Membrane disrupting antimicrobial peptide dendrimers with multiple amino termini

Abstract

Antimicrobial peptide dendrimer H1 Leu₈(Lys-Leu)₄(Lys-Phe)₂Lys-LysNH₂ (Lys = branching COMPOUND LINKS

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Download mol file of compoundlysine) was identified by screening a 6750-membered combinatorial library by the bead-diffusion assay. Sequence variations also revealed dendrimer bH1 Leu₈(Dap-Leu)₄(Dap-Phe)₂Dap-LysNH₂ (Dap = branching COMPOUND LINKS

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Download mol file of compound2,3-diaminopropanoic acid) as a more potent analog. H1 and bH1 showed good antimicrobial activities mediated by membrane disruption (MIC = 2–4 μg mL⁻¹ on Bacillus subtilis and Escherichia coli) but low hemolytic activity (MHC = 310 μg mL⁻¹ respectively >2000 μg mL⁻¹).

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Many antimicrobial peptides (AMPs) act by disrupting microbial membranes.¹ These include linear α-helical amphipathic peptides,²cyclic peptides and peptoids,³ foldamers,⁴ various amide oligomers,⁵ and multivalent COMPOUND LINKS

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Download mol file of compoundlysine dendrimers appended with linear AMPs of various lengths.^6,7 In all of these cases the multiple positive charges necessary for membrane disruption are brought about by the side chains of basic amino acids.⁸ Herein we report the discovery of antimicrobial peptide dendrimers such as H1 and bH1 in which positive charges are provided by the multiple amino termini at the dendrimer periphery (Fig. 1). This new type of AMP acts as membrane disrupting agent and shows remarkably low hemolytic activity.

Fig. 1 Structure of antimicrobial peptide dendrimers H1 and bH1.

We recently developed the chemistry of peptide dendrimers incorporating variable amino acids within the dendrimer branches.⁹ In contrast to the polylysine dendrimers which are limited to the multivalent display of linear peptides,¹⁰ our peptide dendrimers vary in the nature and length of the dendrimer core, intermediate and terminal branches. This diversity can be exploited to obtain peptide dendrimers acting as enzyme¹¹ and metalloprotein models,¹² and in glycosylated version as biofilm inhibitors¹³ and drug delivery agents.¹⁴

We wondered if the multiple amino termini created by the ramified structure of our peptide dendrimers might serve as the source of positive charges in a new type of dendritic AMPs in which the nature of the amino acids within the branches would enable a membrane disrupting activity by bringing about hydrophobic groups. To test this hypothesis, a diverse 6750-membered combinatorial library L of G3 peptide dendrimers X⁴₈(KX³)₄(KX²)₂KX¹ (X = variable amino acid, K = branching COMPOUND LINKS

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Download mol file of compoundL-lysine, Fig. 2A), a sequence type which is particularly resistant to proteolysis,¹⁵ was prepared by split-and-mix solid phase peptide synthesis (SPPS)¹⁶ on a photolabile resin suitable for off-bead assays.¹⁷

Fig. 2 Screening for dendrimer AMPs. (A) Structure of peptide dendrimer libraries L and AcL. (B) Antimicrobial screening showing active beads from library L after staining the B. subtilis bacteria with MTT (COMPOUND LINKS

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Download mol file of compoundthiazolyl blue tetrazolium bromide). (C) Inactive beads in library AcL. (D) Overall composition of hits and non-hits at variable positions X¹–X⁴, calculated from the total occurrence of each amino acidT_AA = 8X⁴ + 4X³ + 2X² + X¹. The sequences of 15 hits and 51 non-hits were determined. Sequences are shown in Tables S1 and S2†.

Library L with free amino termini and the corresponding N-acetylated library AcL were subjected to antimicrobial activity screening¹⁸ using a bead-diffusion assay¹⁹ with an adapted protocol²⁰ compatible with bead decoding by amino acid analysis (AAA).^21,22 In this assay the beads are photolyzed in the absence of solvent and spread onto an agar plate inoculated with bacterial lawn. Peptides diffuse into the agar resulting in a clearing zone around beads carrying AMPs, whose sequence can be determined by AAA of the peptide left on the bead.

Upon screening of dendrimer library L, three to five beads per agar plate (0.1% of approximately 5000 beads) were surrounded by a clearing zone (Fig. 2B). The assay with acetylated library AcL did not give any hits (Fig. 2C). The active beads from library L contained predominantly the hydrophobic residues Leu and Phe (>10-fold enrichment over control), with a consensus for sequence Leu₈(Lys-Leu/Phe)₄(Lys-Phe/Pro)₂Lys-LysNH₂, while control inactive beads gave random sequences (Fig. 2D and Tables S1 and S2†). Taken together, these results showed that the combination of free amino termini with hydrophobic side chains was essential for obtaining an antimicrobial activity in these dendrimers.

The positive hits H1, H2 and H3, all carrying eight N-terminal leucines at position X⁴, were resynthesized by SPPS, together with their N-acetylated analogs as controls Ac1, Ac2 and Ac3. All products were obtained in good yields after purification by preparative HPLC. The following analogs of the strongest hits H1 and H2 were also prepared: (a) diastereoisomers dH1 and dH2 with D-amino acids at all non-branching positions; (b) COMPOUND LINKS

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Download mol file of compoundisoleucine analogs iH1 and iH2; (c) an alanine scan series AX1–AX4 corresponding to hit H1; (d) analogs with the shorter 2,3-diaminopropanoic (Dap) branching unit instead of COMPOUND LINKS

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Download mol file of compoundlysine bH1 and bH2 (Table 1).

Table 1 Synthesized library hits and analogs

No	Sequence^a	Yield^b	MS calc./obsd^c
a B indicates a branching 2,3-diaminopropanoic acid and K a branching lysine. Standard one-letter codes are used with L-amino acids in upper case and D-amino acids in lower case. b Yield given taking the amino acid content determined by amino acid analysis into account. c Sequence: ILPWKWPWWPWRRNH2.
H1	(L)8(KL)4(KF)2KKNH2	45.6 mg/8%	2693.9/2694.0
Ac1	(AcL)8(KL)4(KF)2KKNH2	9.5 mg/6%	3030.0/3031.0
H2	(L)8(KF)4(KP)2KKNH2	62.5 mg/14%	2729.8/2730.0
Ac2	(AcL)8(KF)4(KP)2KKNH2	18.3 mg/12%	3065.9/3066.7
H3	(L)8(KQ)4(KF)2KKNH2	73.1 mg/12%	2753.8/2754.0
Ac3	(AcL)8(KQ)4(KF)2KKNH2	76.8 mg/44%	3089.9/3091.0
dH1	(l)8(Kl)4(Kf)2KkNH2	76.2 mg/12%	2693.9/2694.0
iH1	(I)8(KI)4(KF)2KKNH2	33.6 mg/1%	2693.9/2694.0
dH2	(l)8(Kf)4(Kp)2KkNH2	42.0 mg/10%	2729.8/2730.0
iH2	(I)8(KF)4(KP)2KKNH2	38.2 mg/5%	2729.8/2730.0
AX1	(L)8(KL)4(KF)2KANH2	27.2 mg/5%	2638.6/2637.7
AX2	(L)8(KL)4(KA)2KKNH2	33.4 mg/10%	2543.5/2542.5
AX3	(L)8(KA)4(KF)2KKNH2	14.8 mg/4%	2527.3/2526.6
AX4	(A)8(KL)4(KF)2KKNH2	23.2 mg/8%	2359.0/2358.2
bH1	(L)8(BL)4(BF)2BKNH2	21.8 mg/7%	2401.1/2400.0
bH2	(L)8(BF)4(BP)2BKNH2	33.5 mg/12%	2437.1/2436.0
indo	Indolicidin ^c	47.5 mg/9%	1906.1/1906.0

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The purified hits H1 and H2 showed the expected antimicrobial activity against B. subtilis. Dendrimer H3 with COMPOUND LINKS

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Download mol file of compoundglutamine instead of Leu/Phe in position X³ was not active showing the importance of a hydrophobic internal shell for activity. The N-acetylated controls Ac1–Ac3 were also inactive, consistent with the absence of active beads in the N-acetylated library, once more highlighting the importance of the multiple free N-termini preferentially from N-terminal leucine residues for antimicrobial activity. Although they were slightly less active, the diastereoisomers dH1/dH2 and isomers iH1/iH2 showed comparable activities to H1/H2. In the alanine scan series, mutation of the outer eight or four leucines in the G3 (AX4) or G2 (AX3) branches reduced activity, confirming that hydrophobic residues were needed for activity in combination with the multiple positive charges provided by the free N-termini (Table 2).

Table 2 Antimicrobial and hemolysis activity profiles^a

Cpd.	MIC^aB. subtilisBR151	MIC^aE. coliDH5α	MIC^aP. aeruginosaPA01	MHC ^b erythrocytes
a Minimal inhibitory concentration (MIC) values are given in μg mL^−1 and were determined following ref. 23. b Minimal hemolytic concentration (MHC) values are given in μg mL^−1 and were determined following ref. 24. All measurements were done in triplicate for MIC and duplicate for MHC. Shown are average values. Ac1, Ac2 and Ac3 showed MIC > 100 μg mL^−1 against B. subtilisBR151 and were not evaluated further.
H1	2.0	21	>72	310
H2	2.0	19	66	>10 000
H3	>100	>100	>100	250
dH1	2.5	15	60	>10 000
iH1	4.4	79	>100	20
dH2	4.4	11	60	>10 000
iH2	2.5	18	61	>10 000
AX1	2.0	15	≥100	31
AX2	4.5	>100	67	>2000
AX3	67	>100	>100	>2000
AX4	>100	>100	67	1000
bH1	2.9	3.9	18	>2000
bH2	3.1	15	20	>2000
indo	3.0	22	>100	16

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Several of the dendrimers also showed good activities against E. coli and P. aeruginosa, in particular the Dap-branched analogs bH1 and bH2, which were also more compact (r_H ≈ 1.15 nm from diffusion NMR) than the lysine branched dendrimers (r_H = 1.30–1.40 nm, see ESI†). Profiling against an additional 17 strains showed further interesting activities for H1, iH1 and bH1 (Table 3). Hemolysis by the dendrimers was generally weak compared to the linear control peptide indolicidin, except in AX1 lacking the core lysine residue and iH1 containing isoleucines. The Dap branched analog bH1 combined a broader activity than H1 with a weaker hemolytic activity, suggesting a useful therapeutic window. Similarly low hemolysis has been observed previously in antimicrobial COMPOUND LINKS

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Download mol file of compoundlysine dendrimers.⁷

Table 3 Antimicrobial profiling of selected dendrimers^a

Bacterium	PB	AP	H1	iH1	bH1
a Minimal inhibitory concentration (MIC) values are given in μg mL^−1 of the weighed lyophilized solid as TFA salt and were determined following the standard broth microdilution method recommended by the Clinical and Laboratory Standards Institute (M7-A7). The concentration range was 0.06 to 32 μg mL^−1. Polymyxin B (PB) and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundampicillin (AP) were used as positive controls. The peptides did not show measurable inhibition (MIC > 32 μg mL^−1) on the following strains: S. aureus (42080, ATCC29213), En. faecalis (ATCC29212, Van B E80-8), E. coli (ATCC25922), P. aeruginosa (ATCC27853, PA01(HS), 8S/H, K799/WT, K799/61). Peptides Ac1, H3, Ac2, H2, iH2 and bH2 were not active in any of the strains tested (MIC > 32 μg mL^−1). AX1–AX4 were not measured in this series.
S. aureus ATCC25923	≥32	0.13	24	>32	≥32
S. aureus 887	16	>32	12	16	32
S. epidermidis ATCC14990	>32	1	32	32	24
S. epidermidis J147	>32	1.5	24	>32	24
En . faecium ATCC19434	>32	4	≥32	24	>32
En . faecium Van B E38-10	>32	>32	32	8	>32
E. coli HB101 (PAT266)	2	>32	32	>32	32
E. coli DC2	4	1	>32	>32	32

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The peptide dendrimers were generally less active on Gram negative bacteria, which together with the activity of the dendrimer stereoisomers and the requirement for a combination of cationic charges and hydrophobic residues pointed towards membrane disruption as a possible mechanism of action. The effect was evidenced for dendrimer H1 using synthetic unilamellar vesicles assembled from E. colilipids. Dendrimer H1 at 25 μg mL⁻¹ effected a complete disassembly of the vesicles as observed by electron microscopy, while the vesicles were stable in the presence of the N-acetylated analog Ac1 at the same concentration (Fig. 3).

Fig. 3 Negative-stain transmission electron microscopy of untreated and dendrimer-treated E. colilipid vesicles: untreated (A), 25 μg mL⁻¹H1-treated (B) and Ac1-treated (C) liposomes. The scale bars represent 100 nm.

The interaction of the peptide dendrimers with the membrane probably involves electrostatic binding of the cationic N-terminal protonated amines with the anionic phospholipid head group and insertion of hydrophobic amino acid side chains into the lipid bilayer. A molecular dynamics (MD) simulation of dendrimer H1 in the presence of a phosphorylcholine (POPC) lipid bilayer over 100 ns indeed supports this hypothesis (Fig. 4). A more extended simulation will be required to see if the interaction leads to the formation of pores as observed in MD studies of amphiphilic membrane disrupting AMPs, cell penetrating peptides, and G3–G7 PAMAM dendrimers.²⁵

Fig. 4 MD simulation of peptide dendrimer H1:POPC membrane interaction. The model represents the last frame obtained from 100 ns MD simulation using NPγT ensemble at 300 K using the OPLS-AA force field. Six of the eight N-terminal leucine protonated amines form H-bonds with anionic phospholipid head groups, while their side chains have inserted into the lipid bilayer.

In summary, G3 peptide dendrimers with hydrophobic amino acids in their branches and multiple free amino termini as the sole source of positive charges possess significant antimicrobial activities against Gram positive and Gram negative bacteria. The activities of H1 and bH1 compare well with those of linear peptides such as indolicidin, but also of previously reported dendritic peptides (best MIC ≈ 1–20 μg mL⁻¹, various bacterial strains, see ref. 6 and 7). These peptide dendrimers represent a new class of AMPs since previous peptide based linear or dendritic AMPs all relied on amino acid side chains as the source of positive charges. The evidence for significant activity variations upon amino acid exchanges suggests that it should be possible to optimize activities against specific strains by further exploration of the sequence space, which might open a new opportunity to develop non-toxic antimicrobial agents.

Acknowledgements

This work was supported financially by the University of Berne, the Swiss National Science Foundation, and the European Union FP7-ITN-238434.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Details of library design, synthesis and assays. See DOI: 10.1039/c1md00272d

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