Structure and antimicrobial properties of multivalent short peptides

Anne W. Young; Zhigang Liu; Chunhui Zhou; Filbert Totsingan; Nikhil Jiwrajka; Zhengshuang Shi; Neville R. Kallenbach

doi:10.1039/C0MD00247J

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DOI: 10.1039/C0MD00247J (Concise Article) Med. Chem. Commun., 2011, 2, 308-314

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Structure and antimicrobial properties of multivalent short peptides†

Anne W. Young ^a, Zhigang Liu ^a, Chunhui Zhou ^a, Filbert Totsingan ^a, Nikhil Jiwrajka ^a, Zhengshuang Shi *^ab and Neville R. Kallenbach *^a
^aDepartment of Chemistry, New York University, 100 Washington Square East, New York, NY 10003, USA. E-mail: nrk1@nyu.edu; zs212@nyu.edu; Fax: +(212)-260-7905; Tel: +(212)-998-8757
^bSchool of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, PR China

Received 3rd December 2010 , Accepted 18th January 2011

First published on 28th February 2011

Abstract

The emergence of multidrug-resistant (MDR) strains continues to threaten human health. Along with many efforts directed towards the development of antimicrobial peptides (AMPs) as potential antibiotics, we have designed and tested several series of multivalent AMPs in comparison with the natural AMP, indolicidin. Constructs include multivalent displays with different sequences, repeats and scaffolds, including dendrimers, brush-like structures and polymeric displays constructed by linking various peptides to polymaleic anhydride (PMA). A branched tetramer of dipeptides (RW)_4D demonstrates the highest level of effectiveness among the constructs of this study. The results also show that under extended exposure to sublethal doses of (RW)_4D and indolicidin over a 400 generations period, MDR strains elicit much lower levels of resistance than to traditional antibiotics or disinfectants such as ciprofloxacin, vancomycin, chlorhexidine and gentamicin.

Introduction

The emergence of multidrug-resistant (MDR) strains^1–5 continues to threaten human health.^6–8 Within the past 60 years, resistance to almost every widely used commercial antibiotic in either community-associated or hospital-acquired strains has appeared shortly after their introduction.^9–11 The rapid acquisition of resistance reflects the tremendous diversity of resistance genes present in a population such as that of the human microbiome.¹² Discovering new classes of antibiotics able to overcome MDR bacteria may thus entail a search for rare targets able to evade this enormous pool.¹³ Faced with this situation, peptides that are secreted in the host defense of many organisms, antimicrobial peptides or AMPs, have been considered as a possible class of antibiotics for treating infectious diseases.^14–17

A wide variety of naturally occurring antimicrobial peptides (AMPs) are expressed in both the animal and plant kingdoms;^18–22 they act in the innate and acquired immunity of vertebrates and are part of the humoral defense of invertebrates against infections from pathogenic microorganisms.^23–26 Many efforts have been directed to develop natural AMPs as new antibacterial drugs^14–17 with limited success to date.¹⁵ The main problems are that they are hemolytic, expensive to produce, and not orally bioavailable. Recently we described the design of cationic antimicrobial peptides with repeating sequences of arginine and tryptophan, (RW)_n,²⁷ based on the observation that short chains of R and W comprise a pharmacophore for AMPs derived from the natural AMP.^28,29 Our previous studies have demonstrated enhanced anti-bacterial activity by multivalent displays of dipeptides or tetrapeptides on different scaffolds.^30,31 Natural AMPs such as polymyxins or gramicidins tend to display high antimicrobial potency, but cytotoxicity to the host cells. In this study, we report on the antimicrobial properties of multivalent displays of short peptide sequences (Fig. 1). These include multivalent dendrimeric displays with different sequences, repeats and number of branches, brush-like structures such as Ac-[GK(RWRW)]₄_/₈-NH₂, in which RWRW tetrapeptides are grafted onto linear scaffolds Ac-(GK)₄_/₈-NH₂, and polymeric displays, PMA-RWRW, constructed by linking various ratios of the tetrapeptide RWRW to polymaleic anhydride (PMA). As judged from values of the hemolytic index (HI) (HI is the ratio of HD₅₀ to IC₅₀; HD₅₀, is the concentration of agent that lyses 50% red blood cells and IC₅₀, the concentration of the agent that inhibit 50% of bacterial growth), (RW)_4D emerged as the optimal anti-bacterial agent (Fig. 2). Further resistance studies show that the dendrimer (RW)_4D and natural indolicidin from bovine neutrophils offer clear advantages over conventional antibiotics or disinfectants such as ciprofloxacin, vancomycin, chlorhexidine and gentamicin, against MDR bacterial strains tested. Both (RW)_4D and indolicidin induce extremely low levels of bacterial resistance after 400 generations of growth relative to the conventional drugs tested (Fig. 3). The dendrimer is less cytotoxic than indolicidin, and more potent as an antimicrobial.


	Fig. 1 (A) Synthetic scheme for constructing polydisperse multivalent PMA-RWRWs based on RWRW and PMA. Unreacted groups on the polymer are capped by carboxymethylation. (B) Structure of PMA-RWRWs with different grafting ratios. (C) Structures of Ac-[GK(RWRW)_4/8]-NH₂. (D) Multivalent dendrimer peptides in this study. (E) The structure of (RW)_4D as a representative; structures of other dendrimer peptides are displayed with different sequences/repeats and number of branches as shown in (D).


	Fig. 2 Comparison of hemolytic index of different multivalent AMPs. HI value is calculated as the ratio of HD₅₀ (HI = HD₅₀/IC₅₀). Please note the logarithmic scale used in the plot.


	Fig. 3 Development of resistance among different bacteria strains over 400 generations against (RW)_4D and control antibacterial agents: (A) Gram-negative A. baumannii against (RW)_4D, (RW)₄, indolicidin, gentamicin and ciprofloxacin; (B) Gram-negative E. coli D31 against (RW)_4D, (RW)₄, indolicidin, chlorhexidine and gentamicin; (C) Gram-positive S. aureus against (RW)_4D, (RW)₄, indolicidin, vancomycin and chlorhexidine. Note for figure 3A and 3B, the figure is re-plotted with a log scale as an inset in which the effects around the low value portion of y axis are emphasized.

Results

Antimicrobial activities of PMA-RWRW and Ac-[GK(RWRW)]₄_/₈-NH₂

In a previous study, we created multivalent antimicrobial agents by covalently linking antimicrobial tetrapeptides (RWRW and RRWW) onto reactive polymaleic anhydride (PMA) chains.³⁰ In the initial study PMA chains containing ∼40 monomer units on average were fully grafted. However does the activity of multivalent AMP require complete substitution with peptides? To answer the question, we tested 50% and 25% grafts of the same polymer, containing on average ∼20 and ∼10 RWRW units per chain, respectively (Table 1). The antibacterial activity of the latter products is roughly the same as that of the 100% grafted polymer if IC₅₀ is expressed in micrograms per unit volume (Table 2). The fact that as few as ten RWRW units per chain retain full biological activity prompted us to investigate multivalent designs using a much shorter linear scaffold. Accordingly we constructed two multivalent AMPs Ac-[GK(RWRW)_4/8]-NH₂ (Fig. 1C) in which RWRW tetrapeptides were tethered to the lysine side chains of linear scaffolds Ac-(GK)₄_/₈-NH₂. Antibacterial and hemolytic assays revealed that Ac-[GK(RWRW)₄]-NH₂ was more potent and less hemolytic than Ac-[GK(RWRW)₈]-NH₂ (Table 2). The IC₅₀ and HD₅₀ values showed that Ac-[GK(RWRW)₄]-NH₂ was more active than PMA-RWRW against Gram-negative bacteria, but less active than PMA-RWRW against Gram-positive bacteria.

Table 1 Molecular weight and monomer content in PMA-RWRW

PMA-RWRWs	Molecular weight (Da)		Monomer content
PMA-RWRWs	Calculated	Observed	Monomer content
a The peak average molecular weight (Mp).
PMA-RWRW (100% graft)	33254	30500	39
PMA-RWRW (50% graft)	20320	18600	19
PMA-RWRW (25% graft)	13774	12600	10

Table 2 Summary of IC₅₀/HD₅₀ results for studied multivalent AMPs

Peptides	IC₅₀, μg mL⁻¹			HD₅₀, μg mL⁻¹
Peptides	E. coli	S. aureus	A. baumannii	HD₅₀, μg mL⁻¹
a The IC₅₀ results are the mean of three independent experiments performed in parallel. b HD₅₀ determined from dose-response curve is peptide concentrations corresponding to 50% hemolysis.
(KW)_4D	30.6	23.3	52	1459.3
(KF)_4D	77	145	175	2500
(KY)_4D	84.4	240	360	3740
(RW)_4D	3.9	15	42	1961.8
(RF)_4D	30	180	160	2780
(RY)_4D	12.6	45.7	193	2151
(HW)_4D	30.6	43.3	82.7	1860.2
(RWRW)_4D	16.4	23.6	/	130.6
(RWRWRW)_4D	20.8	31.7	/	365.2
(RWRW)_8D	32.6	70.5	/	277.1
PMA-RWRW 100%	34.1	24.6	/	425.7
PMA-RWRW 50%	33.9	24.0	/	366.5
PMA-RWRW 25%	34.4	25.0	/	598.4
Ac-[GK(RWRW)]₄-NH₂	15.0	75.4	/	1123
Ac-[GK(RWRW)]₈-NH₂	65.6	82.0	/	885.8

Antimicrobial activities of multivalent dendrimer peptides with different display of amino acids and different number of branches

Comparison of short linear scaffolds with dendrimers showed that the tetramer of dipeptides RW, (RW)_4D, is more effective than linear constructs against both Gram-negative and Gram-positive bacteria.³¹ In contrast to the polymeric species, PMA-RWRW or PMA-RRWW, whose cytotoxicity increases with antimicrobial activity,³⁰ (RW)_4D has much lower hemolytic activity: its HI values are about 5 to 30 fold higher than those of PMA-RWRW/PMA-RRWW and 10 to 50 fold higher than those of indolicidin against S. aureus and E. coli, respectively.³¹ Analysis of the sequences of linear AMPs with extended structure reveals that they contain a high frequency of cationic and bulky non-polar amino acids, including not only Arg and Trp, but also Lys, Tyr, Phe, His and Pro.^{22,28,29,32–37} We therefore constructed six new dendrimers, on which different dipeptide combinations of cationic and hydrophobic amino acids are linked to a four-branched lysine dendritic core. In addition to RW, these dipeptides include RF, RY, KW, KF, KY and HW as summarized in Fig. 1D and 1E.

The IC₅₀ and HD₅₀ values of these products are shown in Table 2. A general trend for the charged side chains indicates that Arg has higher activity against Gram-negative bacteria when compared to His and Lys. For the nonpolar side chains, Trp and Tyr display higher activity than Phe, with Trp being more potent than Tyr. A different trend can be seen for the hydrophobic side chains, in that all the peptides containing Trp have a low HD50 value: (KY)4D has the highest HD50 value and (KW)4D the lowest. The results indicate that (RW)4D has the best overall antimicrobial activity (Fig. 2).

While promising results are found for the dendrimer (RW)_4D we need to determine the role of size of the tethered peptide units. To this end, RW, RWRW and RWRWRW were covalently attached to a four-branched lysine dendritic core. In this set of dendrimers, the order of activities is (RWRWRW)_4D < (RWRW)_4D < (RW)_4D; the RW dipeptide again offers the best choice in terms of antibacterial activity and selectivity (Table 2). To further test how the number of dendrimer branches affect their antibacterial activity, we constructed (RWRW)_8D in which eight RWRW peptides are tethered to an eight-branched lysine dendrimer core. It turns out (RWRW)_4D is more active than (RWRW)_8D against either Gram-negative or Gram-positive bacteria (Table 2). Thus unexpectedly branched arrays of relatively inert dipeptide sequences confer greater biological activity on the dendrimer product than more active tetrapeptides or hexapeptides. No linear arrangement is more active than (RW)_4D.

Topological effects in multivalent scaffolds

If we compare constructs that contain the same number of RW monomers, (RWRW)_4Dvs. Ac-[GK(RWRW)₄]-NH₂ and (RWRW)_8Dvs. Ac-[GK(RWRW)₈]-NH₂, for example, we find that dendrimeric structures such as (RWRW)_4D have a significantly lower IC₅₀ value than Ac-[GK(RWRW)₄]-NH₂ for Gram-positive bacteria while the HI value for the latter is higher; dendrimeric (RWRW)_8D has a significantly lower IC₅₀ value than Ac-[GK(RWRW)₈]-NH₂ for Gram-negative bacteria while the HI for the latter is again higher. Comparing (RWRW)_8D and Ac-[GK(RWRW)₈]-NH₂ to PMA-RWRW (25% graft, approximately 10 RWRW units per molecule), the relative activity for Gram-negative bacteria has the following order: Ac-[GK(RWRW)₈]-NH₂ < PMA-RWRW ≲ (RWRW)_8D; while for Gram-positive bacteria the order is: Ac-[GK(RWRW)₈]-NH₂ ≲ (RWRW)_8D < PMA-RWRW. The corresponding HI values are in the following order: (RWRW)_8D < Ac-[GK(RWRW)₈]-NH₂ < PMA-RWRW. Overall PMA-RWRWs are more effective against Gram-positive relative to Gram-negative bacteria, consistent with the results for the monomeric peptide unit RWRW itself; in contrast to RWRW, Ac-[GK(RWRW)_4/8]-NH₂ and dendrimeric constructs are more effective against Gram-negative bacteria. Thus there appears to be no common mode of action for different constructs of monomeric peptides on scaffolds with different topology. Nevertheless, the dendrimer (RW) _4D offers the most effective display against either Gram-negative or Gram-positive bacteria as revealed by either IC₅₀ or HI value (Table 2 and Fig. 2). Whether this is true for RW displays with different topology remains to be seen.

Low levels of bacterial resistance to (RW)_4D and indolicidin as compared to conventional antibiotics ciprofloxacin, vancomycin, chlorhexidine and gentamicin

Since (RW)4D proved to be an effective dipeptide dendrimer, we subjected this agent to resistance studies in comparison with indolicidin, a natural antimicrobial peptide from bovine neutrophils.²⁸ A linear analog of (RW)4D with the same composition of Arg and Trp, (RW)₄ and ciprofloxacin, vancomycin, chlorhexidine and gentamicin were tested as controls. Acquisition of resistance to these agents was measured for two Gram-negative strains, E. coli D31 and A. baumannii, and a Gram-positive strain, S. aureus (Fig. 3). The development of resistance was monitored for 400 generations of serial transfers in the presence of sublethal concentrations of the agents.

All tested AMPs maintained potent inhibitory activity against the multi-drug resistant strains over the 400 generation period monitored. This is in marked contrast to conventional antibiotics or disinfectants. We observed a 43-fold increase in resistance of A. baumannii to ciprofloxacin, while vancomycin resistance increased 2.5-fold in S. aureus over the time period tested. In the case of the disinfectant chlorhexidine, a 5-fold and 2.5-fold increase in resistance of E. coli D31 and S. aureus were observed, respectively. E. coli (D31) and A. baumannii showed 13- and 8-fold increases in resistance to gentamicin, respectively. By contrast, indolicidin, (RW)₄ and (RW)_4D showed far lower levels of induced resistance; and (RW)_4D displayed slightly lower levels of resistance as compared to its linear analog (RW)₄ (Fig. 3). The levels of resistance observed demonstrate that indolicidin, (RW)4 and (RW)4D offer a clear advantage in comparison to the conventional antibiotics tested.

Discussion

Multivalent designs enhance AMP activities, the effectiveness depending on the strain of bacteria and the structures of scaffolds

A wealth of data shows that AMP activity is strongly concentration dependent. Formation of critically sized clusters appears essential for bacterial membrane lethality as revealed by the observation of a critical threshold concentration for known AMPs.^38–40 Proposed mechanisms focus on formation of “pores” or vesicular structures that lead finally to permeabilization of the bacterial membrane.^41–45 Our motivation for multivalent designs to enhance AMP activity originally derived from the idea that covalently pre-assembling AMP monomers might facilitate nucleation of AMP assemblies^46–49 and thereby reduce the critical concentration for lysis. All the multivalent constructs of this study show enhanced antibacterial activity relative to the monomeric peptides.²⁷ Another study has also reported enhanced membrane pore formation by multimeric/oligomeric antimicrobial peptide.⁵⁰

A more important issue of direct relevance to the development of antibacterial drugs is whether or not a significant gain in bacterial killing effect can be achieved without increasing hemolysis. Thus PMA-RWRW's with different extents of grafting are poor candidates as antibacterial agents since hemolytic activity increases with the enhancement of antibacterial effects relative to the free monomeric peptides (Table 2). For Ac-[GK(RWRW)_4/8]-NH₂, IC₅₀ and HD₅₀ data show that Ac-[GK(RWRW)₄]-NH₂ might have potential as an antimicrobial agent selectively for Gram-negative bacteria. Among all the designed dendrimer AMPs of this study, (RW) _4D provides the best lead for antibacterial agent development. However we note that the effectiveness of different designed multivalent AMPs depends on the bacterial target (Gram-negative or Gram-positive), the length of the peptide, as well as the structure and topology of the scaffold. Thus shape and flexibility of scaffolds, the size and the composition of the monomeric units, and the effective valence of multivalent designs all play a role yet to be fully defined.

Dipeptide displays on the lysine dendrimer core point to Arg and Trp as the best natural side chain combination

The group of naturally occurring AMPs related to indolicidin lacks apparent secondary structural constraints and contains a high frequency of amino acids including Trp, Arg, Lys, Tyr, Phe, His and Pro.^22,34,35 Display of seven different dipeptides on the four-branched lysine dendritic core reveals that RW offers the best choice in terms of antimicrobial activity and selectivity. This result is consistent with previous reports by Svendsen and coworkers that very short peptides-dimers and trimers based on R and W represent a minimum cationic antimicrobial pharmacophore.²⁹ The results of this study also corroborate later findings by Hancock and coworkers.^51–53 In several recent studies, they generated libraries of small cationic antimicrobial peptides either free in solution or tethered to surfaces; high-throughput screening revealed that the sequences of potent antimicrobial peptides are rich in Trp and Arg.^51–53 Another study has also reported that the antimicrobial activity of peptides containing Arg is higher than that of peptides containing Lys⁵⁴ although Lys tends to be less hemolytic.

Why should combinations of R and W be the most effective antimicrobial agent? Studies have shown that Arg plays a key role in membrane insertion since electrostatic interactions of the Arg side chains with the phosphate head group serves to stabilize the peptide-membrane interaction.⁵⁵ The guanidinium group of Arg has a more dispersed positive charge and higher pK_a than the single amine of Lys, which may facilitate electrostatic attraction between peptides and the negatively charged bacterial surface. Trp also has a clear preference for the interfacial regions of lipid bilayers.^56–59 It is not strongly hydrophobic; its extensive π-electron system is asymmetric in both shape and charge distribution. Finally the π-electron system of Trp can participate in cation-π interactions that are energetically favorable in aqueous solution allowing the residues to stabilize their interactions with the membrane.^58–62

Comparing different dendrimer peptides [(RW)_n]₄_/8D indicates there are threshold effects from the valence number and monomer charge

The activity order for dendrimers that we observe—(RWRWRW)_4D < (RWRW)_4D < (RW)_4D and (RWRW)_8D < (RWRW)_4D—is notable because the observed orders differ from those seen in linear RW peptides, for which longer chains lower the IC₅₀.²⁷ One traditional hypothesis has been that cationic antimicrobial peptides interact electrostatically with negatively charged bacterial surfaces, such as lipopolysaccharide or lipoteichoic acid. Since bacterial killing by AMPs is cooperative we hypothesized that higher valence should increase potency. However this does not appear to be true. Instead activity might be lost because of potential immobilization of cationic peptides on the surfaces of bacteria, impeding passage to the membrane itself. Perturbation of the integrity of bacterial membrane via interactions of cationic peptides with negatively charged membrane surfaces may contribute only partially to a combination of different killing mechanisms as noted by Hancock and coworkers.⁶³

Very slow development of resistance to (RW)_4D and indolicidin suggests that surface mutations might be costly in terms of bacterial fitness

Given appropriate exposure, bacteria can evolve resistance to almost any known antibiotic or treatment.^64,65 Facile gene transfer allows resistance genes to spread rapidly.⁴ Bacteria evade antibiotics by inactivating the drug, modifying the target, or by pumping out the drug.^4,6–8 As Groisman et al. demonstrated for polymyxin resistance, AMP resistance tends to reflect changes in surface charge rather than efflux.⁶⁶ Our resistance data suggest that alterations in surface charge may be costly for fitness. However the genes that confer polymyxin resistance in Salmonella are triggered also by changes in pH and/or Mg²⁺ concentration.⁶⁶ Thus we anticipate that resistance to agents such as (RW)_4D can and will develop depending on the environment. Natural AMPs have evolved in living organisms for billions of years,¹⁴ confronted by pathogens with access to an enormous pool of resistance genes.¹² One tactic to suppress resistance is to kill rapidly, even at the expense of collateral damage to host cells. This is characteristic of AMPs involved in mammalian innate immunity: high local concentrations secreted by pursuing macrophages ensure rapid inactivation of pathogens. A second strategy would be to make the evading process so costly that the survival of resistant genotypes is compromised. Recent studies have indicated that after 700 passages in the presence of increasing concentrations of AMPs, bacteria indeed develop resistance⁶⁴ in contrast to previous reports by Ge et al.⁶⁷ Among the mechanisms are reduction of the negative charge of LPS via transfer of amino sugars and phosphatidyl ethanolamine. The fitness cost of such modifications does not appear to impair virulence of Salmonella strains. Our conclusion that resistance to cationic (RW)_4D or indolicin is much slower to develop than to conventional antibiotics does not preclude development of such resistance. Bacteria will surely evolve resistance to (RW)_4D and indolicin once sufficiently stringent challenges are applied.

In summary, we have evaluated several series of multivalent AMPs as well as the natural small AMP, indolicidin. (RW)_4D demonstrates the highest level of effectiveness among the constructs of this study, being more potent and less cytotoxic than indolicidin. The results also show that under extended exposure to sublethal doses of (RW)_4D and indolicidin over the 400 generations period, MDR strains elicit much lower levels of resistance than traditional antibiotics or antimicrobials such as ciprofloxacin, vancomycin, chlorhexidine and gentamicin.

Abbreviations list

AMP	antimicrobial peptide;
CFU	Colony Forming Units
DIEA	diisopropylethylamine
DMF	Dimethylformamide
Fmoc	9-Fluorenylmethyloxycarbonyl
HBTU	Benzotriazole-N,N,N′,N′,-tetramethyl-uronium-hexafluoro-phosphate
HD	Hemolytic Dose
HI	hemolytic index;
HOBT	N-Hydroxybenzotriazole
IC	Inhibitory Concentration
MHB	Mueller Hinton Broth
MALDI-TOF	Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
MDR	multidrug-resistant;
OD	Optical Density
Pbf	NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PBS	Phosphate Buffered Saline
PMA	polymaleic anhydride;
TFA	Trifluoroacetic Acid
THF	Tetrahydrofuran
TIS	Triisopropylsilane
TMDM	trimethylsilyldiazomethane
TSB	Tryptic Soy Broth

Acknowledgements

This work was supported by a grant from ONR (N00014-03-1-0129). We acknowledge the NCRR/NIH for a Research Facilities Improvement Grant (C06 RR-16572) at NYU.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c0md00247j