Structure-Activity and a'Toxicity Relationships of the Antimicrobial Peptide Tachyplesin-1

Article abstract of DOI:10.1021/acsinfecdis.7b00123

Tachyplesin-1 (TP1; 1) is a cationic β-hairpin antimicrobial peptide with a membranolytic mechanism of action. While it possesses broad-spectrum, potent antimicrobial activity, 1 is highly hemolytic against mammalian erythrocytes, which precludes it from further development. In this study, we report a template-based approach to investigate the structure-function and structure-toxicity relationships of each amino acid of 1. We modulated charge and hydrophobicity by residue modification and truncation of the peptide. Antimicrobial activity was then assessed against six key bacterial pathogens and two fungi, with toxicity profiled against mammalian cells. The internal disulfide bridge Cys7-Cys12 of 1 was shown to play an important role in broad-spectrum antimicrobial activity against all pathogenic strains tested. Novel peptides based on the progenitor were then designed, including 5 (TP1[F4A]), 12 (TP1[I11A]), and 19 (TP1[C3A,C16A]). These had 26-to 64-fold improved activity/toxicity indices and show promise for further development. Structural studies of 5 (TP1[F4A]) and 12 (TP1[I11A]) identified a conserved β-hairpin secondary structure motif correlating with their very high stablility in mouse and human plasma. Membrane binding affinity determined by surface plasmon resonance confirmed their selectivity toward bacterial membranes, but the degree of membrane binding did not correlate with the degree of hemolysis, suggesting that other factors may drive toxicity.

Full text of DOI:10.1021/acsinfecdis.7b00123

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Article
Structure-activity and -toxicity relationships
of the antimicrobial peptide tachyplesin-1
Ingrid Alexia Edwards, Alysha G Elliott, Angela Maria Kavanagh,
Mark Arnold Thomas Blaskovich, and Matthew A. Cooper
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00123 • Publication Date (Web): 29 Sep 2017
Downloaded from http://pubs.acs.org on September 30, 2017
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Structure-activity and -toxicity relationships of
the antimicrobial peptide tachyplesin-1
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Ingrid A. Edwards, Alysha G. Elliott, Angela M. Kavanagh, Mark A.T. Blaskovich and Matthew A.
Cooper*
306 Carmody Road (Building 80), Institute for Molecular Bioscience, The University of Queensland,
Brisbane, Queensland 4072 Australia
Phone: +61 7 33462044
Fax: +61 7 33462090
Correspondence to: m.cooper@uq.edu.au
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Tachyplesinꢀ1 (1) is a cationic βꢀhairpin antimicrobial peptide with a membranolytic mechanism of
action. Whilst it possesses broadꢀspectrum, potent antimicrobial activity, 1 is highly hemolytic against
mammalian erythrocytes, which precludes it from further development. In this study we report a templateꢀ
based approach to investigate the structureꢀfunction and structureꢀtoxicity relationships of each amino
acid of 1. We modulated charge and hydrophobicity by residue modification and truncation of the
peptide. Antimicrobial activity was then assessed against six key bacterial pathogens and two fungi, with
toxicity profiled against mammalian cells. The internal disulfide bridge Cys7ꢀCys12 of 1 was shown to
play an important role in broadꢀspectrum antimicrobial activity against all pathogenic strains tested.
Novel peptides based on the progenitor were then designed, including 5 (TP1[F4A]), 12 (TP1[I11A]) and
19 (TP1[C3A,C16A]). These had 26ꢀ to 64ꢀfold improved activity/toxicity indices, and show promise for
further development. Structural studies of 5 (TP1[F4A]) and 12 (TP1[I11A]) identified a conserved βꢀ
hairpin secondary structure motif correlating with their very high stablility in mouse and human plasma.
Membrane binding affinity determined by surface plasmon resonance confirmed their selectivity toward
bacterial membranes, but the degree of membrane binding did not correlate with the degree of hemolysis,
suggesting that other factors may drive toxicity.
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KEY WORDS
Tachyplesinꢀ1; antimicrobial peptide; βꢀhairpin; antimicrobial activity/toxicity index; amphiphilicity,
membrane, antibiotic, resistance
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Increasing rates of bacterial resistance to antibiotics has fueled a dire need for the discovery of new
replacement drugs for obsolete antibiotics.1 Antimicrobial peptides (AMPs) are common in nature, being
essential components of the innate immune system in the majority of multicellular organisms. AMPs have
been well studied over three decades with more than 2000 AMPs reported from eukaryotes
(http://aps.unmc.edu/AP/main.php). AMPs, being short, cationic and amphipathic, are characterized with
broad range antimicrobial activity resulting from a membranolytic mechanism of action, which
consequently offers a low rate of resistance development. Tachyplesinꢀ1 (TP1) 1 is a member of the
closely related tachyplesin and polyphemusin families, both originating from the horseshoe crab.2,3 1 is a
17 amino acid AMP (KWCFRVCYRGICYRRCR) extracted from the hemocytes of the horseshoe crab,
Tachypleus tridentatus. 1, constrained by two disulfide bridges (Cys3ꢀCys16, Cys7ꢀCys12), forms a βꢀ
hairpin structure (Figure 1), both in aqueous solution and in lipidꢀmimicking environments.4,5 1 exhibits
potent activity against Gramꢀpositive (G+ve) and Gramꢀnegative (Gꢀve) bacteria, as well as fungi. 1 did
not induce resistance in short term studies6, but caused decreased susceptibility under longꢀterm
continuous selection conditions7. 1 has been shown to compromise the integrity of both the outer and
cytoplasmic cell membranes of Escherichia coli.8 In addition to its high antimicrobial activity, 1
unfortunately also displays high toxicity towards mammalian cells, a common detrimental characteristic
of many AMPs that renders them unsuitable for therapeutic development. Analogs of 1 have been
reported in the past, with amino acid substitutions around the disulfide bridge regions.9ꢀ13 However, no
systematic study has been reported to ascertain a clear structureꢀfunction relationship for each amino acid
in the sequence. In this report, we have conducted an alanine scan of 1 followed by modifications of the
overall hydrophobicity by replacement at select positions that the Ala scan indicated were amenable to
change. We assessed the effects of amino acid replacements on antimicrobial activity, cytotoxicity, and
hemolytic activity. Furthermore, we evaluated the membrane binding affinity of the most interesting
peptides by surface plasmon resonance (SPR). We then assessed the stability of the most promising
peptides in plasma, and conducted solution phase NMR structrural studies.
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RESULTS AND DISCUSSION
Peptide design
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In this study, we systematically determined the importance of each amino acid in 1 in a search for
therapeutically valuable analogs of 1. A schematic representation of 1 (Figure 1) illustrates the structural
amphipathicity of the peptide. There are two distinct faces; one βꢀsheet (top) composed mainly of
hydrophobic residues and the other one (bottom) being more hydrophilic and charged. Thus, 1 has been
identified as one of the most amphipathic βꢀhairpin AMPs.14
Figure 1 - Schematic representation
of 1 (TP1). Hydrophobic residues in
green and charged residues in blue.
To modulate the properties of a peptide several methodologies are commonly exploited (i) alteration of
the hydrophobicity/amphipathicity by replacing hydrophobic residues with less hydrophobic (e.g. Ala,
Gly) or more hydrophilic (e.g. Ser) residues, (ii) modification of the charge by replacing basic or acidic
residues, (iii) truncation of the peptide, (iv) combination of (i) to (iii).15,16 Here we have chosen a panel of
peptide modifications that encompass all these methods. An alanine scan is commonly employed to
ascertain the importance of individual residues within a peptide. Alanine is the most commonly used
amino acid to study the amino acid function in a peptide because it eliminates sideꢀchain interactions
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without altering the mainꢀchain conformation. It has the ability to form αꢀhelices and/or βꢀsheets, is
flexible and has low hydrophobicity.
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We studied the physicochemical properties of all 1 analogs (see Table 1). The peptides ranged from 2009
Da (29, 16 residues) to 2293 Da (23, 17 residues). The ExPASy ProtParam tool17 was used to calculate
the physicochemical properties of the peptide analogs. The charge of the analogs varied from +4 (29 and
33) to +6 (1), while their pI varied from 9.25 (29 and 33) to 12.71 (9 and 14). GRAVY score (grand
average of hydropathicity index) is a method used to calculate the hydrophobicity/solubility of a peptide.
A negative GRAVY score indicates hydrophilicity and a positive GRAVY score indicates
hydrophobicity. All peptides were hydrophilic except for two, 31 with GRAVY = 0.000, and 33 with a
positive hydrophobic GRAVY = 0.224.
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Table 1 - Amino acid sequences and physicochemical properties of tachyplesin-1 and its analogs
Number
Peptide
H-Sequence-NH2
MW (Da) Charge
pI
GRAVY
1
2
3
4
5
6
7
8
TP1
TP1[K1A]
TP1[W2A]
KWCFRVCYRGICYRRCR
AWCFRVCYRGICYRRCR
KACFRVCYRGICYRRCR
KWAFRVCYRGICYRRSR
KWCARVCYRGICYRRCR
KWCFAVCYRGICYRRCR
KWCFRACYRGICYRRCR
KWCFRVAYRGISYRRCR
KWCFRVCARGICYRRCR
KWCFRVCYAGICYRRCR
KWCFRVCYRAICYRRCR
KWCFRVCYRGACYRRCR
KWCFRVSYRGIAYRRCR
KWCFRVCYRGICARRCR
KWCFRVCYRGICYARCR
KWCFRVCYRGICYRACR
KWSFRVCYRGICYRRAR
KWCFRVCYRGICYRRCA
KWAFRVCYRGICYRRAR
KWCFRVAYRGIAYRRCR
KWAFRVAYRGIAYRRAR
KWCFRRCYAGICYRRCR
RWCFRVCYRGICYRRCR
KWCGRVCYRGICYRRCR
KWCSRVCYRGICYRRCR
KWCFRVCGRGICYRRCR
KWCFRVCYRGGCYRRCR
KWCARVCARGACYRRCR
2263.7
2206.7
2148.6
2217.6
2187.7
2178.6
2235.7
2217.7
2170.0
2178.6
2277.8
2221.7
2217.7
2171.7
2178.7
2178.7
2217.7
2178.7
2203.7
2203.7
2140.5
2239.8
2292.7
2174.6
2204.6
2158.6
2208.6
2054.4
+6
+5
+6
+6
+6
+5
+6
+6
+6
+5
+6
+6
+6
+6
+5
+5
+6
+5
+6
+6
+6
+6
+6
+6
+6
+6
+6
+6
12.29
12.28
12.29
12.29
12.29
12.12
12.29
12.29
12.71
12.12
12.29
12.29
12.29
12.71
12.12
12.12
12.29
12.12
12.29
12.29
11.84
12.29
10.16
9.93
ꢀ0.518
ꢀ0.182
ꢀ0.359
ꢀ0.753
ꢀ0.576
ꢀ0.147
ꢀ0.659
ꢀ0.753
ꢀ0.335
ꢀ0.147
ꢀ0.388
ꢀ0.676
ꢀ0.753
ꢀ0.335
ꢀ0.147
ꢀ0.147
ꢀ0.753
ꢀ0.147
ꢀ0.600
ꢀ0.600
ꢀ0.682
ꢀ0.659
ꢀ0.553
ꢀ0.706
ꢀ0.729
ꢀ0.465
ꢀ0.806
ꢀ0.553
TP1[C3A,C16S]
TP1[F4A]
TP1[R5A]
TP1[V6A]
TP1[C7A,C12S]
TP1[Y8A]
TP1[R9A]
TP1[G10A]
TP1[I11A]
TP1[C7S,C12A]
TP1[Y13A]
TP1[R14A]
TP1[R15A]
TP1[C3S,C16A]
TP1[R17A]
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
TP1[C3A,C16A]
TP1[C7A,C12A]
TP1[C3A,C7A,C12A,C16A]
TP1[V6R,R9A]
TP1[K1R]
TP1[F4G]
TP1[F4S]
TP1[Y8G]
TP1[I11G]
9.93
10.16
9.93
TP1[F4A,Y8A,I11A]
10.16
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KWC ꢀ FVCYRGICYRRCG
AWCARVCYRGICYRRCR
AWCFRVCARGICYRRCR
AWCFRVCYRGACYRRCR
KWCFRVCYAGICYRRCA
TP1[ꢀR5,R17G]
TP1[K1A,F4A]
TP1[K1A,Y8A]
TP1[K1A,I11A]
TP1[R9A,R17A]
2009.4
2131.5
2115.5
2165.5
2094.5
+4
+5
+5
+5
+4
9.25
9.69
9.88
9.69
9.25
ꢀ0.013
ꢀ0.241
0.000
ꢀ0.341
0.224
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Antimicrobial activity, toxicity and cell selectivity
1 was determined previously to possess potent and broadꢀspectrum antimicrobial activity.14 Here we
assessed the antimicrobial activity of 32 analogs of 1 against a broad range of bacteria, both Gꢀve and
G+ve, as well as fungi. Table 2 reports the results of testing against a panel of ATCC reference strains
and drug resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas
aeruginosa (strain details in supplemental Table S1). The majority of the analogs tested retained potent
activity across the range of microbes, even the most resistant ones, as compared to 1, though no analog
displaying increased antimicrobial activity. All peptides, including 1, showed a marked reduction in
activity against Candida albicans as compared to other microbes, with at least 8ꢀfold reduction in
antifungal activity as compared to Cryptococcus neoformans. Similarly, for G+ve bacteria the peptides
had lower potency against Staphylococcus aureus compared to Bacillus subtilis, with antibacterial activity
consistently at least 4ꢀfold lower. For K. pneumoniae, all peptides demonstrated a reduction in activity
between the reference strain, and the multidrugꢀresistant (MDR) strains of 2ꢀ to 8ꢀfold, with no difference
between the various MDR strains. Another 4ꢀ to 32ꢀfold reduction in activity was noted when tested
against an extensively drugꢀresistant (XDR) strain across all peptides. This difference in activity was not
demonstrated in the case of A. baumannii or P. aeruginosa with the MIC activity remaining within a
tight range (~2ꢀfold, i.e. within accepted assay fluctuation range), between reference strains and resistant
strains, including XDR and polymyxin resistant isolates.
The alanine scan showed that single Ala replacements of amino acids at positions 1, 2, 4, 5, 6, 8, 9, 10,
11, 13, 14, 15 or 17 (producing peptides 2, 3, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16 or 18, respectively) gave
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analogs that retained the broadꢀspectrum potent antimicrobial activity of 1; therefore these positions are
presumably not key for the antimicrobial activity of the peptide. An NMR and tryptophan fluorescence
study performed by Kushibiki et al5 concluded that Trp2 inserted into the hydrophobic acyl chains of LPS
and suggested that the residues located at the Nꢀ and Cꢀtermini of 1 are involved in the binding to LPS,
indicating their importance in the mode of action of 1. Our results do not correlate well with those
findings as 3 (TP1[W2A]) showed only 3ꢀfold reduction in antimicrobial activity across all tested strains,
with 2 and 18 also showing good activity. Positions 3, 7, 12 and 16 of 1 correspond to Cys residues,
forming two disulfide bridges (3ꢀ16 and 7ꢀ12) that create the disulfide bridge constrained βꢀhairpin
structure as characterized by several earlier studies.4,5 Removal of the disulfide bridge was achieved via
two different approaches: (i) by Ala replacement of one Cys while the partnering Cys residue was
replaced by Ser, therefore retaining the same hydrophilic properties of CysꢀSH without potential
misfolding of the peptide through mismatched disulfide bond formation18 and (ii) by complete abolition
of the disulfide bridge properties by Ala replacement of both Cys residues. The deletion of the internal
disulfide bridge (7ꢀ12) in 8, 13 or 20 reduced the antimicrobial activity of 1 by 3ꢀ to 12ꢀfold. However,
the deletion of the external disulfide bridge (3ꢀ16) in 4, 17 or 19 more significantly reduced the
antimicrobial activity of 1, by 4ꢀ to 128ꢀfold. Deletion of both disulfide bridges, 21, dramatically reduced
the overall antimicrobial activity by 64ꢀfold across all tested strains, in accordance with the work
performed by Tamamura et al.9 This reduction in antimicrobial activity is presumably due to the
subsequent loss of βꢀsheet stacking, as a direct result of the removal of the disulfide bridges.7,8,15 In our
study, there were several unexpected results. Of the three analogs that removed the external disulfide
bond, 19 (TP1[C3A,C16A]) and 17 (TP1[C3S,C16A]) had similar antimicrobial activities across all
strains tested, losing only 2 to 16ꢀfold activity as compared to 1. However, 4 (TP1[C3A,C16S]) showed
around 100ꢀfold reduction in antimicrobial activity for most strains tested, except for B. subtilis and C.
neoformans where the reduction was only 10ꢀfold. Cys3 is flanked by hydrophobic residues, Trp2 and
Phe4, and Cys16 is flanked by polar residues, Arg15 and Arg17. By inserting the hydrophobic residue
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Ala into position 3 and hydrophilic residue Ser into position 16, the amphipathicity of the peptide was
increased considerably, potentially creating a repulsive effect between the βꢀsheets that led to an unfolded
structure (1H NMR in supplementary information, Figure S13).
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Several
positions
were
selected
for
further
modifications.
The
modulation
of
hydrophobicity/amphipathicity at position 4 was studied by mutating Phe with Ala (F4A) 5, Gly (F4G) 24
or Ser (F4S) 25, resulting in reduced antimicrobial activity, around 2ꢀfold at each mutation, as
hydrophobicity decreased.
A
selection of five peptides (4 (TP1[C3A,C16S]), 19
(TP1[C3A,C7A,C12A,C16A]), 22 (TP1[V6R,R9A]), 28 (TP1[F4A,Y8A,I11A]) and 29 (TP1[ꢀ
R5,R17G])) showed a considerable loss, at least 100ꢀfold, in antimicrobial activity across the full panel of
bacteria and fungi. These five peptides were designed with a range of modifications that comprise
physicochemical properties from all regions of the spectrum of MW, charge, pI and hydrophobicity. No
trend was identified that correlated physicochemical properties with antimicrobial activity, and it is
therefore more likely that the overall peptide structure and sites specific to mode of action are the key
elements to increasing and decreasing antimicrobial potential of the 1 peptide scaffold.
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Table 2 - Antimicrobial activity of tachyplesin-1 and its analogs
MIC (µg/mL)
Bacteria G-ve
Bacteria G+ve
Fungi Yeast
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8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
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Compound
name
colistin
0.015
≤0.03
0.125
0.03
0.03
0.06
0.125
>32
>32
>64
0.06
1
>32
>64
0.125
0.5
>32
>32
meropenem
vancomycin
0.125
1
fluconazole
0.5
0.25
2
8
0.15
0.125
0.25
0.5
4
amphotericin B
1
0.06
0.03
0.25
4
0.06
0.06
0.5
0.25
0.125
1
0.25
0.125
1
2
2
0.06
0.125
0.5
0.125
0.125
0.5
0.125
0.25
0.5
>16
0.5
0.25
1
0.25
0.5
4
0.06
0.03
0.5
0.25
0.5
8
2
8
3
>16
>16
16
8
8
4
16
16
>16
0.5
0.5
1
>16
0.5
>16
0.5
>16
2
0.5
>16
4
>16
8
5
0.125
0.125
0.25
0.5
0.5
0.5
0.5
1
0.25
0.03
0.25
0.25
0.25
0.125
0.03
0.125
0.125
0.06
0.125
0.03
0.125
0.015
0.125
0.125
1
0.5
0.25
1
6
0.125
0.25
0.5
0.06
0.25
0.25
0.125
0.06
0.06
0.25
0.5
0.125
0.5
0.5
2
0.5
4
4
7
16
16
8
8
8
4
2
0.25
0.5
2
4
2
8
0.5
0.5
0.25
0.06
0.5
0.5
0.5
0.25
0.25
0.25
0.25
1
9
0.06
0.125
0.06
0.125
1
0.125
0.03
0.03
0.25
4
0.25
0.25
0.25
0.5
16
0.5
0.125
0.25
0.25
8
1
2
4
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
2
0.125
0.06
0.25
1
0.25
0.25
0.25
8
1
1
2
4
0.5
1
0.5
2
2
8
2
16
8
16
2
16
4
2
0.25
0.125
0.25
0.25
0.015
0.25
0.125
8
0.25
0.06
0.25
1
0.5
0.5
2
0.5
0.25
1
0.125
0.125
0.25
0.25
0.125
0.25
0.25
4
0.25
0.125
0.25
0.25
0.125
0.5
0.5
0.125
4
4
1
0.25
4
0.5
4
8
16
16
4
2
2
1
1
4
4
4
0.125
1
0.25
1
0.25
1
0.25
2
0.5
8
0.5
8
4
>16
16
>16
>16
4
8
0.25
16
2
1
0.25
4
1
2
2
8
1
16
16
>16
>16
0.25
1
>16
>16
0.5
16
>16
16
4
16
>16
1
>16
>16
4
1
0.5
>16
0.125
2
16
>16
0.25
2
16
>16
0.25
2
0.25
0.125
0.5
4
0.125
0.25
0.5
0.5
1
0.125
0.5
0.25
2
>16
>16
>16
8
16
16
16
4
8
2
4
4
0.5
2
4
0.5
>16
>16
8
4
0.5
2
8
8
1
2
8
0.5
4
0.25
8
0.25
>16
>16
4
0.5
>16
>16
4
2
0.5
0.5
1
0.25
>16
1
>16
>16
4
>16
>16
>16
8
>16
16
>16
>16
16
>16
>16
8
>16
>16
>16
4
>16
>16
>16
8
>16
>16
>16
>16
>16
8
>16
4
8
1
2
2
16
>16
2
0.125
0.125
0.03
0.25
0.125
0.06
0.5
0.25
0.5
1
0.25
0.25
0.25
0.5
1
0.5
0.5
0.5
4
0.25
0.5
0.5
0.5
2
0.125
0.06
4
1
4
2
2
0.5
colistin, meropenem, vancomycin, fluconazole and amphotericin B were used as positive inhibitor controls for Gꢀve, G+ve and
fungi, respectively. Data: n = 4. XDR (extensively drug resistant), PmxR (polymyxin resistant)
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The toxicity of the analogs of 1 was tested against Human Embryonic Kidney cells (HEK293) and
human red blood cells (RBC). None of the peptides showed cytotoxicity against HEK293 cells at the
highest tested concentration, 100 ꢁg/mL. Hemolytic activity (MHC) is reported in Table 3 as the
minimum hemolytic concentration that caused 10% hemolysis of fresh RBCs. 1 possessed reasonably
potent hemolytic activity (MHC = 0.25 ꢁg/mL) and most of the peptide analogs did not improve this
quality by decreasing hemolytic potency. However, nine of the analogs (3 (TP1[W2A]), 4
(TP1[C3A,C16S]), 5 (TP1[F4A]), 9 (TP1[Y8A]), 12 (TP1[I11A]), 19 (TP1[C3A,C16A]), 20
(TP1[C7A,C12A]), 22 (TP1[V6R,R9A]) and 30 (TP1[K1A,F4A])) did show promising 32 to 160ꢀfold
reductions in MHC. Similarly to antimicrobial activity, this did not correlate with the physicochemical
properties of the peptides and no trend or correlation could be discerned. Additionally, for some analogs
the reduction in hemolytic activity correlated to the loss of antimicrobial activity, meaning these peptide
sequences essentially lost all biological activity. This is the case for 22, a peptide designed to reduce the
hydrophobicity of 1 without change of charge, reducing the overall amphipathicity of 1. This change was
not beneficial, emphasizing again the importance of peptide amphipathicity to its activity.
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19
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Commonly, the overall therapeutic potential of a peptide is assessed via a cell selectivity or antimicrobial
activity/toxicity index (ATI). The ATI, Table 3, can be improved in one of the following ways (i) increase
in antimicrobial activity, (ii) decrease in hemolytic activity, or (iii) a combination of both, increasing
antimicrobial activity while decreasing hemolytic activity. Here we calculated the ATI from the ratio of
the minimum hemolytic concentration (MHC) to the median of the minimum inhibitory concentrations
(MIC) determined towards all tested bacteria and fungi (MM). Therefore, a larger ATI value indicates
greater desirable cell selectivity. From these ATI calculations, three peptides (5 (TP1[F4A]), 19
(TP1[C3A,C16A]) and 12 (TP1[I11A])) stood out. These analogs had ATI values that were 26ꢀ, 40ꢀ and
64ꢀfold improved, respectively, retaining relatively high antimicrobial activity while dramatically
decreasing hemolytic activity.
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Table 3 - Biological activity and
activity/toxicity indices of tachyplesin-
1 and its analogs
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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50
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60
MMa
(µg/mL)
0.125
0.125
0.4
MHCb
(µg/mL)
0.25
2.1
8
Peptide
ATIc
1
2
3
4
5
6
7
8
2.0
16.8
20.0
0.5
80.0
16.0
4.8
8
40
2
1.2
1
8
2
0.5
32
0.6
1
0.5
0.45
3
0.45
40
8
0.24
11
16
0.5
0.125
0.25
0.5
0.25
0.125
0.125
0.25
1.5
0.25
0.163
0.25
0.5
0.125
0.75
0.375
8
16
0.163
1
2
3
0.5
16
16
6.4
2.0
9
32.0
16.0
4.0
128.0
0.4
4.0
3.1
1.8
6.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
3.6
53.3
21.3
0.0
0.7
6.2
0.5
0.1
0.1
0.4
0.1
0.0
5.0
0.5
0.4
0.5
1
0.47
0.23
0.4
0.2
0.9
0.3
32
0.4
0.2
0.2
0.75
0.5
0.375
aThe median of the peptide Minimum Inhibitory
Concentrations (MICs) against all bacteria and fungi
tested (MM).
bThe MHC was determined as the minimum
hemolytic concentration that caused ≤10%
hemolysis of fresh red blood cells.
cThe activity/toxicity index (ATI) is the ratio of the
MHC to the MM. Larger values indicate a greater
selectivity of the peptides for microbe cells.
The Ala scan peptides that showed an improved ATI, 5 (TP1[F4A]), 9 (TP1[Y8A]) and 12 (TP1[I11A)],
were subsequently substituted with Gly instead of Ala, to see if further reduction in side chain
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hydrophobicity was beneficial. The F4G substitution 24 had similar antimicrobial activity to F4A 5 but
almost 100ꢀfold increase in hemolytic activity. Similarly, while Tyr8 and Ile11 substituted by Ala (9 and
12) retained 1’s antimicrobial activity and reduced its hemolytic activity; the analogs with Gly (26 and
27) had reduced antimicrobial activity while retaining similar levels of hemolytic activity. In a study by
Panteleev et al19, Tyr8 and Ile11 of 1 were substituted by Arg or Ser, in both cases dramatically reducing
the hemolytic activity without detriment to the antimicrobial activity, hence making the compounds very
promising for development as antibiotic candidates. In our study, the replacement of the Tyr8 or Ile11 by
a less hydrophobic amino acid was also shown to be highly beneficial for the cell selectivity of the
peptide. However a fine balance needs to be found, as if the amino acid composition of the peptide
becomes too hydrophilic the toxicity increases significantly. This may be influenced by the overall
conformation of the peptide as well as the specific conformation of the peptide turn. Residues Tyr8 and
Ile11 both form part of the βꢀturn and the replacement of either residue to a Gly would change the type of
structural turn.
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The beneficial mutations seen in analogs 5, 9 and 12 were then combined to see if the effects were
additive. Unfortunately, none of 28, 30, 31, 32 and 33 showed the desired improvements; while they kept
relatively potent antimicrobial activity (except for 28 and 30), they also concomitantly retained high
hemolytic activity (except for 30).
Another method to increase the activity/toxicity index of 1 was reported by Saravanan et al,12 who deleted
the four Cys residues of 1 without replacement (peptide named CDT). In this case, the βꢀhairpin structure
was less rigid but conserved in solution and in the presence of LPS, due to hydrogen bonding and packing
interactions across the sheets, allowing the peptide to maintain potent antimicrobial activity while
reducing its hemolytic activity. Wood et al,20 studied the SAR of CDT, finding that two analogs [desꢀ
Arg12,13] CDT and [Ile2,3,6,10] CDT increased the selectivity index.
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Peptide membrane binding followed by Surface Plasmon Resonance
6
7
To investigate the cell selectivity, we determined the peptide membrane binding affinity of select peptides
8
9
by SPR. We selected two model membranes, one mimicking the outer leaflet of the Gꢀve bacteria E. coli
(taken as representative of the AMP antimicrobial activity) and the other one mimicking the outer leaflet
of the human red blood cell (taken as representative of the AMP hemolytic activity). Bacterial membranes
are more negatively charged than mammalian cells due to their lipid composition. Moreover, the cell
composition of the outer leaflet of bacterial membranes differs between classes. E. coli is predominantly
composed of palmitoyloleoylꢀphosphatidylethanolamine (POPE), palmitoyloleoylꢀphosphatidylglycerol
(POPG) and cardiolipin, and was therefore modelled as POPE/POPG/cardiolipin (15:4:1) in our study.21
In contrast, about half of the mass of the RBC membrane is comprised of protein and the other half is
lipids, predominantly phospholipids with the addition of cholesterol.22 Palmitoyloleoylꢀ
phosphatidylcholine (POPC), a zwitterionic phospholipid forming a fluid bilayer at room temperature, is
the major component of eukaryotic cells. However, cholesterol also plays a key role in the membrane
composition of RBCs. Therefore, POPC/cholesterol (4:1) was used here as a simplistic model to reflect
the charge and fluidity of the outer leaflet of RBCs. Matsuzaki et al.23 stated that hemolytic peptides
exhibited strong interactions with zwitterionic phospholipids, whereas nonꢀhemolytic peptides did not.
We thus investigated the relative membrane binding affinities of the three peptides possessing the highest
ATI values (5, 12 and 19), as well as less active 1 and 27, to assess this hypothesis. In our study (Figure
2), all five peptides exhibited a stronger interaction to POPE/POPG/cardiolipin, modelling the outer
leaflet of E. coli, as opposed to POPC/cholesterol, modelling the outer leaflet of RBCs. The cell
selectivity was relatively similar amongst the five peptides. These data do not correlate with the findings
of Matsuzaki et al.23 and did not correspond with our observed antimicrobial and hemolytic activity and
hemolytic activity testing, which showed that all five peptides had similar antimicrobial potency but
differed by their hemolytic activity. 1 and 27 exhibited high hemolytic activity compared to the other
three peptides. We would therefore have expected to see an increase in binding affinity to RBC with 1
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and 27. This lack of correlation between the model membrane binding affinities and the biological data
could be due to several hypotheses (i) our RBC model is too simplistic and does not reflect the full
complexity of what occurs in vitro with live cells, as the RBC membrane is a very complex ensemble of
two asymmetric layers,24,25 composed mainly of phosphatidylcholine (PC), sphingomyelin (SM) and
cholesterol, or (ii) the affinity for the peptide to bind to the membrane is only the first step in the mode of
action for activity. It is likely that the peptide not only needs to bind to the membrane but must also act to
disrupt the cell to exhibit activity and cause cell death. This is in accordance with the findings of Imura et
al,26 suggesting a lipid flipꢀflop with toroidal pore formation mechanism of action.
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Figure 2 - Membrane binding affinity of tachyplesin-1 and its analogs as monitored by surface
plasmon resonance. The response to the total amount of lipid deposited on the chip surface was
normalized by calculating the peptideꢀtoꢀlipid ratio (P/L mol/mol) (1RU = 1 pg.mmꢀ2). Sensorgrams
obtained upon injection of 64 ꢁg/mL of peptide over (A) POPC/Cholesterol (4:1) [RBC] or (B)
POPE/POPG/cardiolipin (15:4:1) [E. coli] bilayers. (C) Comparison of the P/L obtained for the five
peptides at the reported point at the end of the association phase over both model bilayers. Error bars
represent standard error of the mean of three replicates.
Plasma stability
Plasma stability is an important parameter for potential peptidic drug candidates. Compounds with certain
functional groups are more susceptible to hydrolysis by plasma enzymes than others. These include esters,
amides, lactones, lactams, carbamides, sulfonamides and peptide mimetics. The plasma stability assay is
designed to focus on these classes of compounds. To evaluate the stability of the three peptides
possessing the highest ATI values (5, 12 and 19), we performed a plasma stability assay against mouse
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and human plasma over 2 hours. The percentage of peptide remaining in plasma was followed by LCꢀMS,
as shown in Figure 3. Interestingly, 1, 5 and 12 remained fully stable in both mouse and human plasma,
however, 19 was rapidly degraded to leave only 13% or 52% after two hours, in mouse and human
plasma, respectively. Loss of the disulfide bridge in 19 TP1[C3A,C16A] improved the activity/toxicity
relationship but made 19 TP1[C3A,C16A], more susceptible to hydrolysis in the plasma environment.
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19
20
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
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60
Figure 3 – Plasma stability of tachyplesin-1 and its analogs as monitored by LC-MS. Stability of 1
TP1(black), 5 TP1[F4A] (red), 12 TP1[I11A] (blue) and 19 TP1[C3A,C16A] (green) in mouse (dotted
line) and human (full line) plasma over 120 min. Points represent the average of two replicates.
Structural characterization
To better understand the observed variation of peptide stability in plasma, the structural characterization
of the four peptides was investigated. NMR spectroscopy was used to confirm the folding of the TP1
analogs and to compare their structures. The NMR assignment of 1 was comparable to the chemical shift
assignment reported previously, BMRB 5486.27 A comparison of the Hα chemical shifts of the four TP1
analogs (Figure 4A) illustrates that the structures are conserved for 1, 5 and 12. However, 19 shows very
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different Hα chemical shifts as compared to the other three analogs, demonstrating that its secondary
structure is not conserved. In Figure 4B, secondary chemical shifts were calculated by comparing the
experimentally observed Hα chemical shifts (HA) for each residue with the chemical shifts observed for
the corresponding residues in random coil (RC) peptides28, to compare 1 and its analogs in relation to
sheet formation. 1, 5 and 12 show a similar pattern, with defined βꢀhairpin structure as compared to 19,
where the βꢀhairpin motif is lost due to the reduction of length of the first βꢀsheet region, from Trp2ꢀCys7
to Arg5ꢀCys7. It is logical to attribute this to the loss of the disulfide bridge Cys3ꢀCys16, rendering the Nꢀ
and Cꢀterminal more flexible and reducing the size of the Nꢀterminal βꢀsheet, changing the overall βꢀ
hairpin secondary structure motif.
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A
5
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1.5
1.0
1 TP1
B
5 TP1[F4A]
12 TP1[I11A]
19 TP1[C3A,C16A]
0.5
0.0
I11A
C16A
C3A
-0.5
-1.0
F4A
Figure 4 – TP1 and analogs chemical shifts. (A) Hα chemical shifts and (B) Hα secondary chemical
shifts of TP1 1 (black) and analogs 5 (red), 12 (blue) and 19 (green). Secondary chemical shifts were
calculated by comparing the experimentally observed Hα chemical shifts (HA) for each residue with the
chemical shifts observed for the corresponding residues in random coil (RC) peptides.28 The sequence
mutations are annotated.
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CONCLUSION
5
6
This work presents a structureꢀfunction relationship study of tachyplesinꢀ1 by a systematic amino acid
7
8
replacement strategy with characterization of the resultant antimicrobial, cytotoxicity and hemolytic
9
activity. We identified three modified
peptides: 5 (TP1[F4A]), 12 (TP1[I11A]) and 19
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
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(TP1[C3A,C16A]), that possessed substantially improved therapeutic indexes (26ꢀ to 64ꢀfold) over the
progenitor. With high potency against broadꢀspectrum microbes and considerably less hemolytic toxicity,
these compounds may be considered as promising hit compounds for further development towards new
treatments for infections caused by Gramꢀnegative and Gramꢀpositive bacterial pathogens, as well as
potential fungal infections. Structural studies and preliminary ADME testing support further
investigations of two of the three promising novel antimicrobial peptides, as 5 and 12 not only conserve
the βꢀhairpin secondary structure motif but are highly stable in mouse and human plasma. Additional
studies examining bioavailability, pharmacokinetics, toxicity, mutagenicity and in vivo efficacy of 5 and
12 are required to further validate these interesting novel antibiotics.
MATERIALS AND METHODS
Materials
Tachyplesinꢀ1 and tachyplesinꢀ1 analogs were synthesized by Mimotopes Pty Ltd (Clayton, Australia)
and WuXi AppTech (Shanghai, China) using Fmocꢀbased solid phase peptide synthesis and orthogonal
Cys protection (Fmoc Cys(Acm)ꢀOH for Cys3, Cys16, and Fmoc Cys(Trt)ꢀOH for Cys7, Cys12); the first
disulfide bridge was formed by air oxidation following cleavage from the resin, with iodine treatment
generating the fully cyclized peptide that was purified by RPꢀHPLC to > 90% purity. Identity and purity
were confirmed by LCꢀMS, HRMS and 1H and 2D TOCSY NMR (Figures S1 to S104). Proton chemical
shifts of 1 (TP1) were comparable of previously reported tachyplesinꢀ1 (PDB ID 1ma2 and BMRB 5486)
and taken as reference for the identity confirmation of 1 analogs.27 Synthetic lipids palmitoyloleoylꢀ
phosphatidylcholine (POPC), palmitoyloleoylꢀphosphatidylglycerol (POPG), palmitoyloleoylꢀ
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phosphatidylethanolamine (POPE) and cardiolipin were purchased from Avanti Polar Lipids (Alabaster,
AL). All chemicals were used without further purification. The tachyplesin analogs used in this study do
not contain any structures with potential PAINS (Pan Assay Interference Compounds) liabilities.
9
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Antimicrobial activity
Both antibacterial and antifungal assays were performed by a broth microꢀdilution plate based method as
per CLSI guidelines for antimicrobial susceptibility testing.29, 30 The assay was performed in Mueller
Hinton Broth (MHB, Bacto laboratories, 211443) for antibacterial testing and Yeast ExtractꢀPeptone
Dextrose (YPD, SigmaꢀAldrich, Y1500) for antifungal testing, and the Minimum Inhibitory
Concentration (MIC) was determined as the lowest concentration of compound that prevented all visible
microorganism growth after 18ꢀ24 h. Compounds were prepared at 0.64 mg/mL solution in water:DMSO
(4:1).
For antimicrobial assays, the tested strains, Table S1, were cultured either in Luria broth (LB) (In Vitro
Technologies, USB75852), Nutrient Broth (NB) (Becton Dickson, 234000) or MHB at 37 °C overnight.
A sample of culture was then diluted 40ꢀfold in fresh MHB and incubated at 37 °C for 1.5ꢀ2 h. The
compounds were serially diluted twoꢀfold across the wells of 96ꢀwell plates (Corning 3641, Nonꢀbinding
Surface [NBS]), with compound concentrations ranging from 0.015 ꢁg/mL to 64 ꢁg/mL, plated in
duplicate. The resultant midꢀlog phase cultures were diluted to the final concentration of 1×106 CFU/mL,
then 50 µL was added to each well of the compoundꢀcontaining plates giving a final compound
concentration range of 0.008 ꢁg/mL to 32 ꢁg/mL and a cell density of 5x105 CFU/mL. All plates were
then covered and incubated at 37 °C for 18 h. Resazurin was added at 0.001% final concentration to each
well and incubated for 2 h before MICs were read by eye. Resazurin was added to assist MIC
determination as small spindle tissue or precipitate was present in otherwise clear wells.
For the antifungal assay, fungi strains, Table S1, were cultured for 3 days on YPD agar at 30 °C. A yeast
suspension of 1x106 to 5x106 CFU/mL was prepared from five colonies. These stock suspensions were
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diluted with Yeast Nitrogen Base (YNB) (Becton Dickinson, 233520) broth to a final concentration of
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2.5×103 CFU/mL. The compounds were serially diluted twoꢀfold across the wells of 96ꢀwell plates
(Corning 3641, Nonꢀbinding Surface [NBS]), with compound concentrations ranging from 0.015 ꢁg/mL
to 64 ꢁg/mL and final volumes of 50 ꢁL, plated in duplicate. Then, 50 ꢁL of the fungi suspension that
was previously prepared in YNB broth to the final concentration of 2.5×103 CFU/mL, was added to each
well of the compoundꢀcontaining plates, giving a final compound concentration range of 0.008 ꢁg/mL to
32 ꢁg/mL. Plates were covered and incubated at 35 °C for 24 h. C. albicans MICs were determined by
measuring the absorbance at OD530. For C. neoformans resazurin was added at 0.006% final concentration
to each well and incubated for a further 3 h before MICs were determined by measuring the absorbance at
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OD570ꢀ600
.
Hemolytic assay
The ability of the peptides to hemolyse human red blood cells was determined by a previously reported
method.31 Human red blood cells were separated from serum and washed in phosphateꢀbuffered saline
(PBS, pH 7.4) with 4–5 times centrifugation at 4000 rpm. Erythrocytes were then resuspended in
phosphateꢀbuffered saline as a 0.25% (v/v) solution. Stock solutions (300 ꢁM) of compounds were
prepared. Twoꢀfold serial dilutions were prepared yielding eight solutions in total, which were aliquoted
(20 ꢁL) into a 96ꢀwell plate (NUNC 96 wells polypropylene plates round bottom, Sigma Aldrich P6866).
The erythrocyte solution (100 ꢁL) was dispensed into each well and incubated with diluted peptide
solution at 37 °C for 1 h. Triton Xꢀ100 solution (1%, v/v, 20 ꢁL) was used as a positive control to
represent 100% cell lysis, and PBS solution (20 ꢁL) was used as a negative control (spontaneous lysis
after 1 h incubation). The 96ꢀwell plate was centrifuged to pellet intact red blood cells, and the
supernatant of each well was collected in a fresh plate (NUNC 96 wells polypropylene plates flat bottom,
Sigma Aldrich P7366) and measured by visual absorption spectroscopy at 415 nm. The percentage of
hemolysis was calculated according to the following equation:
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ꢈ
ꢉ
ꢈ
ꢉ
ꢋꢌꢍꢎꢏ ꢆꢐꢂꢑꢄꢁ − ꢋꢌꢍꢎꢏ ꢒꢁꢓꢃ ꢄꢅꢆꢇꢆ
ꢈ ꢉ
ꢀꢁꢂꢃꢄꢅꢆꢇꢆ % = ꢊ
ꢔꢕ100%
ꢈ
ꢉ
ꢈ
ꢉ
ꢋꢌꢍꢎꢏ 100% ꢄꢅꢆꢇꢆ − ꢋꢌꢍꢎꢏ ꢒꢁꢓꢃ ꢄꢅꢆꢇꢆ
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All peptide solutions were assayed in triplicate.
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Cytotoxicity
Cytotoxicity to HEK293 cells was determined using the resazurin assay.32,33 In brief, HEK293 cells were
seeded as 4000 cells/well in blackꢀwall, clearꢀbottom 384ꢀwell plates (Corning, Australia) and incubated
for 24 h at 37 °C, 5% CO2. Compounds were then added into each well. After 24 h incubation, 5 µM
resazurin was added per well and incubated at 37 °C for 2 h. Fluorescence intensity was then read using a
Polarstar Omega with excitation/emission 560/590. The data were then analyzed using GraphPad Prism 6
software. CC50 values were calculated using the following equation: Percentage Viability = (FITEST
FINegative/FIUNTREATED –FINegative)*100.
–
Surface Plasmon Resonance
The binding affinity of 1 and its analogs for model membranes composed of POPC/Cholesterol (4:1) and
POPE/POPG/cardiolipin (15:4:1) were compared using SPR. Solutions were freshly prepared and filtered
(0.22 µM pore size). L1 sensor chip and BIAcore 3000 system (Biacore, GE Healthcare) were used.
Small unilamellar vesicles (diameter, 50 nm) were prepared by freezeꢀthaw fracturing and sized by
extrusion. Vesicle suspensions were deposited on an L1 chip over 40 min at a flow rate of 2 µL/min to
allow the vesicles to bind, fuse and form a stable lipid bilayer. The association of the peptide sample to
the lipid bilayer was evaluated by injection for 240 s at flow rate of 5 µL/min and the dissociation was
followed for 600 s at the same flow rate. The L1 chip was regenerated after each injection cycle, by
injection of 3ꢀ[(3ꢀcholamidopropyl)dimethylammonio]ꢀ1ꢀpropanesulfonate (CHAPS) (5 µL/min, 60 s),
followed by an injection of 10 mM sodium hydroxide in 20 % methanol (w/w) (50 µL/min, 60 s) and to
finish an injection of 10 mM sodium hydroxide (50 µL/min, 36 s) buffer. All measurements were
conducted at 25 °C. 10 mM HEPES buffer pH7.4 containing 150 mM NaCl (to mimic the physiological
conditions) was used as running buffer and to prepare samples and lipid vesicles.34,35 The peptide samples
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were prepared at 7 different concentrations, starting from 64 to 1 µg/mL following 2ꢀfold dilution steps.
The lipid deposited onto the chip surface is dependent on the lipid mixture, therefore, the response units
were converted into peptide to lipid ratio (mol/mol).
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Plasma stability
Test compound (final concentration 2 ꢁM) was combined with preꢀwarmed CDꢀ1 mouse or human
plasma at 37 °C (100 ꢁL volume). At each time point (t = 0, 15, 30, 60 and 120 min), 100 ꢁL of 4%
H3PO4/H2O was added and the reaction was quenched with 600 ꢁL of cold stop solution (200 ng/mL
tolbutamide plus 20 ng/mL buspirone in 50% ACN/MeOH). Upon centrifugation at 4,000 g for 10 min,
the clear supernatants (100 ꢁL) were transferred to a 96ꢀwell plate and mixed with 100 ꢁL of ultra pure
water. The samples were shaken at 800 rpm for 10 min before being analysed by LCꢀMS/MS. The
percentage of remaining parent compound at each time interval relative to the t = 0 min sample is
calculated from peak area ratios in relation to the internal standard. All samples were run in duplicate with
propantheline bromide as a positive control and tolbutamide as internal standard.
NMR characterization
1H and 2D TOCSY NMR were acquired on a Bruker Avance III spectrometer equipped with a
1
cryogenically cooled triple resonance probed operating system at a normal H frequency of 600 MHz at
298 K. Water suppression was performed via excitation sculpting. Secondary chemical shifts were
calculated by comparing the experimentally observed Hα chemical shifts (HA) for each residue with the
chemical shifts observed for the corresponding residues in random coil (RC) peptides.28 The NMR
assignment of 1 TP1 was compared to the chemical shifts reported previously, BMRB 5486.27
ANCILLARY INFORMATION
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Supporting Information. Bacterial strain descriptions and QC data of each compound by HRMS, LC–MS
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and, 1H and 2D NMR. This information is available free of charge via the Internet at http://pubs.acs.org/.
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Abbreviations Used: TP1, tachyplesin-1; ADME, Adsorption Distribution Metabolism Elimination; AMP,
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Antimicrobial peptide; GRAVY, grand average of hydropathicity index; MIC, Minimum inhibitory
concentration; MM, Median of the peptide MICs against all bacteria and fungi tested
; MHC, Minimum
hemolytic concentration; ATI, activity/toxicity index; ATCC, American Type Culture Collection; G-ve,
Gram-negative; G+ve, Gram-positive; HEK293, human embryonic kidney 293; RBC, Red Blood Cell;
RC, Random Coil.
Corresponding Author Information: Matthew A Cooper, Institute for Molecular Bioscience, University
of Queensland, Brisbane, Australia. E-mail: m.cooper@uq.edu.au, Fax +61-7-3346-2090, Phone +61-7-
3346-2044
Author contribution: IAE, AGE, MAC and MATB conceived the study. IAE, AGE and AMK performed
the experiments and analysed the data. IAE wrote the paper with input from all authors. MAC oversaw
the research program.
Acknowledgment: This project is supported by NHMRC project grant APP1106590. MAC is a NHMRC
principle research fellow (APP1059354). IAE is supported by an Australian Postgraduate Award (APA)
PhD scholarship. AGE, AMK and MATB are supported in part by Wellcome Trust Strategic Grant
WT1104797/Z/14/Z. MAC currently holds a fractional Professorial Research Fellow appointment at the
University of Queensland with his remaining time as CEO of Inflazome Ltd. a company headquartered in
Dublin, Ireland that is developing drugs to address clinical unmet needs in inflammatory disease by
targeting the inflammasome. We thank Geraldine Kaeslin and Janet Reid for technical contribution in
performing the cytotoxicity assays. We thank Ilias Karaiskos and Helen Giamarellou (6th Dept. of
Internal Medicine, Hygeia General Hospital, Athens, Greece) and Roger L. Nation (Institute of
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Pharmaceutical Sciences, Monash University, Australia) for providing the Gram-negative clinical
isolates for antimicrobial testing.
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REFERENCES
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.
2.
Butler, M. S., Blaskovich, M. A. T., and Cooper, M. A. (2016) Antibiotics in the clinical pipeline at
the end of 2015, J. Antibiot. DOI:10.1038/ja.2016.72
Nakamura, T., Furunaka, H., Miyata, T., Tokunaga, F., Muta, T., Iwanaga, S., Niwa, M., Takao, T.,
and Shimonishi, Y. (1988) Tachyplesin, a class of antimicrobial peptide from the hemocytes of
the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure, J. Biol. Chem.
263, 16709-16713.
Miyata, T., Tokunaga, F., Yoneya, T., Yoshikawa, K., Iwanaga, S., Niwa, M., Takao, T., and
Shimonishi, Y. (1989) Antimicrobial peptides, isolated from horseshoe crab hemocytes,
tachyplesin II, and polyphemusins I and II: chemical structures and biological activity, J. Biochem.
(Tokyo, Jpn.) 106, 663-668.
Kawano, K., Yoneya, T., Miyata, T., Yoshikawa, K., Tokunaga, F., Terada, Y., and Iwanaga, S.
(1990) Antimicrobial peptide, tachyplesin I, isolated from hemocytes of the horseshoe crab
(Tachypleus tridentatus). NMR determination of the beta-sheet structure, J. Biol. Chem. 265,
15365-15367.
Kushibiki, T., Kamiya, M., Aizawa, T., Kumaki, Y., Kikukawa, T., Mizuguchi, M., Demura, M.,
Kawabata, S., and Kawano, K. (2014) Interaction between tachyplesin I, an antimicrobial peptide
derived from horseshoe crab, and lipopolysaccharide, Biochim. Biophys. Acta 1844, 527-534.
DOI:10.1016/j.bbapap.2013.12.017
J. Hong, J. G. D., W.T. Guan, G. Jin, Zh.L. Huang, L.J. Zhang, J. Zh. Dang and Y. Zhang. (2012)
Tachyplesin I Induce Drug Resistance in Bacteria in vitro., J Anim Vet Adv 11, 939-945. DOI:
10.3923/javaa.2012.939.945
Hong, J., Hu, J., and Ke, F. (2016) Experimental Induction of Bacterial Resistance to the
Antimicrobial Peptide Tachyplesin I and Investigation of the Resistance Mechanisms, Antimicrob.
Agents Chemother. 60, 6067-6075. DOI:10.1128/AAC.00640-16
Hong, J., Guan, W., Jin, G., Zhao, H., Jiang, X., and Dai, J. (2015) Mechanism of tachyplesin I injury
to bacterial membranes and intracellular enzymes, determined by laser confocal scanning
microscopy and flow cytometry, Microbiol. Res. 170, 69-77. DOI:10.1016/j.micres.2014.08.012
Tamamura, H., Ikoma, R., Niwa, M., Funakoshi, S., Murakami, T., and Fujii, N. (1993)
Antimicrobial activity and conformation of tachyplesin I and its analogs, Chem. Pharm. Bull. 41,
978-980.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Rao, A. G. (1999) Conformation and antimicrobial activity of linear derivatives of tachyplesin
lacking disulfide bonds, Arch. Biochem. Biophys. 361, 127-134. DOI:10.1006/abbi.1998.0962
Tam, J. P., Lu, Y. A., and Yang, J. L. (2000) Marked increase in membranolytic selectivity of novel
cyclic tachyplesins constrained with an antiparallel two-beta strand cystine knot framework,
Biochem. Biophys. Res. Commun. 267, 783-790. DOI:10.1006/bbrc.1999.2035
Saravanan, R., Mohanram, H., Joshi, M., Domadia, P. N., Torres, J., Ruedl, C., and Bhattacharjya,
S. (2012) Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with
lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and
12.
endotoxin
neutralization,
Biochim.
Biophys.
Acta
1818,
1613-1624.
DOI:10.1016/j.bbamem.2012.03.015
24
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Page 25 of 27
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
13.
14.
Holland-Nell, K., and Meldal, M. (2011) Maintaining biological activity by using triazoles as
disulfide bond mimetics, Angew. Chem. 50, 5204-5206. DOI:10.1002/anie.201005846
Edwards, I. A., Elliott, A. G., Kavanagh, A. M., Zuegg, J., Blaskovich, M. A. T. and Cooper, M. A.
(2016) Contribution of Amphipathicity and Hydrophobicity to the Antimicrobial Activity and
Cytotoxicity of β-Hairpin Peptides, ACS Infect. Dis. 2, 442-450. DOI:10.1021/acsinfecdis.6b00045
Brogden, N. K., and Brogden, K. A. (2011) Will new generations of modified antimicrobial
peptides improve their potential as pharmaceuticals?, Int. J. Antimicrob. Agents 38, 217-225.
DOI:10.1016/j.ijantimicag.2011.05.004
Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) Rational
design of alpha-helical antimicrobial peptides with enhanced activities and
specificity/therapeutic index, J. Biol. Chem. 280, 12316-12329. DOI:10.1074/jbc.M413406200
Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel,
V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti, R.,
Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I., and Stockinger, H. (2012)
ExPASy: SIB bioinformatics resource portal, Nucleic Acids Res. Suppl. 40, W597-603.
DOI:10.1093/nar/gks400
15.
16.
17.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18.
19.
Nagano, N., Ota, M., and Nishikawa, K. (1999) Strong hydrophobic nature of cysteine residues in
proteins, FEBS letters 458, 69-71.
Panteleev, P. V., and Ovchinnikova, T. V. (2015) Improved strategy for recombinant production
and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity,
Biotechnol. Appl. Biochem. DOI: 10.1002/bab.1456. DOI:DOI: 10.1002/bab.1456
Wood, S. J., Park, Y. A., Kanneganti, N. P., Mukkisa, H.R., Crisman, L. L., Davis, S. E.,
Vandenbosch, J.L., Scaglione, J. B. and Heyl, D. L. (2014) Modified Cysteine-Deleted Tachyplesin
(CDT) Analogs as Linear Antimicrobial Peptides: Influence of Chain Length, Positive Charge, and
Hydrophobicity on Antimicrobial and Hemolytic Activity, Int. J. Pept. Res. Ther. 20, 519-530.
DOI:10.1007/s10989-014-9419-7
Soblosky, L., Ramamoorthy, A., and Chen, Z. (2015) Membrane interaction of antimicrobial
peptides using E-coli lipid extract as model bacterial cell membranes and SFG spectroscopy,
Chem. Phys. Lipids 187, 20-33. DOI:10.1016/j.chemphyslip.2015.02.003
Cooper, G. (2000) The Cell: A Molecular Approach, 2nd ed., Sinauer Associates.
Matsuzaki, K. (2009) Control of cell selectivity of antimicrobial peptides, Biochim. Biophys. Acta
1788, 1687-1692. DOI:10.1016/j.bbamem.2008.09.013
Virtanen, J. A., Cheng, K. H., and Somerharju, P. (1998) Phospholipid composition of the
mammalian red cell membrane can be rationalized by a superlattice model, Proc. Natl. Acad. Sci.
U. S. A. 95, 4964-4969.
Salvioli, G., Rioli, G., Lugli, R., and Salati, R. (1978) Membrane lipid composition of red blood cells
in liver disease: regression of spur cell anaemia after infusion of polyunsaturated
phosphatidylcholine, Gut 19, 844-850.
20.
21.
22.
23.
24.
25.
26.
Imura, Y., Nishida, M., Ogawa, Y., Takakura, Y., and Matsuzaki, K. (2007) Action mechanism of
tachyplesin
I and effects of PEGylation, Biochim. Biophys. Acta 1768, 1160-1169.
DOI:10.1016/j.bbamem.2007.01.005
27.
28.
Laederach, A., Andreotti, A. H., and Fulton, D. B. (2002) Solution and micelle-bound structures of
tachyplesin I and its active aromatic linear derivatives, Biochemistry 41, 12359-12368.
Wishart, D. S., Bigam, C. G., Holm, A., Hodges, R. S., and Sykes, B. D. (1995) (1)H, (13)C and (15)N
random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-
neighbor effects, J. Biomol. NMR 5, 332. DOI:10.1007/BF00211764
25
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7
8
9
29.
30.
31.
32.
33.
34.
Cockerill, F. R., Wikler, M. A. (2012) Methods for dilution Antimicrobial susceptibility tests for
bacteria that grow aerobically; Approved standard, Vol. 32, 9th ed., Clinical and laboratory
standards institute, Wayne, PA.
Rex, J. H., Alexander, B. D. (2008) Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Yeasts; Approved Standard, Vol. 22, 3rd ed., Clinical and laboratory standards
institute, Wayne , PA.
Ravipati, A. S., Henriques, S. T., Poth, A. G., Kaas, Q., Wang, C. K., Colgrave, M. L., and Craik, D. J.
(2015) Lysine-rich Cyclotides: A New Subclass of Circular Knotted Proteins from Violaceae, ACS
Chem. Biol. 10, 2491-2500. DOI:10.1021/acschembio.5b00454
McMillian, M. K., Li, L., Parker, J. B., Patel, L., Zhong, Z., Gunnett, J. W., Powers, W. J., and
Johnson, M. D. (2002) An improved resazurin-based cytotoxicity assay for hepatic cells, Cell Biol.
Toxicol. 18, 157-173.
O'Brien, J., Wilson, I., Orton, T., and Pognan, F. (2000) Investigation of the Alamar Blue
(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity, Eur. J. Biochem.
267, 5421-5426.
Henriques, S. T., Huang, Y. H., Rosengren, K. J., Franquelim, H. G., Carvalho, F. A., Johnson, A.,
Sonza, S., Tachedjian, G., Castanho, M. A., Daly, N. L., and Craik, D. J. (2011) Decoding the
membrane activity of the cyclotide kalata B1: the importance of phosphatidylethanolamine
phospholipids and lipid organization on hemolytic and anti-HIV activities, J. Biol. Chem. 286,
24231-24241. DOI:10.1074/jbc.M111.253393
10
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15
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
35.
Craik, D. J., Henriques, Sonia Troeira, Mylne, Joshua S. and Wang, Conan K. (2012) Cyclotide
isolation and characterization, Vol. 516, David A. Hopwood, Burlington, USA. DOI:
10.1016/B978-0-12-394291-3.00024-1
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