Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity

Article abstract of DOI:10.1002/cbic.201700599

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18–29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In t

Full text of DOI:10.1002/cbic.201700599

Accepted Article
Title: Alanine and lysine scans of the LL-37 derived peptide fragment
KR-12 reveal key residues for antimicrobial activity
Authors: Sunithi Gunasekera, Taj Muhammad, Adam A. Strömstedt, K.
Johan Rosengren, and Ulf Göransson
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10.1002/cbic.201700599
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Manuscript
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Alanine and lysine scans of the LL-37 derived peptide fragment KR-12 reveal key residues
for antimicrobial activity
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Sunithi Gunasekera1*, Taj Muhammad1*, Adam A. Strömstedt1*, K. Johan Rosengren2, Ulf Göransson1
1Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574,
SE-75123 Uppsala, Sweden.
2The University of Queensland, School of Biomedical Sciences, Brisbane, QLD 4072, Australia.
*These authors contributed equally.
Corresponding author: Ulf Göransson, Pharmacognosy, Department of Medicinal Chemistry, Uppsala
University, Biomedical Centre, Box 574, SE-75123 Uppsala, Sweden. E-mail: ulf.goransson@ilk.uu.se
Keywords: antimicrobial peptide, LL-37, KR-12, membrane permeabilization, antibacterial, antifungal
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ABSTRACT
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The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions.
Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains
antibacterial activity with decreased cytotoxicity. In this study, we generated analogues of KR-12 by Ala
and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens
and for membrane permeabilization on liposomes. Replacements of hydrophobic and cationic residues
with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity as long as the
increase in cationic density did not disrupt the amphiphilic disposition of the helical structure.
Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5
with Lys and Asp9 with Ala or Lys improved broad-spectrum activity the most, each resulting in up to
eight-fold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida
albicans. The improved analogues displayed no significant toxicity against human cells, depicting KR-12
as a tuneable template for antibiotic development.
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INTRODUCTION
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The human cathelicidin LL-37 is a host defence peptide that has attracted widespread interest for drug
development.[1] It displays broad-spectrum antibacterial activity against both Gram-positive and Gram-
negative bacteria by membrane disruption.[2] Furthermore, LL-37 modulates the inflammatory response
and counteracts sepsis by binding lipopolysaccharide (LPS) and lipoteichoic acid (LTA);[3] it recruits
immune cells through chemotaxis,[4] augments wound healing[5] and displays antifungal and anti-parasitic
activities.[6] Native LL-37 and peptide derivatives thereof are currently in preclinical and clinical
development. Phase I/II trials for treatment of venous leg ulcers show that low dose topical treatment with
LL-37 has significant effect on wound healing.[7] Moreover, the 24-mer synthetic LL-37 derivate OP-145
that binds LPS or LTA is in Phase II clinical trials for chronic bacterial middle-ear infection.[8]
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LL-37 has previously been truncated into smaller fragments to dissect its bioactive regions and to identify
sequence-activity relationships. The antibacterial and antineoplastic activity of LL-37 has been located to
its C-terminal region, specifically residues 13-37.[9] A 13-residue core antibacterial and anticancer peptide
corresponding to residues 17-29 of LL-37 has been identified, and referred to as FK-13.[9] The
cytotoxicity of this peptide triggered the search for derivatives, resulting in a peptide comprising residues
18-29 of LL-37, referred to as KR-12 (Figure 1). This exhibited similar activity as LL-37 and FK-13
against E. coli, with a minimum inhibitory concentration (MIC) of 40 μM in a standard broth
microdilution assay.[9] Contrary to FK-13, KR-12 displayed no cytotoxicity on human cells up to 100
μg/mL (63 μM).[10] The retained antibacterial potency and low cytotoxicity has made KR-12 an attractive
starting point for further antimicrobial peptide (AMP) development.
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LL-37 and KR-12 are both linear peptides with disordered conformations in aqueous solution, but are
known to adopt an amphiphilic helix in the presence of salt or in membrane-like environments.[10a, 11]
Several lines of evidence suggest that oligomerization of LL-37 is promoted by membrane interaction,
which in turn is critical for the mechanisms of membrane permeabilization.[12] LL-37 forms
transmembrane pores of the toroidal type also exhibited by magainin and other pore-forming peptides.[13]
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The aim of the present work was to analyse the relationship between the primary structure and bioactivity
of KR-12, and to identify modifications that would improve the potential of this peptide as a drug lead.
Alanine and lysine scans were conducted to examine the contribution of individual residues in KR-12 to
antimicrobial activity and membrane permeabilization. The identification of modifications that improve
activity as a broad spectrum antimicrobial demonstrates the potential utility of KR-12 as an antibiotic
drug lead.
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EXPERIMENTAL SECTION
Reagents
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All Fmoc protected amino acids were purchased from PepChem (Tubingen, Germany) or Iris Biotech
(Marktredwitz, Germany). The following N-Fmoc amino acids were used: Fmoc-Ala-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Asn(trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(otBu)-OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Val-OH. Following resins were used in synthesis: Fmoc-Arg(Pbf)-Novasyn
TGT (0.2 mmol/g, Novabiochem, Stockholm, Sweden). Fmoc-Lys(Boc)-Novasyn TGT (0.2 mmol/g,
Novabiochem), Fmoc-Ala-Novasyn TGT (0.2 mmol/g, Novabiochem) and Fmoc-Ser(tBu)-Novasyn TGT
(0.2 mmol/g, Novabiochem). 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetrametyluronium hexafluorophosphate)
HBTU) was purchased from PepChem. Dichloromethane (DCM), N,N-dimethylformamide was from
Thermo Fisher Scientific (Waltham, MA) or Saveen & Werner (Limham, Sweden). HPLC grade
acetonitrile (ACN) was from VWR (West Chester, PA). Diethyl ether was purchased from Merck
(Darmstadt, Germany). Diisopropylethylamine (DIPEA), trimethylsilylisopropane (TIPS), piperidine,
trifluoroacetic acid (TFA), formic acid (FA) and phosphate buffered saline (PBS) were purchased from
Sigma-Aldrich, Stockholm, Sweden. Tryptic soy broth (TSB) was from Merck KGaA (Darmstadt,
Germany).
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Peptide synthesis
Peptide synthesis was performed on a CEM Liberty 1 (CEM corporation, North Carolina, USA)
automated microwave-assisted peptide synthesizer using Fmoc/tBu chemistry with piperidine (20% v/v)
as the Fmoc deprotecting agent. All peptides were synthesized on a 0.1 mmol scale using a 5-fold molar
excess of Fmoc-protected amino acids, activated by a 5-fold excess of HBTU in the presence of DIPEA
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(10 equiv., 2 M). Preloaded resins were used for the synthesis. Following synthesis the resin was washed
well with dry DCM and dried under N2. A sample of the dried resin was taken out and cleaved with a
mixture containing TFA/TIPS/water (95:2.5:2.5, 1.5 h). The cleaved peptide was filtered from resin and
dried down to 0.5 mL under N2, precipitated by addition of cold diethyl ether and centrifuged at 600 g for
10 min. The precipitate was re-dissolved in 50% ACN/0.05% TFA and freeze dried.
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HPLC and MS
RP-HPLC was performed on an Äkta Basic (GE Healthcare) and Shimadzu LC10 AD (Shimadzu, Japan)
with detection at 215 nm and 254 nm. Preparative HPLC was done on a 250 × 10 (i.d.) mm Phenomenex
(Torrance, CA) C18 column (5 μm) at a flow rate of 4 mL/ min, and analytical HPLC was done on a
100 × 60 (i.d.) mm Phenomenex C18 column (2.6 μm) at a flow rate of 0.5 mL/min. Solvents A (10%
ACN, 0.05% TFA in water) and B (60% ACN, 0.05% TFA in water) were used in a linear gradient from
0–100 % solvent B over 70 min for the preparative HPLC. Solvents A (0.05% TFA in water) and B
(0.05% TFA in ACN) was used in a linear gradient from 5 to 90% B over 35 min was used for analytical
HPLC. LC–MS was performed on a nanoAcquity UPLC coupled to a micromass Q-TOF detector. A
flow rate of 0.3 μL/min was used on a nanoAcquity UPLC BEH C18 column (1.7 μm, 250 × 0.075 mm)
using solvents A (0.1% FA in water) and B (0.1 % FA in ACN). A linear gradient from 0 to 90% B in
solvent A was used for analysis over 75 min.
Antimicrobial assay
Antibacterial and antifungal activities of the peptides were evaluated using the two-step microdilution
assay optimized for AMP-activity testing.[14] The method assesses MIC in buffer conditions. Four
organisms, representing frequent pathogens involved in wound infections, were chosen from clinical
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isolated strains obtained from the Department of Clinical Bacteriology at Lund University Hospital. These
were the Gram-negative bacteria Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC
27853, the Gram-positive Staphylococcus aureus ATCC 29213, and the pathogenic yeast Candida
albicans ATCC 90028. In short; the microbes are grown in 3% (m/m) tryptic soy broth (Merck KGaA,
Darmstadt, Germany) at 37°C to mid logarithmic phase, washed and transferred to 96-well plates (U-
shaped, untreated polystyrene) prepared with peptides in a 2-fold serial dilution. The resulting wells have
50 000 cfu suspended in 100 μL of 10 mM Tris buffer (Tris base adjusted by HCl to pH 7.4 at 37°C).
After 5 h incubation with the peptides, 5 μL of 20% (m/m) TSB added to each well and the plates were
re-incubated. Growth was monitored at 5, 8, 10 and 12 h for E. coli, S. aureus, P. aeruginosa and C.
albicans, respectively, at which time the control growth had produced pellets of approximately 2 mm in
diameter. The MIC was defined as the lowest peptide concentration that fully inhibited visible bacterial
growth by the indicated time. The resulting MIC-values are median values from triplicate experiments.
Deviations between experiments did not exceed one peptide dilution factor. Bactericidal vs bacteriostatic
activity was identified from samples of re-suspended MIC-well contents taken after the first Tris buffer
incubation step of the microdilution assay. Samples were diluted to 20% and transferred to LB-agar plates
resulting a theoretical 1000 cfu per plate. From the same stage in the microdilution assay, membrane
disruption on C. albicans was identified by concentrating the well contents by centrifugation and using
trypan blue staining and microscopy.
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Liposome leakage assay
An E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL), with a mass composition in terms of
headgroups of 67% phosphatidylethanolamine, 23% phosphatidylglycerol and 10% cardiolipin, was used
for the bacterial membrane model. The liposome production and the liposome leakage assay were
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performed as previously described.[15] In brief, dry lipid films of E. coli polar lipid extract (Avanti Polar
Lipids, Alabaster, AL) were formed on round-bottom flask walls and re-suspended in Tris buffer
containing of 100 mM 5(6)-carboxyfluorescein. Multilamellar structures and polydispersity were reduced
by repeated extrusion through 100-nm polycarbonate membranes mounted in a LipoFast mini-extruder
(Avanti Polar Lipids). Un-trapped carboxyfluorescein was removed by separation on Sephadex PD-10
columns (GE Healthcare, Little Chalfont, UK). Membrane permeability was measured by monitoring
carboxyfluorescein efflux from the liposomes to the external low concentration environment, resulting in
loss of self-quenching and an increased fluorescence signal. The leakage experiments were performed in a
96-well plate format. Wells were prepared with a two-fold serial dilution of the peptides in Tris buffer, as
well as controls without peptides (background) and the detergent Triton X-100 (maximum leakage). The
plates were pre-heated to incubation temperature (37°C) and liposomes subsequently administered (to a
final lipid concentration of 10 μM in 200 μL) by an automated dispenser. The effect of each peptide
concentration on liposome integrity was monitored for 45 min, at which point the leakage had largely
subsided. Results shown represent the mean from triplicate experiments with standard deviations, and are
presented as percent of total leakage generated with Triton X-100 after subtraction of the baseline value.
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Cytotoxicity assay
The cytotoxicity of the peptides was tested on the human lymphoma cell line U-937 GTB in a
fluorometric microculture cytotoxicity assay, described in detail previously.[16] The cell line was
maintained in RPMI 1640 complete medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat
inactivated fetal bovine serum, 2 mM glutamine, 50 μg/mL streptomycin and 60 μg/mL penicillin (all
from Hy Clone, Cramlington, GB) and kept under standard incubating conditions (humidified atmosphere
of 37°C, 5% CO2) and continuously grown at log phase. Peptide two-fold serial dilutions were prepared in
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96-well plates to which 20 000 cells in fresh growth medium were added (to a total volume of 200 μL)
and incubated for 72 h. Control wells without peptide or with Triton X-100 (representing complete cell
death) were used to quantify the viability interval. The cells were washed with PBS, and fluorescein
diacetate was added to each well. After incubation for 40 min the fluorescence was measured at 485 nm
excitation and 538 nm emission using a Fluoroscan II (Labsystems OY, Helsinki, FI). The IC50-value was
calculated using sigmoidal dose response curves with nonlinear regression and 0/100 constraints in
GraphPad Prism (GraphPad Software, San Diego). Each data point in Figure 3 represents the mean
viability as a percentage of the mean uninhibited cell growth, with standard deviations from triplicate
experiments.
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Haemolysis assay
A 2-fold serial dilution of the peptides in PBS was prepared in 96-well V-bottom microplates (Thermo
Scientific). Human single donor red blood cells (RBCs) were washed with PBS and centrifuged at 3600
rpm so that a clear supernatant was obtained. A 0.25% suspension of washed RBCs in PBS (100 μL) was
added to the peptide solutions. The plates were incubated at 37 °C for 1 h and centrifuged at 150 g for
5 min. Aliquots of 100 μL were transferred to a 96-well U-bottom microplate (Greiner Bio-One) and the
absorbance was measured at 405 nm with a Varioskan Flash Multimode Reader (Thermo Fisher
Scientific, Waltham, MA). The amount of haemolysis was calculated as the percentage of maximum lysis
(1% Triton X-100 control) after adjusting for background lysis (RBC in PBS).
Serum stability assay
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Peptide stability was assayed in diluted human serum. The serum was centrifuged at 16 000 × g for 10
min to remove lipids, and the supernatant was collected and incubated at 37°C for 15 min. Here 8 μL of
peptide stock solutions (200-250 μM) were incubated in 25% human serum (80 μL) at 37°C for the
desired time points (0, 3, 6 10 min). 80 μL of 6 M urea was added and incubated at 4°C for a further 10
min. 80 μL of 20% TCA was added and incubated at 4°C for a further 10 min. The samples were
centrifuged at 16 000 × g for 10 min and the supernatant was analyzed by LC-MS.
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RESULTS
Ala and Lys scans identify substitutions improving antimicrobial activity of KR-12
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To identify the residues important for bioactivity, each amino acid in KR-12 was systematically replaced
by Ala or Lys, producing two series of twelve and ten analogues, respectively. In addition, several other
analogues with double and triple substitutions with Ala or Lys were synthesized in order to identify
cumulative effects on activity and target selectivity. All peptides were assembled using Fmoc-based solid
phase peptide synthesis, and purity and identity were confirmed by RP-HPLC and MS (Supporting
Information Fig. S1-S3). Each peptide sequence, net charge, hydropathy index and molecular weight are
given in Supporting Information Table S1. The peptide-adapted two-step microdilution assay[14] was used
to determine the MIC against four clinical strains of human pathogens, the Gram-negative Escherichia
coli and Pseudomonas aeruginosa, the Gram-positive Staphylococcus aureus and the fungus Candida
albicans. The assay determines antimicrobial activity in buffer conditions, in order to avoid AMP
inhibition by factors associated with rich growth media.[14] MICs of each peptide were then compared
with that of native KR-12. Figure 2 shows the relative activity of the analogues compared to the MIC of
KR-12. A complete list of MIC values is available in Supporting Information Table S2.
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Notably, residues Asp9 and Gln5 in KR-12 stand out as positions suitable for modification from the
modified peptide series. Asp9 was the only residue for which overall activity increased when replaced
with Ala. Although D9A displayed increased antimicrobial activity against all organisms, the effect was
most pronounced against S. aureus and P. aeruginosa with a four- and eight-fold decrease in MIC,
respectively. When replaced with a Lys, the resulting peptide D9K displayed four- to eight-fold
improvement on all organisms, which for S. aureus meant it reached an activity level equivalent to the
parent peptide LL-37. Q5K also displayed increased antibacterial activity against all organisms, although
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the improvement in antibacterial activity was not as substantial as for D9K. Substitution of Gln5 with
Ala, in contrast, had only marginal effect on activity.
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A substantial decrease in antimicrobial activity was observed when cationic Arg or Lys residues in native
KR-12 were substituted with Ala. The antibacterial activity against E. coli decreased 16-fold for K1A,
R2A, R6A, K8A and R12A compared to KR-12. The activity of the above analogues against other
organisms also decreased, although not to the same degree (Figure 2). Substitution of the Arg located at
positions 2, 6 and 12 by Lys had little effect on activity. Replacements of hydrophobic residues with
either Ala or Lys were found to have an overall negative effect on antimicrobial activity, e.g. I7A
displaying a 16-fold higher MIC on E. coli. Similarly, peptides I3A, V4A, I7K, F10A, F10K, L11A and
L11K all displayed reduced antibacterial and antifungal activities. These results support the previous
observation that the interactions between the hydrophobic side chains of LL-37 and the acyl chains of the
target membrane are as important for its ability to disrupt lipid bilayers as its electrostatic interactions
with the phospholipid head groups[17]. Changes in selectivity between pathogens were observed primarily
when I3 and V4 were substituted with Lys. Antibacterial activity against E. coli and P. aeruginosa
increased two- to four-fold for I3K, and similarly for V4K on P. aeruginosa. In contrast, activity on S.
aureus was unchanged or diminished, and activity on Candida was particularly reduced when these
hydrophobic residues were substituted by either Lys or Ala.
Combinations of substitutions reveal additive effects primarily on antifungal activity
Double and triple substitutions were synthesized to identify additive or synergistic effects between key
positions identified from the Ala/Lys scans that would lead to further enhancement of antimicrobial
activity. Relative to the activity of the single-substituted Q5A and D9A, the double-substituted Q5A,D9A
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exhibited marginal improved activity, for instance against C. albicans. The Lys-substituted Q5K,D9K
displayed the same activity as Q5A,D9A and absence of additive effects from combining the two single
Lys substitutions was apparent. However, it is interesting to note that Q5K,D9K displayed an activity
equivalent to LL-37 against both S. aureus and C. albicans. The combined Ala and Lys substituted
Q5K,D9A, displayed eight-fold improvement in activity compared to KR-12 for all organisms except E.
coli, and complementary effects from both substitutions were evident. The triple and double substitutions
the D9A substitution with Ala substitutions of hydrophobic residues (Q5A,D9A,F10A, V4A,D9A and
I3A,V4A,D9A) reversed the positive effect from the D9A mutation. Notably, I3K,Q5K,D9K specifically
decreased the activity on the Gram-positive S. aureus by eight-fold while preserving or even increasing
activity for the other organisms when compared with Q5K,D9K. The data show a similar trend when
involving other Ile3 and Val4 substitutions. Double and triple Lys substitutions of hydrophobic residues
on the C-terminal half of the sequence (I7K,L11K and I7K,F10K,L11K) displayed broad decrease in
antimicrobial activity, which corresponded with the single substitutions.
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Antimicrobial enhanced KR-12 analogues retain low cytotoxic activity
To ensure that the beneficial substitutions identified from the Ala and Lys scans for KR-12 do not
compromise selectivity towards bacterial cells compared to human cells, lead peptides were assayed
against a human lymphoma cell line. In line with published reports, KR-12 showed weak cytotoxic
activity with only a 13% loss of cell viability at the maximum concentration of 80 μM (Figure 3). In
comparison, LL-37 displayed distinct cytotoxicity in this assay, having an IC50 of 10 μM. Importantly, the
antimicrobial enhancing substitutions of Gln5 and Asp9 are well tolerated, and neither of the double
substitutions Q5A,D9A and Q5K,D9K showed any substantial cytotoxicity within the concentration
interval used (Figure 3).
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In agreement with the cell line cytotoxicity data, KR-12 and the double substitutions Q5A,D9A and
Q5K,D9K did not exhibit any dose-dependent haemolytic activity against human erythrocytes within the
tested concentration interval (0.6-80 μM) (Fig. S4). In contrary, LL-37 did exhibit an incrementing
haemolytic activity over the same concentration interval reaching 15% at 80 μM.
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E. coli liposome permeabilization reflects trends in bioactivity
Follow-up analysis of the microdilution assays on bacteria and Candida showed that when MIC was
reached, these pathogens could not be resuscitated by diluting below MIC and transfer to LB-agar plates.
These bactericidal and fungicidal properties were further characterized by the gradual increment of trypan
blue uptake ratios for Candida cells when approaching MIC-levels and sampled immediately after the
peptide pre-incubation step in the microdilution assay. MIC thus gave 100% stained cells. Both results
agree with a membrane disruptive mechanism for KR-12, Q5A,D9A and Q5K,D9K, much like the parent
peptide LL-37. Hence a liposome leakage assay was used to assess the effects on membrane disruption by
the amino acid substitutions. E. coli phospholipids were reconstituted into large unilamellar vesicles
trapping a self-quenching fluorescent marker. KR-12 induced leakage from the E. coli liposomes, but
approximately thirty-fold higher concentration of KR-12 was required to reach 50% leakage than for LL-
37 (Figure 4). The importance of hydrophobic residues for membrane disruptive activity was highlighted
by the Ala scan of KR-12, with a substantial decrease in leakage observed when hydrophobic residues
were substituted. Substitutions of cationic residues by Ala did not decrease activity to the same extent.
Ala substitution of Asp9 gave the most significant increase in activity, most noticeable in the lower
peptide concentration range and consistent with the antimicrobial activity data.
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The Lys scan of KR-12 showed similar effects as the Ala scan when hydrophobic residues were
substituted, with Ile7 and Leu11 the most important for membrane activity. Arg to Lys substitutions
showed no marked difference in leakage, while Q5K and D9K showed increase in activity at lower
peptide concentrations. In terms of membrane permeabilization efficacy, favourable and unfavourable
substitutions at different positions cancelled each other out when combined in double and triple
substitutions, and multiple favourable substitutions exhibited only a marginal, if any, additive effect on
activity. Noteworthy is that double substitutions of Q5 or D9 showed the same increase in activity when
replaced with either Ala or Lys. This activity increase was cancelled-out if combined with the negative
influence of substituting hydrophobic residues in triple substitutions.
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The KR-12 analogues are unstable in serum
Proteolytic stability of the peptides were determined in diluted human serum (1:4 v/v serum:PBS) at 37
°C followed by LC-MS analysis. To examine the cleavage patterns of the peptides in serum, aliquots were
taken at various time points for MS-MS analysis. The peptides with observable intermediate fragments
are shown in Figure 5, together with cleavage products identified. Overall the linear peptides showed poor
stability and none could be observed after 10 min of incubation in serum; less than 10% of the peptides
were detected in serum after 10 min for KR-12 and the tested Ala/Lys analogues. KR-12 rapidly degraded
within the first three min and analysis of cleavage products indicated that the peptide bonds following the
R6 and R12 residues were the most susceptible cleavage sites (Figure 5). Peptides with Lys substitutions
introduced at the above sites (R6K and R12K) likewise degraded rapidly in serum producing similar
degradation patterns. The corresponding Ala substitutions (R6A and R12A) conferred a slight
improvement in stability. In particular, the R12A variant persisted with close to 85% intact peptide within
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the first three min. The R12A analogue exhibited P1 cleavage sites of R1, R11 and K13 as identified by
the analysis of degradation products in serum.
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DISCUSSION
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Activity is enhanced by increased amphiphilicity and cationic moment in a helical conformation
It is well known that an overall positive charge is generally important for the bioactivity of antimicrobial
peptides, thus it was perhaps not surprising to see that substitution of any of the Lys or Arg residues with
Ala resulted in reduced activity. Similarly, replacement of the anionic Asp9 with Ala resulted in an
increase in activity against all tested pathogens. For a more detailed analysis of the data from our Ala and
Lys scans it is useful to consider the likely active conformation of KR-12. Whereas KR-12 is unstructured
in solution, it adopts a well-defined three turn amphiphilic α-helical structure in dioctanoyl
phosphatidylglycerol (D8PG) micelles,[10a] displaying distinct hydrophilic and hydrophobic surfaces as
shown in Figure 6. A similar conformation is likely adopted in phospholipid bilayers, such as bacterial
membranes, which presumably is important for membrane perturbing properties with resulting
bactericidal activity.
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Asp9 is positioned centrally along the polar side of an α-helical KR-12. Replacing it with either Ala or
Lys has a major influence on the overall cationic moment. This rationale also accounts for the benefit of
the Gln to Lys modification at position 5, with Gln5 positioned adjacent to Asp9 on the positively
charged surface. Ala substitutions of large hydrophobic residues forming the hydrophobic sector along
KR-12 helical axis also reduce activity: notably at Ile7, Phe10 and Leu11 rather than Ile3 and Val4. This
suggests that the C-terminal region of the helix is more important than the N-terminal part for activity.
The significance of the position of the substitution is best demonstrated by the I3K and I7K variants,
which have the same overall charge and hydropathy index (+5, -1.408), but differ substantially in
antibacterial activity. Introduction of a positive charge in the middle of the hydrophobic surface, at
position 7, is detrimental for activity. However, at position 3 a similar substitution of a hydrophobic
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amino acid instead controls selectivity as it increases activity against E. coli and P. aeruginosa, but no
activity change against S. aureus. The importance of the positively charged residues and their position in
the amphiphilic helix of KR-12 has been reported previously. Substitutions of Arg6 and Lys8 located in
the interface of the amphiphilic helical structure have been reported to not only lead to reduced activity,
but in their absence the interactions between the peptide and the lipids are less stabilizing for the
structure, evident from a drop in the helicity of this KR-12 variant in D8PG micelles.[18]
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The overall most activity enhanced analogue of the double substitutions was Q5K,D9A, which shows an
eight-fold improvement in activity against S. aureus, P. aeruginosa and C. albicans and a two-fold
improvement against E. coli. The Q5K,D9K variant, which confers an even higher increase in dipole
moment, was slightly less active than Q5K,D9A against S. aureus and C. albicans. This may suggest that
too high density of the repulsive positive charges, which is detrimental to helix formation, also has a
negative influence on bioactivity.[19]
Strain specific effects of Ala and Lys substitutions
KR-12 itself displays the highest antimicrobial activity against E. coli (Supporting Information Table 2).
Substitutions changed both general potency and susceptibility between targets. For example, substitutions
of Lys and Arg to Ala in the monomers lead to weaker activity against Gram-negative compared to Gram-
positive bacteria. This may be explained by the high anionic phospholipid content in these bacterial
membranes that augment the importance of charge interactions in Gram-negatives. Lys to Arg
substitutions of KR-12 have been shown previously to be favourable for activity against E. coli,[18] and we
did observe a marginal decrease in activity against E. coli for all three Arg-to-Lys substitutions.
Interestingly, an R6A mutation (with amidated C-terminal) was reported to gain in activity against E. coli
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K-12 strain by Mishra et al,[18] but in our hands this change resulted in reduced activity against all
organisms. A possible reason for these seemingly conflicting results is the +2 difference in net charge
resulting from the terminal amidation. The R6A substitution for the native sequence results in a
proportionally greater decrease in net charge, while for the amidated variant, the more marginal decrease
in cationicity is likely outbalanced by an increase in helicity conveyed by Ala. Residues Ile3 and Val4
appear to be key positions to modulate bacterial selectivity, introduction of a positive charge at either
position improves activity against Gram-negative, while the absence of hydrophobic residues on these
positions has a negative effect on C. albicans in particular.
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Activity on liposomes suggests variations in mode of action
Activity of KR-12 was weaker on the E. coli liposomes compared to activity on the corresponding
bacteria. KR-12 was also substantially less potent than LL-37 on this phospholipid composition, and did
not share its steep sigmoidal dose-response behaviour. Plateauing dose-responses not dissimilar from the
present data have been observed for EFK17 (a KR-12 overlapping LL-37 fragment) on other membrane
liposome systems [20] and for other AMPs on the same liposome system [21]. However, both LL-37 and the
KR-12 analogues show similar leakage kinetics resembling a steep logarithmic function with an
immediate effect that gradually subsides within minutes until leakage reaches a plateau (for leakage
kinetics of LL-37, see Strömstedt et al 2017 [14]). The liposomes used in the current assay were based on
an E. coli polar lipid extract, and are comparable in terms of charge and phospholipid composition to the
outer membranes of both P. aeruginosa and E. coli. Both these bacteria and the E. coli liposomes have
negative zeta potentials in the range of 41-48 mV [21-22], due to a high proportion of anionic head groups
among the phospholipid. However, the model system does not account for inter-leaflet heterogeneity of
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phospholipids in bacteria, or the presence of LPS and other non-phospholipid components that influence
specific interactions and mechanisms.
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Similar to the results from the antibacterial assay, substitutions of Gln5 to Lys and Asp9 to either Lys or
Ala increased potency the most (especially in the low concentration range). Substitutions by either Ala or
Lys of hydrophobic residues Ile7, Leu11 and Phe10 proved particularly detrimental to membrane
permeabilization properties in the liposome assay, followed by residues I3 and V4. The positive effects of
substitutions at positions 5 and 9 were subordinate to the negative influence of hydrophobic substitutions.
Hence, these hydrophobic residues proved to be essential for activity. These results may be explained by
the helical wheel projection that illustrates the distinct hydrophobic and hydrophilic sectors of KR-12:
interruptions of these properties have substantial effects on potency (Figure 6).
As described above, the KR-12 analogues followed the same trends in the liposome assay as in the
antimicrobial assay regarding effects of substitutions within each scan. This suggests that membrane
disruption is indeed the primary mode of action that is dissected by the sequence variables in this study.
However, substitutions of hydrophobic residues had the greatest negative influence on activity in the
liposome assay, whereas in the bacterial assay cationic residues appear more important. This, and overall
weak liposome leakage potency, suggests that factors not represented in this liposome system may be
involved in the antibacterial activity. In this context, it is interesting to note that the parent peptide LL-37
has been suggested to have intracellular targets in C. albicans.[23]
Activity enhancing substitutions of KR-12 are achieved without elevated cytotoxicity
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In this study, KR-12 and analogues showed low toxicity against the human lymphoma cell-line, and the
difference to the high toxicity of the parent peptide LL-37 was striking. Similarly, KR-12 and analogues
did not exhibit dose-dependent haemolysis, in contrast to LL-37, within the same concentration range (up
to 80 μM). This is in agreement with previous studies that have attributed low levels of haemolytic
activity (10% haemolysis at 100 μM) and toxicity against mammalian cells (TC50 of >63 μM) to KR-
12.[10b, 18] Cancer cells are often more sensitive to AMPs than healthy cells, due to enrichment of anionic
phosphatidylserine in the outer leaflet,[24] making them easier to target by cationic peptides. Importantly,
for our lymphoma cell line, the low toxicity of KR-12 was retained also for the Lys and Ala double
substitutions, highlighting that the KR-12 template is amenable to modifications that improve activity
without compromising target selectivity.
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KR-12 is unstable under biological conditions but the template has potential for further
improvement in stability
KR-12 itself was found to have poor stablility in serum, but Ala substitutions at the susceptible cleavage
sites, mainly R12A and to a lesser significance R6A, were found to display enhanced biological stability.
These results indicate the need and potential to improve stability of KR-12-based AMPs in future
antimicrobial drug designs.
KR-12 peptides as potential therapeutics
The apparent low commercial success rate in the development of new antibiotics has likely at least in part
been due to research mainly being focused on tweaking of traditional antimicrobials to meet the growing
resistance problem. The realization that new mechanisms and targets are required has again increased the
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interest and efforts focusing on AMPs. From that perspective, KR-12 is promising because it appears to
be highly selective for pathogens compared to host cells. Moreover, it is small and easy to synthesize.
Analogues of KR-12 have also shown promise for combination therapy; synergistic effects have been
reported for Lys-substituted variants of KR-12 when tested together with three conventional antibiotics
against multi-drug-resistant Pseudomonas aeruginosa (MDRPA), presumably due to their facilitation of
intracellular uptake of antibiotics.[25] With future work focusing on stability and distribution in vivo it
represents a promising template for further development.
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CONCLUSION
The design of novel host defence peptides requires the optimization of multiple parameters. In this study
the balance of hydrophobic to cationic residues, location of specific amino acids in the peptide three-
dimensional structure, and their contribution to peptide’s helicity were assessed as important factors for
antimicrobial efficacy of KR-12. We have confirmed the importance of the amphiphilic nature of KR-12
for activity and identified positions suitable for modifications to improve activity, Gln5 and Asp9, and
positions that may be explored to control selectivity towards different microbial strains, Ile3 and Val4.
These data may provide a basis for development of further improved antimicrobial peptide leads based on
KR-12.
ACKNOWLEDGMENT
This work was supported by the Swedish Research Council (#2011-3403) awarded to UG and a
postdoctoral research grant to SG from the Carl Tryggers Institute, Stockholm (#CTS 10: 126 and CTS
11: 169). KJR is an Australian Research Council Future Fellow. AAS was funded in part by The Swedish
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Society of Medicine (Grant #SLS-254511). We thank Camilla Eriksson and Damon Wallin Fahimi for
assistance with peptide synthesis.
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FIGURE LEGENDS
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Figure 1. LL-37 and its active fragment KR-12. Sequence and three-dimensional structure of LL-37
(PDB code 2k6o) with the C-terminal region identified as the minimal antibacterial region KR-12
highlighted.[10a] The standard numbering system for amino acid residues was followed for KR-12, thus the
sequence was numbered from N-terminal Lys1 to C-terminal Arg12. Each peptide analogue was named
according to the single letter symbol of the original residue at the substitution site, followed by its
sequence position number (e.g. I3 or D9) followed by the substituting residue symbol (A or K).
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Figure 2. Relative MICs for LL-37 and KR-12 analogues. The relative MIC was derived by dividing
the median MIC value for each peptide variant against a specific organism by the median MIC of native
KR-12 against that organism. These KR-12 MICs are 2.5 μM for Escherichia coli (EC) and 10 μM for -
Pseudomonas aueroginosa (PA), Staphylococcus aureus (SA), and Candida albicans (CA). A) Ala
substitutions, B) Lys substitutions, C) double and triple Ala/Lys substitutions.
Figure 3: Cytotoxicity data for LL-37 and KR-12 analogues. The cell toxicity data is generated from
hydrolysis of the probe fluorescein diacetate by esterases in cells with intact plasma membranes using the
human lymphoma cell line U-937 GTB. Results are means from triplicates with standard deviation and
the IC50 level is highlighted by a double line. The dose-response curve for LL-37 has been constrained
within 100% to compensate for elevated fluorescein hydrolysis by intermediate LL-37 concentrations.
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Figure 4. E. coli liposome leakage activity. Liposomes made from E. coli polar lipid extract were
monitored for fluorescent marker leakage. The data represent the leakage levels reached after 45 min of
peptide incubation. A) Ala substitutions B) Lys substitutions C) double/triple substitutions. Results are
shown as means from triplicate experiments with standard deviations.
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Figure 5. Serum stability of selected KR-12 analogues with corresponding sites of proteolytic
digestion. Analysis of cleavage patterns reveal that R12 and R6 are primary cleavage sites.
Figure 6. Helical wheel projection of KR-12. In an α-helical conformation the separation of polar and
nonpolar residues, on each side of the helix-normal, is distinct. The Gln5 (diamond) and Asp9 (triangle),
represent the prominent activity-increasing substitution sites, where Lys and Ala substitutions either
increase the positive net charge of the polar side, or where the helix propensity of Ala could have a
positive influence. Substitutions that interrupt this polar-nonpolar separation in the helical arrangement
have negative effect on activity.
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REFERENCES
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