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 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; 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: john.bowie@adelaide.edu.au 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 GLMSVLGHAVGNVLGGLFKPKS-OH Neither lesueurin nor frenatin 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 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 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 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 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 and 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 Concentrations of peptide tested were 100, 50, 25, 12.5, 6, and 1.5 lgỈmL)1 The maximum error in the antibiotic results listed in Table is ± dilution factor: e.g., if the MIC is 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 Citropin 1.1 and synthetic modifications Modifications are shown in bold Relative molecular mass Citropin Sequence 1.1 1.1.2 Modified 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 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 at MIC ẳ 100 lgặmL)1 b Compounds so marked failed the initial NCI tests against three cancer types Many of these compounds show activity, but not below concentrations of 10)4 M For NCI test results, see Table provided in this paper The summary data recorded in Table 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 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 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 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 25 100 50 100 25 12 25 25 100 100 50 25 25 25 12 100 12 25 12 25 25 12 12 100 100 100 25 100 25 12 100 25 25 100 25 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 6 3 1.5 12 12 12 6 12 25 100 50 25 12 12 12 25 50 100 25 100 25 50 12 25 25 50 25 100 12 25 100 100 100 100 100 100 100 100 100 100 100 100 25 100 12 25 25 25 25 50 25 50 50 50 100 25 100 12 6 12 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 12 50 50 Ó FEBS 2003 1144 J Doyle et al (Eur J Biochem 270) Table Anticancer activites of citropin 1.1 and synthetic analogues (IC50 values) Averaged concentration for a particular group of cancers, e.g 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 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 55 5 5 5 5 56 >6 6 6 6 6 53 >4 5 5 5 5 43 5 5 5 5 59 5 5 5 5 59 5 5 5 5 60 5 5 5 5 57 5 5 5 5 53 5 5 5 5 60 5 5 5 5 56 >5 5 5 5 5 38 5 5 6 5 58 >5 >5 5 5 5 46 >5 5 5 5 5 49 >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 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 Hz, / ¼ )60 ± 30°; < 3JNHCaH 6 Hz, / ¼ )60 ± 40° Where 3JNHCaH > 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 nNOS activities of citropin peptides, citropin synthetic modifications, and some related peptides IC50 mean error ± 1.3% Citropin 1.1 modification 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 and Modifications are shown in bold IC50 Hill slope Peptide Sequence Relative molecular mass Citropin 1.1 Citropin 1.1.2 Modified peptide 10 11 12 13 14 15 16 17 18 19 20 21 22 Retro 23 Lesueurin Modified peptide 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 +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 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 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 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 (anticancer activities), the numbers and 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 · 10)4 M Table lists the data for nNOS inhibition by 32 peptides Twenty-five of these peptides are citropin 1.1 and synthe- Fig 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 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 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 ± 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 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 Structural statistics of A4K14-citropin following RMD/SA calculations 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 ˚ 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 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 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 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 and 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 (± 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 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 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 H3N + H3N+ NH2 O Fig 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 H3N+ H 3N + OH -O H 2N O O Fig 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 (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 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, 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 [Lys11 (charge +3)], [Gln11 (+2)] and [Glu11 (+1)] give IC50 values of 17.8, 49.0 and > 100 lgỈmL)1, respectively, and also that in citropin (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 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 or 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 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