, it prevented the growth of fungus during the first few hours of incubation, then hold down its growth. Similar biological activity profile was observed for peptide 1d at 1MIC (Fig. 3C). The higher activity of peptide 1d was found at concentration of 2MIC. Peptide 1d also exerted strong

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fungicidal activity, whereas 2e (2MIC) caused decrease of the amount of population during the first few hours of incubation, not allowing for growth of Candida albicans spp. The most important is fact that the peptide 1e exhibited a very strong fungicidal effect after 4-h incubation time. This effect at 8MIC is interesting due to the similar properties obtained for Nystatin. In summary, we have described the preliminary in vitro antimicrobial activity of novel Gly-His-Lys conjugates, which helped to select nine compounds marked 1b–e, 2a, 2b, 2e, 4b, 5b for further studies. These compounds seemed to be safe in our preliminary in vitro studies on human skin cells. The obtained results correspond with the literature review of toxicology of Gly-His-Lys analogues and fatty acids, and they will be described elsewhere with their encouraging proliferative activity. These data make them, in the future, very interesting candidates for topical therapeutics. Acknowledgments This work was supported by the Gdansk University of Technology (Grant DS No. 030386 and Grant DS No. 030861). We would like to thank the group of the Department of Microbiology at the Gdansk University of Technology (Ph.D. Beata Krawczyk) for cooperation providing us with several microorganism strains and PPST (Pomeranian Science and Technology Park in Gdynia) for help in some microbiological assays. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 12.029. References and notes 1. Laverty, G.; McLaughlin, M.; Shaw, C.; Gorman, S. P.; Gilmore, B. F. Chem. Biol. Drug Des. 2010, 75, 563. 2. Benard, J. J.; Gallo, R. L. Cell. Mol. Life Sci. 2011, 68, 2189. 3. Ki, V.; Rotstein, C. Can. J. Infect. Dis. Med. Microbiol. 2008, 19, 173. 4. Namjoshi, S.; Caccetta, R.; Benson, H. A. E. J. Pharm. Sci. 2008, 97, 2524. 5. Steenbergen, J. N.; Alder, J.; Thorne, G. M. J. Antimicrob. Chemother. 2005, 55, 283. 6. Jerala, R. Expert Opin. Investig. Drugs 2007, 16, 1159. 7. Tally, F. P.; Debruin, M. F. J. Antimicrob. Chemother. 2000, 46, 523. 8. Dawgul, M.; Baran´ska-Rybak, W.; Bielin´ska, S.; Nowicki, R.; Kamysz, W. Alerg. Astma Immunol. 2010, 15, 220. 9. Dawgul, M.; Maciejewska, M.; Jaskiewicz, M.; Karafova, A.; Kamysz, W. Acta Pol. Pharm. Drug Res. 2014, 71, 39. 10. Hilpert, K.; Elliott, M. R.; Volkner-Engert, R.; Henklein, P.; Donini, O.; Zhou, Q.; Winkler, D. F. H.; Hancock, R. E. W. Chem. Biol. 2006, 13, 1101. 11. Kabara, J. J.; S´wieczkowski, D. M.; Conley, A. J.; Truant, J. P. Antimicrob. Agents Chemother. 1972, 2, 23. 12. Kabara, J. J. Cosmet. Perfum. 1975, 90, 21.

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In vitro studies of antimicrobial activity of Gly-His-Lys conjugates as potential and promising candidates for therapeutics in skin and tissue infections.

In this Letter, we presented in vitro studies of antimicrobial activity of Gly-His-Lys conjugates that are important point in preliminary biological e...
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