Báo cáo khoa học: nNOS inhibition, antimicrobial and anticancer activity of the amphibian skin peptide, citropin 1.1 and synthetic modifications pot

Eur. J. Biochem. 270, 1141–1153 (2003) Ó FEBS 2003

doi:10.1046/j.1432-1033.2003.03462.x

nNOS inhibition, antimicrobial and anticancer activity of the
amphibian skin peptide, citropin 1.1 and synthetic modifications
The solution structure of a modified citropin 1.1
Jason Doyle1, Craig S. Brinkworth2, Kate L. Wegener2, John A. Carver3, Lyndon E. Llewellyn1,
Ian N. Olver4, John H. Bowie2, Paul A. Wabnitz2 and Michael J. Tyler5
1

Australian Institute for Marine Science, Townsville MC, Queensland, Australia; 2Department of Chemistry,
The University of Adelaide, Australia; 3Department of Chemistry, University of Wollongong, Wollongong, Australia;
4
Oncology Department, Royal Adelaide Hospital and Department of Medicine, The University of Adelaide,
South Australia, Australia; 5Department of Environmental Biology, The University of Adelaide, South Australia, Australia

A large number of bioactive peptides have been isolated
from amphibian skin secretions. These peptides have a
variety of actions including antibiotic and anticancer activities and the inhibition of neuronal nitric oxide synthase.
We have investigated the structure–activity relationship of
citropin 1.1, a broad-spectrum antibiotic and anticancer
agent that also causes inhibition of neuronal nitric oxide
synthase, by making a number of synthetically modified
analogues. Citropin 1.1 has been shown previously to form
an amphipathic a-helix in aqueous trifluoroethanol. The
results of the structure–activity studies indicate the terminal
residues are important for bacterial activity and increasing

the overall positive charge, while maintaining an amphipathic distribution of residues, increases activity against
Gram-negative organisms. Anticancer activity generally

mirrors antibiotic activity suggesting a common mechanism
of action. The N-terminal residues are important for inhibition of neuronal nitric oxide synthase, as is an overall
positive charge greater than three. The structure of one of the
more active synthetic modifications (A4K14-citropin 1.1)
was determined in aqueous trifluoroethanol, showing that
this peptide also forms an amphipathic a-helix.

Amphibians have rich chemical arsenals that form an
integral part of their defence systems, and also assist with
the regulation of dermal physiological action. In response to
a variety of stimuli, host defence compounds are secreted
from specialized glands onto the dorsal surface and into the
gut of the amphibian [1–4]. A number of different types of
bioactive peptides have been identified from the glandular
skin secretions of Australian anurans of the Litoria genus,
including (a) smooth muscle active neuropeptides of the
caerulein family [5–8], and (b) wide-spectrum antibiotics,
e.g., the caerin peptides from green tree frogs of the genus
Litoria [6–8], the citropins from the tree frog, L. citropa
[9,10], and the aureins from the bell frogs, L. aurea and
L. raniformis [11]. Among the most active of the antibiotic
peptides are caerin 1.1, citropin 1.1 and aurein 1.2:
caerulein 1.1 pEQGY(SO3)TGWMDF-NH2; caerin 1.1

GLLSVLGSVAKHVLPHVVPVIAEHL-NH2; citropin 1.1
GLFDVIKKVASVIGGL-NH2; aurein 1.2 GLFDIIKKI
AESF-NH2.
Aurein 1.2 contains only 13 amino acid residues and is
the smallest peptide from an anuran reported to have
significant antibiotic activity. The aurein peptides have also

been shown to exhibit modest anticancer activity in tests
carried out by the National Cancer Institute (Washington,
WA, USA) [12].
The solution structures of the antibiotic (and anticancer
active if appropriate) peptides shown above have been
investigated by NMR spectroscopy. In d3-trifluoroethanol/
water mixtures, caerin 1.1 adopts two well-defined helices
(Leu2–Lys11 and Val17–His24) separated by a hinge region
of less-defined helicity and greater flexibility, with hydrophilic and hydrophobic residues occupying well defined
zones [13]. The central hinge region is necessary for optimal
antibiotic activity [13]. Similar NMR studies of citropin 1.1
[9] and aurein 1.2 [11] show that these peptides adopt
conventional amphipathic a-helical structures, a feature
commonly found in membrane-active agents [1–4,8]. Interaction occurs at the membrane surface with the charged,
and normally basic peptide adopting an a-helical conformation and attaching itself to charged, and normally
anionic sites on the lipid bilayer. This ultimately causes
disruption of normal membrane function leading to lysis of
the bacterial or cancer cell [14–16].
Many Australian anurans that we have studied conform
to the model outlined above in that they have a variety of

Correspondence to: J. H. Bowie, Department of Chemistry,
The University of Adelaide, South Australia, Australia.
Fax: + 61 8303 4358, Tel.: + 61 88303 5767,
E-mail:
Abbreviations: MIC, minimum inhibitory concentration; NADPH,
nicotinamide adenine nucleotide phosphate, reduced form;
eNOS, endothelial nitric oxide synthase; iNOS, inducible NOS;
nNOS, neuronal NOS; RMD, restrained molecular dynamics;
SA, simulated annealing.

(Received 23 September 2002, revised 28 November 2002,
accepted 15 January 2003)

Keywords: citropin; antibacterial; anticancer; nNOS activity.


Ó FEBS 2003

1142 J. Doyle et al. (Eur. J. Biochem. 270)

host defence peptides in the skin (and gut) glands including a
neuropeptide that acts on smooth muscle and at least one
powerful wide-spectrum antibiotic and/or anticancer active
peptide like those described above [8]. However there are
some species of anuran that divert markedly from this
scenario. For example, the Australian stony creek frog
(L. lesueuri) [17] and the giant tree frog (L. infrafrenata) [18]
both produce the neuropeptide, caerulein, but lack any widespectrum antimicrobial peptide. The major peptides in the
skin secretions of these two Litoria species have been named
lesueurin and frenatin 3, respectively: their sequences are
shown below: Lesueurin GLLDILKKVGKVA-NH2; Frenatin 3 GLMSVLGHAVGNVLGGLFKPKS-OH. Neither
lesueurin nor frenatin 3 show any significant antibiotic or
anticancer activity, but in tests carried out at the Australian
Institute of Marine Science (Townsville, Queensland,
Australia), both peptides were shown to inhibit the formation of nitric oxide by the neuronal isoform of nitric oxide
synthase (nNOS) with IC50 values at lM concentrations [17].
Further nNOS testing on other peptides isolated from tree
frogs of the Litoria genus showed that each species has at
least one major skin peptide that inhibits nNOS and that
there are (at least) three groups of peptides that inhibit

nNOS. Inhibitor group 1 includes citropin type peptides
(that are also antimicrobial and anticancer agents); for the
sequence of citropin 1.1 see above. The second group
comprises peptides with sequence similarity to frenatin 3:
these peptides show no significant antimicrobial or anticancer activity. The third inhibitor group includes the caerin 1
peptides (see the sequence of caerin 1.1 above): these peptides
also show powerful antimicrobial and antifungal activity.
The three nitric oxide synthases, namely neuronal, endothelial (eNOS) and inducible (iNOS), are highly regulated
enzymes responsible for the synthesis of the signal molecule,
nitric oxide. They are among the most complex enzymes
known (e.g., for nNOS see [19,20]). By a complex sequence
involving binding sites for a number of cofactors including
heme, tetrahydrobiopterin, FMN, FAD and NADPDH,
nNOS converts arginine to citrulline, releasing the shortlived but reactive radical NO [21,22]. Nitric oxide synthases
are composed of two domains: (a) the catalytic oxygenase
domain that binds heme, tetrahydrobiopterin and the
substrate arginine, and (b) the electron supplying reductase
domain that binds NADPH, FAD and FMN. Communication between the oxygenase and reductase domains is
determined by the regulatory protein calmodulin which
interacts at a specific site between the two domains. In the
cases of nNOS and eNOS isoforms, but not for iNOS,
calmodulin is regulated by intracellular Ca2+ [23–26]. Dimerization of the oxygenase domain is necessary for catalytic
activity [21,22]. The amphipathic amphibian peptides inhibit
nNOS by interacting with Ca2+-calmodulin, changing the
shape of the regulatory enzyme, thus impeding its interaction at the calmodulin binding site on nNOS [17]. There
are other examples of small helical peptides inhibiting
nNOS in this way [27,28].
The amphibian may have two possible uses for a peptide
that inhibits nNOS. First, on attack by a predator, the
amphibian may use the nNOS inhibitor to regulate its own

physiological state. The second scenario is that the nNOS
inhibitors are front-line defence compounds. A predator
ingesting even a small amount of the nNOS inhibitor could

be seriously affected if only part of its NO messenger
capability is reduced. All animals produce NOS isoforms,
and it has been reported that bacteria also produce NOS
[29–32].
The citropin 1 group of peptides has significant antibiotic, anticancer and nNOS activity, despite being comprised of only 16 amino-acid residues. In this paper we
describe our investigations into the structure/activity relationships for the amphibian peptide citropin 1.1. The
activities of citropin 1.1 are compared with those of a
number of synthetically modified citropins 1 and other
related molecules to gain insight into the sequence requirements for activity. The 3D solution structure of one of the
most potent of the synthetically modified citropins has been
determined using 1H-NMR procedures. This structure is
compared with that already determined for citropin 1.1 [9].

Methods
Preparation of synthetic peptides
All peptides listed in Tables 1 and 4 were synthesized (by
Mimotopes, Clayton, Victoria, Australia) using L-amino
acids via the standard N-a-Fmoc method (full details
including protecting groups and deprotection have been
reported recently [33]). Synthetic versions of naturally
occurring peptides were shown to be identical to the native
form by electrospray mass spectrometry and HPLC.
Bioactivity assays
Bioactivity testing was carried out on citropin 1.1,
D-citropin 1.1 and A4K14-citropin of both 95% and 80%
purities. The activities were the same range for each pair of

samples. Activity tests on all other synthetic modifications
were performed with samples which had  80% purity as
adjudged by HPLC.
Antimicrobial testing
Synthetic peptides were tested for antibiotic activity by the
Microbiology Department of the Institute of Medical and
Veterinary Science (Adelaide, Australia) by a standard
method [34]. The method involved the measurement of
inhibition zones (produced by the applied peptide) on a thin
agarose plate containing the microorganisms listed in
Table 2. Concentrations of peptide tested were 100, 50,
25, 12.5, 6, 3 and 1.5 lgỈmL)1. The maximum error in the
antibiotic results listed in Table 2 is ± 1 dilution factor:
e.g., if the MIC is 3 lgỈmL)1, the maximum possible range
is 1.5–6 lgỈmL)1.
Anticancer activity testing
Synthetic peptides were tested in the human tumour line
testing program of the US National Cancer Institute [12].
All compounds were tested initially against three tumour
lines (breast, lung and CNS cancers), and if activity was
indicated, the peptide was then tested in vitro against 60
human cell lines. If a particular peptide failed the first stage
of the test program it is indicated as inactive (even though it
may have shown some activity). Full test data are not


Ó FEBS 2003

Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1143

Table 1. Citropin 1.1 and synthetic modifications. Modifications are
shown in bold.
Relative
molecular mass

Citropin

Sequence

1.1
1.1.2
Modified
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

19
20
21
22
23
Retro
1.1

GLFDVIKKVASVIGGL-NH2

1614
1297

a,b

DVIKKVASVIGGL-NH2

peptide

KLFAVIKKVAAVIGGL-NH2b
KLFAVIKKVAAVIRRL-NH2b
GLFKVIKKVASVIGGL-NH2
GLFKVIKKVAKVIKKL-NH2

1614
1537
1570
1557
1557
1500

1599
1572
1628
1643
1685
1714
1756
1699
1655
1712
1696
1752
1753
1625
1823
1627
1810

LGGIVSAVKKIVDFLG- NH2

1614

GlfdvikkvasviGGl-NH2
b
GLADVIKKVASVIGGL-NH2
GLFAVIKKVASVIGGL-NH2
b
GLFDVIAKVASVIGGL-NH2
a

GLFDVIKAVASVIGGL-NH2
a,b
GLFDVIAAVASVIGGL-NH2
GLFDVIKKVAAVIGGL-NH2
b
GLFDVIKKVASVIGGA-NH2
b

GLFEVIKKVASVIGGL-NH2
b
GLFDVIKKVASKIGGL-NH2
GLFDVIKKVASVIKGL-NH2
b

GLFDVIKKVASKIKGL-NH2
GLFDVIKKVASVIKKL-NH2
GLFDVIAKVASVIKKL-NH2
GLFAVIKKVASVIKGL-NH2
GLFAVIKKVASVIKKL-NH2
GLFAVIKKVAAVIKKL-NH2
GLFAVIKKVAAVIRRL-NH2
GLFAVIKKVAKVIKKL-NH2

Neuronal nitric oxide synthase inhibition
Inhibition of nNOS was measured by monitoring the
conversion of [3H]arginine to[3H]citrulline. In brief, this
involved incubation of a homogenate of rat cerebella (which
had endogenous arginine removed by ion exchange chromatography) in a reaction buffer (33 mM Hepes, 0.65 mM
EDTA, 0.8 mM CaCl2, 3.5 lgỈmL)1 calmodulin, 670 lM
b-NADPH, 670 lM, dithiothreitol, pH 7.4) containing

20 nM [3H]arginine (NEN Life Sciences, Boston, MA,
USA). The nNOS inhibitor, Nx-nitro-L-arginine (1 mM)
was used to measure background radioactivity. Reactions
were terminated after 10 min with 50 lL of 0.3 M EGTA.
An aliquot (50 lL) of this quenched reaction mixture was
transferred to 50 lL of 500 mM Hepes (pH 5.5). AG50WX8 (Na+ form) resin (100 lL) was added to separate
[3H]arginine from [3H]citrulline. After repeated vortexing,
this slurry was centrifuged at 1200 g for 10 min, and 70 lL
of supernatent was removed and the [3H]citrulline measured
by scintillation counting. Peptides selected for further
examination to determine the mechanism of inhibition were
assayed in the same reaction buffer as used for initial
screening except that it contained 30 nM [3H]arginine
supplemented with 0.3–13.3 mM arginine.
Data analysis for nNOS studies

a

These compounds show no antibiotic activity against the listed
bacteria in Table 2 at MIC ẳ 100 lgặmL)1. b Compounds so
marked failed the initial NCI tests against three cancer types. Many
of these compounds do show activity, but not below concentrations
of 10)4 M. For NCI test results, see Table 3.

provided in this paper. The summary data recorded in
Table 3 indicate the particular groups of cancers tested, the
average IC50 concentration of the peptide against that group
of cancers and the number of tumours, out of 60 tested, that
were affected by the particular peptide. For details of how
the IC50 value is determined from graphical data see [12].

Peptide inhibition curves were fitted using the curve-fitting
routine of SIGMAPLOT (SPSS, Chicago, IL, USA) using a
variation of the Hill equation: fmols [3H]citrulline production ¼ 1/(1 + [inhibitor]/ICn ), where IC50 is the concen50
tration at which the peptide causes 50% inhibition and n is
the slope of the curve and can be considered as a pseudo Hill
coefficient [35]. Lineweaver–Burk plots [36] were generated
using SIGMAPLOT (SPSS, Chicago, IL, USA). The mean
error in the IC50 results listed in Table 4 is ± 1.3%.
NMR spectroscopy of citropin synthetic modification
(A4K14-citropin 1.1)
NMR experiments were performed on a solution of 5.7 mg
of A4K14-citropin 1.1 dissolved in a mixture of water
(0.35 mL) and d3-trifluoroethanol (0.35 mL), that had a
final concentration of 4.9 mM and a measured pH of 4.12.
NMR spectra were acquired on a Varian Inova-600 NMR
spectrometer at a 1H frequency of 600 MHz and 13C

Table 2. Antibiotic activites of Citropin 1.1 and synthetic analogues [MIC values (lgỈmL)1)]. The absence of a figure means the activity is
> 100 lgỈmL)1. For error range see Methods.
1.1 1.1D 2
Bacillus cereus
Escherichia colia
Leuconostoc lactis
Listeria innocua
Micrococcus luteus
Pasteurella multocidaa
Staphyloccus aureus
Staphylococcus epidermidis
Streptococcus uberis

a

Gram-negative organism.

50

50

50

3

4

7

25 100 50

100
6
25
12

3
25
25

100
100
50

3 25 6
25 25
12 100 12

25
12
25

25
12
12

100
100
100

25 100 25
12 100 25
25 100 25

8

9

10

50

11

12

13

14

15

16

17

18

19

20

21

22

23

Retro

50
25 12 25 25 100 50 25 100
50 50 100

50
50 50 100 100
100
100
25
3
6
3
6
3
3 1.5 12
3 12 12
6
6 12
25 100 50 25 12
6 12 12
25 50 100 25 100
25
50 12
6 25 25 50 25 100
12 25 100
100
100 100 100 100 100 100 100 100
100
100
25
100
6 12 25 25 25 25 50
25 50 50
50 100 25 100 12

6
6 12
3 12 12 25 100 12 12 100
100 50 100 25 100 50 12 25 25 12 50 50 100 100 50 50 50
100
6 12
50
50


Ó FEBS 2003

1144 J. Doyle et al. (Eur. J. Biochem. 270)

Table 3. Anticancer activites of citropin 1.1 and synthetic analogues (IC50 values). Averaged concentration for a particular group of cancers,
e.g. 5 means 10)5 M. The number on the bottom line (total) indicates to how many human cancers (out of the test number of 60) that peptide is
cytotoxic.
Cancer

1.1

1.1D

3

5

7

11

13

14

15

16

17

18

19

22

23

retro

Leukaemia
Lung
Colon
CNS
Melanoma
Ovarian
Renal
Prostate
Breast

Total

5
5
5
5
5
5
5
5
5
55

5
5
5
5
5
5
5
5
5
56

>6
6
6
6
6
6

6
6
6
53

>4
5
5
5
5
5
5
5
5
43

5
5
5
5
5
5
5
5
5
59

5
5
5

5
5
5
5
5
5
59

5
5
5
5
5
5
5
5
5
60

5
5
5
5
5
5
5
5
5
57

5
5
5
5
5
5
5
5
5
53

5
5
5
5
5
5
5
5
5
60

5
5
5
5
5
5
5
5

5
56

>5
5
5
5
5
5
5
5
5
38

5
5
5
5
6
6
5
5
5
58

>5
5
>5
5
5

5
5
5
5
46

>5
5
5
5
5
5
5
5
5
49

>5
5
>5
5
5
>5
5
5
>5
18

frequency of 150 MHz. All NMR experiments were
acquired at 25 °C. 1H-NMR resonances were referenced

to the methylene protons of residual d3-trifluoroethanol
(3.918 p.p.m). The 13C (F1) dimensions of the heteronuclear
single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC) spectra were referenced to
the 13CD2 (60.975 p.p.m) and 13CF3 (125.9 p.p.m) resonances of d3-trifluoroethanol, respectively.
Double-quantum-filtered
correlation
spectroscopy
(DQF-COSY) [37]; total correlation spectroscopy (TOCSY)
[38]; and nuclear Overhauser effect spectroscopy
(NOESY) [39]; were all collected in the phase-sensitive
mode using time proportional phase incrementation [40]
in t1. Two hundred and fifty-six t1 increments were used
for each experiment. Thirty-two scans were time averaged
for each increment in the TOCSY and NOESY experiments, while 16 scans were averaged in the DQF-COSY
experiment. The free induction decay in t2 consisted of
2048 data points over a spectral width of 5555.2 Hz. The
transmitter frequency was centred on the water resonance
and conventional low power presaturation from the same
frequency synthesizer was applied during a 1.5-s relaxation delay to suppress the large water signal in the
TOCSY and NOESY spectra. Gradient methods for
water suppression were used in the DQF-COSY spectrum
[41]. The TOCSY spectrum was acquired with the pulse
sequence used by Griesinger et al., 1988 [42] which
minimizes cross relaxation effects, employing a 70-ms
MLEV-17 spin-lock. NOESY spectra were acquired with
mixing times of 80, 150 and 250 ms.
An HSQC experiment [43] was performed to assign the
a-13C resonances via correlations to their attached protons.
The interpulse delay was set to 1/2JCH (3.6 ms corresponding to JCH ¼ 140 Hz). Two hundred and fifty-six t1
increments, each comprising 64 time averaged scans, were

acquired over 2048 data points and 5555.2 Hz in the directly
detected (1H, F2) dimension. The spectral width in the 13C
(F1) dimension was 24133 Hz. An HMBC spectrum [44]
was collected to assign the carbonyl-13C resonances via
correlations through two and three bonds to a, b and NH
protons (with an interpulse delay of 1/2JCH ¼ 62.5 ms for
JCH ¼ 8 Hz). For this experiment, 400 t1 increments, each
comprising 64 scans, were acquired over 4096 data points

and 5555.2 Hz in the 1H (F2) dimension. The spectral width
for the 13C (F1) dimension was 36216 Hz.
All 2D NMR spectra were processed on a Sun Microsystems Ultra Sparc 1/170 workstation using VNMR software
(version 6.1 A). The data matrices were multiplied by a
Gaussian function in both dimensions, then zero-filled to
2048 data points in F1 prior to Fourier transformation
(4096 data points for the HMBC). Final processed 2D
NMR matrices consisted of 2048 · 2048 or 4096 · 4096
real points.
Structural restraints
Cross-peaks in the NOESY (mixing time ¼ 250 ms) spectrum were assigned using the program SPARKY (version 3.98)
[45]. The cross-peak volumes were converted to distance
restraints using the method of Xu et al., 1995 [46]. Briefly, in
this procedure, the weakest and strongest peaks are calibra˚
ted at 5.0 and 1.8 A, respectively, in order to calculate
intensity-dependent proportionality factors. These factors
were then used to determine the upper bound restraints for
the remaining peaks. To be conservative, the final restraints
were increased by 10 percent from these calculated values. All
˚
lower bound restraints were set to 1.8 A. For each symmetric

pair of cross-peaks, the peak of smaller volume was used.
This procedure generated 264 distance restraints, including
115 intraresidue restraints, 52 sequential (i,i + 1) restraints
and 65 medium range restraints (from 2–4 residues distant).
Thirty-two additional restraints were ambiguous. 3JNHCaH
values were measured from a 1D 1H NMR spectrum, where
the free induction decay had been multiplied by a sine-bell
window function to enhance the resolution. Dihedral
angles were restrained as follows: 3JNHCaH 6 5 Hz,
/ ¼ )60 ± 30°; 5 < 3JNHCaH 6 6 Hz, / ¼ )60 ± 40°.
Where 3JNHCaH > 6 Hz, phi angles were not restrained. A
total of 13 dihedral angle restraints were used in the structure
calculations.
Structural calculations
Structures were generated on a Sun Microsystems Sparc 1/
170 workstation using X-PLOR software (version 3.851)
[47,48]. The restrained molecular dynamics (RMD) and


Ó FEBS 2003

Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1145

Table 4. nNOS activities of citropin peptides, citropin synthetic modifications, and some related peptides. IC50 mean error ± 1.3%. Citropin 1.1
modification 6 has a charge of zero, is hydrophobic,and shows minimal solubility in water thus testing was carried out in dimethyl sulfoxide as
solvent, and is not reproducible. Three tests gave IC50 values of 29.6, 33.7 and 39.5 lgỈmL)1, hence we give the IC50 range as 30–40 lgỈmL)1.
Qualitatively, this compound shows less nNOS inhibition than modifications 4 and 5. Modifications are shown in bold.
IC50
Hill
slope

Peptide

Sequence

Relative molecular
mass

Citropin 1.1
Citropin 1.1.2
Modified peptide
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

20
21
22
Retro
23
Lesueurin
Modified peptide
1
2
3
Citropin 1.2.3
Aurein 1.1
Dahlein 1.1
Dahlein 1.2

GLFDVIKKVASVIGGL-NH2
DVIKKVASVIGGL-NH2

1614
1297

13.3
>100

8.2

2.0

+2
+2

GlfdvikkvasviGGl-NH2

49.5
5.1
4.3
3.8
7.0
30–40
8.0
12.4
6.8
11.5
6.8
5.0
3.5
1.6
1.6
1.9
1.9
2.1
2.1
1.9
3.4
2.2

30.7
3.3
2.7
2.4

4.5
20–26
5.0
7.9
4.2
7.0
4.0
2.9
2.0
0.9
1.0
1.1
1.1
1.2
1.3
1.0
2.1
1.2

1.0
1.6
2.1
3.4
2.1

KLFAVIKKVAAVIGGL-NH2
KLFAVIKKVAAVIRRL-NH2
GLFKVIKKVASVIGGL-NH2
GLFKVIKKVAKVIKKL-NH2

1614
1537
1570
1557
1557
1500
1599
1572
1628
1643
1683
1714
1756
1699
1655
1696
1752
1753
1625
1823
1646
1810

1.4
1.7
1.8
2.3
3.0
2.1
2.5

4.0
2.3
3.8
4.6
2.2
3.0
4.4
2.1
3.3

+2
+2
+3
+1
+1
0
+2
+2
+2
+3
+3
+4
+4
+3
+4
+5
+5
+6
+3
+5

+4
+7

LGGIVSAVKKIVDFLG-NH2

1614

24.2

15.0

1.3

+2

GLLDIIKKVGKVA-NH2
GLLDIIKKVGQVA-NH2

1353
1353
1354
1188
1444
1430
1434

17.8
49.0
>100
24.4

49.1
>100
>100

13.2
36.2

2.0
2.0

20.5
34.0

2.2
2.0

+3
+2
+1
+2
+1
+1
+1

GLADVIKKVASVIGGL-NH2
GLFAVIKKVASVIGGL-NH2
GLFDVIAKVASVIGGL-NH2
GLFDVIKAVASVIGGL-NH2
GLFDVIAAVASVIGGL-NH2
GLFDVIKKVAAVIGGL-NH2

GLFDVIKKVASVIGGA-NH2
GLFEVIKKVASVIGGL-NH2
GLFDVIKKVASKIGGL-NH2
GLFDVIKKVASVIKGL-NH2
GLFDVIKKVASKIKGL-NH2
GLFDVIKKVASVIKKL-NH2
GLFDVIAKVASVIKKL-NH2
GLFAVIKKVASVIKGL-NH2
GLFAVIKKVAAVIKKL-NH2
GLFAVIKKVAAVIRRL-NH2
GLFAVIKKVAKVIKKL-NH2

GLLDIIKKVGEVA-NH2
GLFDIIKKVAS-NH2
GLFDIIKKIAESI-NH2
GLFDIIKNIVSTL-NH2
GLFDIIKNIFSGL-NH2

dynamical simulated annealing (SA) protocol was used [49],
which included the use of floating stereospecific assignments
[50]. Sum-averaging was employed to take care of the
ambiguous restraints. The all hydrogen distance geometry
(ALLHDG) force field (version 4.03) was employed for all
calculations [51]. Initially, a family of 60 structures was
generated with random / and w dihedral angles. These
structures were subjected to 6500 steps (19.5 ps) of high
temperature dynamics at 2000 K. The Knoe and Krepel force
constants were increased from 1000–5000 kcalỈmol)1Ỉnm)2
and 200–1000 kcalỈmol)1Ỉnm)4, respectively. This was
followed by 2500 steps (7.5 ps) of cooling to 1000 K with

Krepel increasing from 1000–40000 kcalỈmol)1Ỉnm)4 and the
atomic radii decreased from 0.9 to 0.75 times those in the
ALLHDG parameter set. The last step involved 1000 steps
(3 ps) of cooling from 1000–100 K. Final structures were

lgỈmL)1

lM

Charge

subjected to 200 steps of conjugate gradient energy minimization. The 20 structures produced with the lowest
potential energies were selected for analysis. 3D structures
were displayed using INSIGHT II software (version 95.0,
MSI) and the program MOLMOL [52].

Results
Biological testing
The antibiotic activities [as minimum inhibitory concentration (MIC) values in lgỈmL)1] of two natural citropins (1.1
and 1.1.2) and 23 synthetic modifications of citropin 1.1,
against nine pathogens, are listed in Table 2; summarized in
Table 3 are the IC50 values of the same peptides in in vitro
anticancer tests against 60 human tumour lines as


Ó FEBS 2003

1146 J. Doyle et al. (Eur. J. Biochem. 270)

V5

7.40

D4

7.60

D4

F3
V12
Sll

G14
G15

7.80

I6

K7

I6

8.00

F2
(ppm)

K7

V5

K8
G14

8.20

I13
I13
V12

8.40

F3

L2

8.60

K8

8.80

V9
8.80

A10

8.60

Sll
8.40

8.20

8.00

V9

A10

7.80

7.60

7.40

F1(ppm)

Fig. 1. NH to NH region of the NOESY spectrum (mixing time ¼
250 ms) of A4K41-citropin 1.1 in 50% (v/v) d3-trifluoroethanol in water.
NOEs between sequential NH protons are indicated.

Fig. 2. Summary of NOEs used in structure calculations for A4K14citropin 1.1 in 50% (v/v) d3-trifluoroethanol in water. The thickness of
˚
the bars indicates the relative strength of the NOEs (strong < 3.1 A,
˚
˚
medium 3.1–3.7 A or weak > 3.7 A). Grey shaded boxes represent

NOEs that could not be assigned unambiguously. The 3JNHaCH values
obtained are also shown. The error here is ± 0.5 Hz. A cross-hatch (#)
indicates the coupling constant could not be determined reliably due to
overlap. Due to overlap with the diagonal, the dNN(i,i + 1) NOE
between I6 and K7 could not be determined with certainty, and is not
included in this figure.

determined by the National Cancer Institute. The NCI lists
anticancer activities in molar concentrations and these are
the units used here. In Table 3 (anticancer activities), the
numbers 5 and 6 refer to 10)5 and 10)6 M, respectively. Ten
of the peptides failed the first stage of the anticancer testing
program and are specified as ÔinactiveÕ: essentially this means
that no anticancer activity is noted at peptide concentrations
less than 1 · 10)4 M.
Table 4 lists the data for nNOS inhibition by 32 peptides.
Twenty-five of these peptides are citropin 1.1 and synthe-

Fig. 3. Deviation from random coil chemical shifts [59]. (A) 1H a-CH
resonances, (B) 13C a-C resonances, and (C) 1H NH resonances. Solid
line, A4K14-citropin 1.1 (GLFAVIKKVASVIKGL-NH2). Dotted
line, citropin 1.1 (GLFDVIKKVASVIGGL-NH2). A negative chemical shift difference indicates an upfield chemical shift compared to
random coil, while a positive chemical shift difference indicates a
downfield shift. Deviation values for the a-CH resonances were
smoothed over a window of n ¼ ±2 residues [60].

tically modified analogues. The other seven peptides are
related to citropin 1.1, but have fewer residues. These
include lesueurin [17], dahleins 1.1 and 1.2 [53] and some
synthetic modifications of lesueurin.

The solution structure of citropin 1.1 synthetic
modification (A4K14-citropin 1.1)
The solution structure of the basic peptide citropin 1.1, as
determined by 2D NMR, is that of a well defined a-helical
and amphipathic peptide [9]. A number of synthetically
modified citropin peptides have significantly greater anticancer and antibacterial activity (and also nNOS activity)
than citropin 1.1 itself. We have chosen to investigate the


Ó FEBS 2003

Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1147

structure of one of the more active synthetic modifications
of citropin 1.1 – A4K14-citropin 1.1 (number 15 in
Tables 1–4) – by CD and NMR spectroscopy in order to
see whether there is any major difference between the
solution structure of this peptide and that of citropin 1.1.
NMR spectroscopy
NMR experiments were performed on the synthetically
modified citropin analogue in which the Asp4 residue was
replaced with Ala and the Gly14 residue was replaced with
Lys (A4K14-citropin 1.1). NMR studies were performed
using a 50% d3-trifluoroethanol/H2O solution of A4K14citropin 1.1 as the parent peptide citropin 1.1 has maximal
helicity in this solvent system, as judged by circular
dichroism [9]. d3-Trifluoroethanol is widely thought of as
a helix-inducing solvent, however, Sonnichsen et al., 1992
ă
[54] found that for peptides in triuoroethanol/H2O solutions, helical structure was only observed where there was a
helical propensity in the sequence. In addition, examples of

b-turn [55] and b-sheet [56,57] structures have been observed
in aqueous trifluoroethanol mixtures, demonstrating that
trifluoroethanol does not enforce helical structure but
merely enhances it if the propensity exists. Thus trifluoroethanol/H2O was deemed a suitable solvent system for
structural studies on the citropin 1.1 peptides. The NMR
experiments were carried out at the same temperature as
that used for the experiments on citropin 1.1 [9]. The
NMR sample of A4K14-citropin 1.1 had a pH of 4.1,
compared to pH 2.3 for citropin 1.1. The difference in pH
value was not expected to have an effect on the final
structures as both peptides were fully protonated at their
respective pH values.
The 1H-NMR resonances were assigned using the
sequential assignment procedure of Wuthrich [58], which
ă
involved the combined use of DQF-COSY, TOCSY and
NOESY spectra. The a-13C resonances were assigned from
the one-bond correlations to the assigned a-1H resonances,
recorded in the HSQC spectrum. Similarly, an HMBC
spectrum was employed to make the carbonyl-13C assignments from the two- and three-bond correlations to the
assigned aH, bH and NH 1H resonances. Table 5 lists all
the assignments for the 1H and a-13C resonances.
A qualitative indication of the peptide structure can be
obtained from an examination of the observed NOEs and
chemical shifts. The NH region of the A4K14-citropin 1.1
NOESY spectrum (mixing time ¼ 250 ms), shown in Fig. 1,
reveals a series of sequential NH–NH NOEs [dNN(i,i + 1)]
that occur along the length of the peptide. A series of weaker
dNN(i,i + 2) NOEs can also be observed at a lower contour
level in this region. The various types of NOEs observed for

A4K14-citropin 1.1 are summarized in Fig. 2. Here it can
be seen that, in addition to the NOEs mentioned above, a
number of weak sequential daN(i,i + 1) NOEs occur as well
as a series of NOEs from residues three and four amino
acids apart [daN(i,i + 3), dab(i,i + 3) and daN(i,i + 4)].
Taken together, the observed NOEs and their intensities are
consistent with A4K14-citropin 1.1 having a helical structure along the majority of its sequence. The pattern of NOE
connectivity is also similar to that found for the parent
peptide, citropin 1.1 [9]. However, the patterns extend over
more residues for A4K14-citropin 1.1. This is particularly

noticeable for the daN(i,i + 1) NOEs that cease at residue
10 in citropin 1.1, but continue over the length of the
peptide for A4K14-citropin 1.1. Similarly, the daN(i,i + 3)
NOEs extend right up to residue 16 in A4K14-citropin 1.1
but stop at residue 14 for the parent peptide. Thus, from an
examination of the NOE data, it would seem the modified
citropin peptide has the greater a-helical character beyond
residue 10.
A helical structure for A4K14-citropin 1.1 is also indicated from an examination of the deviation from random coil
chemical shift values of the a-1H and a-13C resonances
determined in water [58,59]. Smoothed over a window of
n ¼ ± 2 residues [60], the plot for the a-CH 1H resonances
shows a distinct upfield shift (Fig. 3A), while those for the
13
C resonances show a distinct downfield shift (Fig. 3B).
The directions of these deviations from random coil
chemical shift values are consistent with the peptide having
a helical structure along its length, with maximal helicity in
its central region and less well-defined structure at its N- and

C-termini [61–63]. For comparison, Fig. 3A,B also show the
deviations from random coil chemical shift for the 1H and
13
C a-CH resonances of citropin 1.1 [9]. Both peptides have
very similar plots over the central region of the peptide
(from residues 4–10), i.e., where there is no difference in
amino acid sequence between the two peptides and they
both have the greatest helicity. However, from approximately residue Ala10 onwards, the 1H and 13C chemical
shifts of A4K14-citropin 1.1 are consistently upfield and
downfield, respectively, of those of the parent peptide. These
differences suggest that A4K14-citropin 1.1 forms a more
stable a-helix than citropin 1.1 in the C-terminal region. The
small differences at the extreme N-terminal region (first
three residues) for the plots of the 1H and 13C a-CH
resonances are opposite in directional trend for structural
conclusions to be drawn. This may reflect the poorly defined
nature of the first turn of the a-helix due to the lack of
hydrogen bonds to their NH protons.
Comparison of the observed NH chemical shifts of
A4K14-citropin 1.1 with the corresponding random coil
NH chemical shifts [59] revealed a periodic distribution such
that those from hydrophobic residues were shifted downfield with respect to the random coil values and those from
hydrophilic residues were shifted upfield (Fig. 3C). This
behaviour is characteristic of amphipathic a-helices [64,65]
and is due to differences in backbone hydrogen bond length
on either face of the peptide, which lead to slight curvature
of the helix. The curvature may not be significant for
A4K14-citropin 1.1, as it consists of only 16 residues,
however, the periodic distribution of NH shifts is consistent
with A4K14-citropin 1.1 forming an amphipathic a-helix.

Furthermore, Fig. 3C also shows that the periodicity of the
NH chemical shifts is very similar between the parent and
modified peptides.
Structural analysis
The conclusions derived from an examination of the
NMR data were confirmed when the NOE data were
used as input for structural calculations. Sixty structures
were generated by restrained molecular dynamics (RMD)
and dynamical SA calculations and the 20 structures of
lowest potential energy were selected for close examination.


Ó FEBS 2003

1148 J. Doyle et al. (Eur. J. Biochem. 270)

Table 5. 1H and 13C NMR chemical shifts for A4K14-citropin in 50% trifluoroethanol in water (by volume), at a measured pH of 4.12 at 25 °C. Data
are shown in p.p.m. Assignments for all the 1H NMR resonances are shown whereas only the a-13C and carbonyl-13C resonances are presented;
NO, not observed.
Chemical shift of
a-13CH

13

42.4
56.4

169.5
176.2

59.5

175.2

1.00
1.23

53.8
65.1
63.8

178.4
176.0
176.1

1.39

58.5

177.1

58.1

177.8

66.0
54.1
60.7
64.8
63.5

176.7
178.6
174.7
176.9
177.2

57.0

177.1

44.7
54.5

173.5
179.2

Residue

NH

a-CH

b-CH

Others

Gly1
Leu2

NO
8.45

3.93, 3.83
4.15

1.64

Phe3

8.12

4.25

3.17

c-CH 1.59
d-CH3 0.99, 0.92
H2,6 7.20
H3,5 7.31
H4 7.26

Ala4
Val5
Ile6

7.71
7.38
7.96

4.02
3.70
3.67

1.57
2.35
1.94

Lys7

8.01

3.90

1.79

Lys8

7.71

4.10

2.21, 2.07

Val9
Ala10
Ser11
Val12
Ile13

8.60
8.83
7.83
7.91
8.28

3.61
4.02
4.17
3.85
3.81

2.22
1.53
4.12, 4.03
2.41
2.01

Lys14

8.18

4.13

1.99, 1.76

Gly15
Leu16

7.85

7.90

4.01, 3.93
4.24

1.89

c-CH3 1.09,
c-CH2 1.72,
c-CH3 0.96
d-CH3 0.88
c-CH2 1.49,
d-CH2 1.68
e-CH2 2.94
NH3+ n.o.
c-CH2 1.56
d-CH2 1.81,
e-CH2 2.96
NH3+ n.o.
c-CH3 1.10,

CO

1.73

1.00

c-CH3 1.13, 1.02
c-CH2 1.73, 1.34
c-CH3 0.99

d-CH3 0.88
c-CH2 1.57
d-CH2 1.62
e-CH2 3.04
NH3+ n.o.
c-CH 1.65
d-CH3 0.95
CONH2 7.24, 6.77

Table 6. Structural statistics of A4K14-citropin following RMD/SA calculations. <SA> is the ensemble of the 20 final structures (SA) is the mean
structure obtained by best-fitting and averaging the coordinates of backbone N, a-C and carbonyl-C atoms of the 20 final structures. (SA)r is the
representative structure obtained after restrained energy minimization of the mean structure. Well-defined residues are those with angular order
parameters (S) > 0.9. For A4K14-citropin 1.1, residues Leu2 to Gly15 are well-defined.
<SA>
˚
RMSD from mean geometry (A)
All heavy atoms
All backbone atoms (N, a-C, carbonyl-C)
Heavy atoms of well-defined residues
Backbone atoms (N, a-C, carbonyl-C) of well-defined residues
)1
X-PLOR energies (kcalỈmol )
Etot
Ebond
Eangle
Eimproper
Erepel
ENOE
Ecdih

(SA)r

0.74
0.34
0.72
0.21

±
±
±
±

0.10
0.09
0.11
0.08

–
–
–
–

75.34
6.76
23.39
4.25
4.39
36.55
0.00

±
±
±
±
±
±
±

1.66
0.15
1.06
0.56
0.34
1.31
0.00

70.12
6.45
21.39
3.29
4.80
34.19
0.00


Ó FEBS 2003

Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1149

Some statistics for the 20 final structures are given in

Table 6.
The superimposition of the 20 structures over the
backbone N, aC and carbonyl-C atoms shows that
A4K14-citropin 1.1 forms a regular a-helix along its
entire length (Fig. 4A). Analysis of the angular order
parameters (S, / and w) [66] of these structures indicated
that, except for the N- and C-terminal residues (Gly1
and Leu16), all residues were well defined (S > 0.9 for
both their / and w angles). A Ramachandran plot [67]
of the average / and w angles of the well-defined
residues reveals these angles are distributed within the
favoured region for a-helical structure (not shown). The
most energetically stable of the 20 final structures is
displayed in Fig. 4B and from this representation it is
apparent that A4K14-citropin 1.1 forms an amphipathic
a-helical structure with well-defined hydrophobic and
hydrophilic faces.

Discussion
Citropin 1.1 is the major wide-spectrum antibiotic peptide
in the secretion of the skin glands of L. citropa [9]. It is one
of the most potent membrane-active antibiotic peptides
isolated from amphibians and is particularly effective
against Gram-positive organisms [8]. Citropin 1.1 is a 16
residue peptide and is one of a number of amphibian
antibiotic peptides containing the characteristic Lys7-Lys8
pattern: a group which includes lesueurin (from L. lesueuri)
[17], the aureins (from L. aurea and L. raniformis) [11] and
the uperins (from toadlets of the genus Uperoleia) [68].
Citropin 1.1 does not cause lysis of red blood cells at a

concentration of 100 lgỈmL)1, but lysis is complete at
1 mgỈmL)1 (B. C. S. Chia & J. H. Bowie, unpublished
results). Citropin 1.1 is thought to be stored in an inactive
form (spacer peptide – citropin 1.1) in the skin glands, but
when the frog is stressed, sick or attacked, an endoprotease
cleaves off the spacer peptide and the active citropin 1.1 is
released onto the skin. Citropin 1.1 must be cytotoxic to the
frog as after about 10 min of exposure on the skin a further
endoprotease removes the first two residues of the peptide
destroying the antibiotic (and anticancer) activity [9].
The solution structure of citropin 1.1 is shown in Fig. 5;
this should be compared with that of the synthetically
modified A4K14-citropin 1.1 depicted in Fig. 4B. The
NMR studies reported here indicate that both peptides
adopt amphipathic a-helices, but that the helicity is more
pronounced for A4K14-citropin 1.1. Each peptide has well
defined hydrophobic and hydrophilic regions. However,
chemical shift and NOE connectivity data suggest that the
C-terminal region of the a-helix may be more stable in the
modified citropin. This is due probably to the replacement
of Gly14 with Lys14. Gly is more conformationally mobile
than other residues, due to its lack of a side chain, and is a
well-known breaker of helical structure [69]. The Lys residue
would therefore be expected to stabilize a helical structure in
this region. In addition, the positively charged side-chain of
Lys would stabilize a C-terminal helix due to its interaction with the negative end of the helix dipole [69]. The
replacement of Asp4 with Ala4 does not have a significant
effect on the structure of the peptide. This may be because
removal of the negatively charged Asp4, which would

stabilize the N–terminal helix by interaction with the
positive end of the helix dipole [69], is compensated by the
introduction of Ala, which has a high helical propensity.
Finally, we believe it is likely that all of the peptides listed in
Tables 1 and 4 adopt such structures when interacting with
either bacterial or cancer cell membranes.
The antibiotic and anticancer activities of peptides of
this type are due to the disruption of the cell membrane
by the peptide. In order to span the lipid bilayer of
bacterial and cancer cells, the peptide needs to have at
least 20 amino acid residues [4,14,70]. Citropin 1.1 has
only 16 residues and thus is unable to fully span the lipid
bilayer. Amphipathic peptides of this type are thought
to operate via the ÔcarpetÕ mechanism, which involves
aggregation of the helical peptides on the surface of the
membrane by interaction of the positively charged sites of
the peptide with negatively charged sites on the membrane
surface. The peptides then insert into the lipid membrane,
weakening the bilayer and making it susceptible to
osmotic lysis [4,24,70]. From the work reported herein, the
greated helicity of A4K14-citropin 1.1 in its C-terminal
region may be responsible for its enhanced antimicrobial
activity.
Antibacterial and anticancer activity
Synthetic modifications of citropin 1.1, shown in Table 1,
were made to investigate the relationship between activity
and sequence. The first point to be made is that the natural
L-citropin 1.1 has, within experimental error (± 1 dilution
factor), the same spectrum of antibiotic activities as the
synthetic all D-citropin 1.1. This is a feature of membrane

active peptides [4,13]. Other synthetic modifications were
made to the following plan: (a) to successively replace the
hydrophilic residues (to ascertain the effect of a particular
hydrophilic residue on the bioactivity), and some hydrophobic residues (certain hydrophobic residues, particularly
terminal residues are often vital for good activity) with Ala,
and (b) to change Gly and some hydrophilic residues to Lys
(to determine the effect on activity of an increase in the
positive charge of the peptide). The spectrum of antibiotic
activities for each synthetic modification is recorded in
Table 2. The following observations can be made. Replacement of the following residues with Ala show (a) little
change in activity for Asp4 and Ser11, and (b) significant
reduction in the activity for Phe3, Lys7, Lys8 and Leu16;
replacement of the following residues with Lys show (a)
reduction in activity for Gly1 and Val12 and (b) significant
increases in activity against Gram-negative organisms for
Gly14 and Gly15. The conclusions from this study are that
(a) modification of either of the terminal residues reduces
the activity, and (b) the activity against Gram-positive
organisms is not significantly improved (in comparison with
citropin 1.1) by synthetic modification, but increasing the
number of basic Lys residues in the hydrophilic zone of the
amphipathic peptide markedly increases the activity against
Gram-negative organisms like E. coli.
Apart from particular detail, the trends in anticancer
activities of the modified citropins 1.1 mirror those outlined
above for the antibiotic activities (Table 3). The citropin 1
peptides are generally cytotoxic toward the majority of the
60 cancers tested in the NCI regime: IC50 values are

Ó FEBS 2003

1150 J. Doyle et al. (Eur. J. Biochem. 270)

A
GLY1

LEU16

+
NH3

B

OH
+NH
3

H3N

+

H3N+
NH2

O

Fig. 4. Most stable structures of A4K14-citroprin 1.1. (A) Superimposition of the 20 most stable structures of A4K14-citropin 1.1 along the
backbone atoms (N, a-C and carbonyl C) (prepared with the program MOLMOL [52]) and (B) the most stable calculated structure of A4K14citropin 1.1. A ribbon is drawn along the peptide backbone in (B).

+NH
3
H3N+

H 3N +

OH

-O

H 2N

O

O

Fig. 5. The most stable calculated structure of citropin 1.1. This figure was originally published by Wegener et al. [9] in Eur. J. Biochem. 265, 627–635.

generally in the moderate 10)5 M range, with synthetic
modification 3 (Asp4 to Ala4) showing the strongest
cytotoxicity (in the 10)6 M range). As was the case with
antibiotic activity, L- and D-citropins 1.1 show almost
identical activity.
The trends observed for antibiotic activity are more
marked when considering anticancer activity. For example,
some synthetic modifications which decrease antibiotic
activity, often destroy the anticancer activity, e.g., the
modifications Gly1 to Lys1, Phe3 to Ala3, Lys7 to Ala7,
Val12 to Lys12, and Leu16 to Ala16. The conclusions from
this study are, that for best anticancer activity of citropin 1.1 type molecules, (a) the residues Gly1, Phe3, Ala4,

Lys7 and Leu16 are essential, and (b) the charge needs to be
P+2. The close correlation between the broad-spectrum

anticancer and antibacterial activity of membrane active
peptides, suggests that the anticancer activity is also due to
penetration and disruption of the membranes of the cancer
cells. The selectivity of these peptides for cancer over
normal cells may be due to the significantly higher levels of
anionic phospholipids present in the outer leaflet of cancer
cells [71–74].
nNOS activity
We have already reported that citropin 1.1 causes the inhibition of nNOS by forming a complex with the regulatory
protein Ca2+-calmodulin, thus impeding the attachment of
this enzyme at the calmodulin binding site on nNOS [17].
The actual nature of the complex is not known, but NMR


Ó FEBS 2003

Solution structure of a modified citropin 1.1 (Eur. J. Biochem. 270) 1151

studies on other peptide Ca2+-calmodulin complexes show
that the dumb-bell shaped calmodulin wraps itself around
and then partially or fully encloses the a-helical peptide,
completely changing the shape of the calmodulin system
[75–79].
The nNOS inhibition data for the various citropins and
related systems are collated in Table 4. The activity/sequencing relationship for effective nNOS inhibition is quite
different from that described above for antibiotic/anticancer
activity. The following observations may be made: (a) L- and

D-citropin 1.1 show quite different activities. Not only is the
IC50 value for D-citropin 1.1 significantly less than that for
L-citropin 1.1, but the Hill slope of 1.0 (2.0 for L-citropin 1.1), may indicate that the inhibition of nNOS by
D-citropin 1.1 involves the Arg substrate site rather than
interaction with Ca2+-calmodulin [36]. (b) Loss of residues
from the N-terminal end of the citropin system destroys the
activity against nNOS, whereas loss of activity is not so
marked when residues are removed from the C-terminal end
of the peptide. For example, lesueurin (13 residues, charge
+3) and citropin 1.2.3 (11 residues, charge +2) show moderate activity with IC50 values of 21.9 lgỈmL)1 (16.2 lm)
and 24.4 lg/mL (20.5 lm), respectively. (c) A change in the
nature of the end groups and some other residues of
citropin 1.1 is not as important as it is for antibiotic or
anticancer activity. For example, compare the data for
changes in Gly1, Phe3, Ser11 and Leu16 (see Table 4, for
citropin 1.1 and citropin modifications 2, 3, 7, 8 and 21). (d)
The extent of positive charge on the peptide is important.
For example, note the change in IC50 in the three lesueurin
modifications, i.e., lesueurin 1 [Lys11 (charge +3)], 2 [Gln11
(+2)] and 3 [Glu11 (+1)] give IC50 values of 17.8, 49.0 and
> 100 lgỈmL)1, respectively, and also that in citropin 6
(charge 0), the IC50 value is reduced to 30–40 lgỈmL)1
(Table 4). Maximum nNOS inhibition by a citropin occurs
when the charge is +3 or greater [e.g., citropin 14, charge
+3, IC50 1.6 lgỈmL)1, and citropin 15 (A4K14–citropin 1.1, charge +4, IC50 1.6 lgỈmL)1]. As long as there is
at least one Lys at residue 7 or 8, it does not seem particularly
important where the other positive charges reside (e.g.,
citropins 12, 14, 15, 20 and 21). Even retro citropin (charge
+2) shows moderate activity.
The prerequisites for maximum nNOS inhibition by a

citropin type peptide are (a) an a-helix; (b) preferably 16
amino acid residues (c); Lys at either residue 7 or 8 and (d)
an overall charge of +3.

Conclusions
Citropin 1.1, the major peptide in the skin secretion of
L. citropa, exhibits multifaceted biological activity within
the 10)6 M concentration range, including widespectrum
antimicrobial and anticancer activity, together with inhibition of nNOS. This concentration is significantly less than
that required to cause lysis of red blood cells. Synthetic
modification of citropin 1.1 can achieve a 10-fold increase in
these activities. Both citropin 1.1 and the more active
synthetic modification, A4K14-citropin 1.1, have been
shown to adopt amphipathic a-helical structures in aqueous
trifluoroethanol. As antibiotic and anticancer activity are the
same for L- and D-citropin 1.1, modified D-citropins could
be useful as pharmaceutical agents, especially as the

citropins 1.1 are active against a number of pathogens that
show resistance towards currently used antibiotics [80,81].
The amphibian uses citropin 1.1 as a primary host-defence
compound against both small and large predators. It is not
clear whether the animal utilizes the anticancer activity of
citropin 1.1, or whether this activity is simply a serendipitous
bonus arising from the membrane activity of this peptide.

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