New alloferon analogues: Synthesis and antiviral properties

Article abstract of DOI:10.1111/cbdd.12020

We have extended our study on structure/activity relationship studies of insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) by evaluating the antiviral effects of new alloferon analogues. We synthesized 18 alloferon analog

Full text of DOI:10.1111/cbdd.12020

Chem Biol Drug Des 2013; 81: 302–309
Research Letter
New Alloferon Analogues: Synthesis and Antiviral
Properties
Mariola Kuczer1,*, Anna Majewska2 and Renata
Viruses are pathogens that cause many serious diseases
Zahorska2
in humans such as flu, HIV ⁄ AIDS, smallpox and haemor-
rhagic fevers, liver cancer, fever blisters, genital herpes,
etc. (1). In addition, viruses can also infect plants and ani-
mals, causing huge losses in agriculture (2).
1Faculty of Chemistry, University of Wrocław,
F. Joliot-Curie 14, 50-383 Wrocław, Poland
2Department of Microbiology, Medical University of
´
Warsaw, Chałubinskiego 5, 02-005 Warsaw, Poland
Effective control of viral infections and diseases is limited,
because of a relatively small number of efficient antiviral
drugs (3,4). Furthermore, many of known antiviral drugs
have a limited spectrum of activity; moreover, drugs that
can eliminate viruses are dangerous to non-infected cells.
The development of new antiviral drugs is difficult
because the replication of viruses is very rapid and
viruses mutate frequently, often rendering antiviral drugs
ineffective. On the other hand in laboratory conditions, it
is often problematic to cultivate viral cultures, thus estab-
lishing reliable tests for potential antiviral drugs is difficult
(5,6).
*Corresponding author: Mariola Kuczer,
mariola.kuczer@chem.uni.wroc.pl
We have extended our study on structure ⁄ activity
relationship studies of insect peptide alloferon (H-His-
Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH) by
evaluating the antiviral effects of new alloferon ana-
logues. We synthesized 18 alloferon analogues: 12 pep-
tides with sequences shortened from N- or C-terminus
and 6 N-terminally modified analogues H-X1-Gly-
Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH, where
X1 = Phe (13), Tyr (14), Trp (15), Phg (16), Phe(p-Cl) (17),
and Phe(p-OMe) (18). We found that most of the evalu-
ated peptides inhibit the replication of Human Herpesvi-
ruses or Coxsackievirus B2 in Vero, HEp-2 and LLC-MK2
cells. Our results indicate that the compound [3-13]-
alloferon (1) exhibits the strongest antiviral activity
(IC50 = 38 lM) among the analyzed compound. More-
over, no cytotoxic activity against the investigated cell
lines was observed for all studied peptides at concen-
tration 165 lM or higher.
To this day, some of the antiviral agents that are based on
natural products, including a variety of polyphenols, flavo-
noids, polysaccharides, anthraquinones, terpenes and pro-
teins (7–9). It seems that natural compounds from plants,
animals and fungi present a promising strategy in the
search for biological active compounds (1,7,10,11).
Recently, many antimicrobial peptides have been discov-
ered in insects (1,12,13). However, relatively little data are
available on insect peptides with the antiviral properties
(13). Until now, only eight peptides with antiviral activity
have been isolated from insects (13–19).
Key words: alloferon, antiviral activity, insect peptides
Abbreviations: The symbols of amino acids, peptides and
their derivatives are in accordance with the IUPAC-IUB Joint
Commission on Biochemical Nomenclature Recommendation,
1983 (Eur. J. Biochem. 1984, 138, 9 and J. Pept. Sci. 2006,
12, 1).Boc, tert-butyloxycarbonyl; CPE, cytopathic effect;
DCC, dicyclohexylcarbodiimide; DMF, dimethylformamide;
EDT, 1,2-ethanedithiol; FBS, foetal bovine serum; Fmoc,
9-fluorenylmethoxycarbonyl; HBTU, O-benzotriazole-N,N,N’,
N’-tetramethyl-uronium-hexafluorophosphate; HOBt, N-hydrox-
ybenxotriazole; HPLC, high-performance liquid chromatogra-
phy; NEM, N-ethylmorpholine; MTT, 3-[4,5-dimethylthiazol
-2-yl]-2,5-diphenyl tetrazolium bromide; TCID, tissue culture
infected dose; TFA, trifluoroacetic acid; TLC, thin layer
chromatography; TFMSA, trifluoromethanesulfonic acid; UV,
ultraviolet.
One of them is alloferon, a novel insect tridecapeptide
(H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH)
isolated in 2002 by Chernych from the blow fly Calliphora
vicina (14). It is interesting that this short peptide contains
four His residues in positions 1, 6, 9 and 12, and five Gly
residues in position 2, 5, 7, 10 and 13. The primary struc-
ture of alloferon is also similar to some functionally relevant
proteins such as precursors of influenza virus B haemag-
glutinin, bovine prion protein I and II and Sarcophaga pere-
grina antifungal protein (14).
The in vitro experiments show that alloferon stimulates nat-
ural killer lymphocytes. In vivo, alloferon induces the inter-
feron (IFN) synthesis in mice (14), probably through
activation of nuclear factor jB (20).
Received 21 May 2012, revised 16 July 2012 and accepted
for publication 2 August 2012
302
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12020

New Alloferon Analogues
Several other biological activities of alloferon have been
discovered. The in vivo experiments in mice indicate that
alloferon has antitumor properties (14,21), it also prevents
mortality of most animals challenged by influenza virus A
(14). Although rimantadine offers better protection, a much
higher dose is required than in case of alloferon. An injec-
tion of alloferon also stimulates resistance to influenza virus
B in dosage much smaller than ribavirin. Moreover, it was
found that alloferon inhibits the replication of Human Her-
pesvirus 8 (Kaposi’s sarcoma-associated herpesvirus) and
Human Papillomaviruses (20,22).
In first group, to determinate the active core of alloferon,
we synthesized 12 analogues partially truncated at N- or
C-terminus. In the second series, histidine in position 1
was replaced: (i) by different proteinaceous aromatic amino
acid residues as: phenylalanine (13), tyrosine (14), trypto-
phan (15); (ii) by non-proteinaceous aromatic amino acid
residues as: phenylglycine (16) and para-substituted phen-
ylalanine derivatives [-Cl (17) or -OMe (18)].
During the biological investigations of the peptides, we
analyzed for their antiviral activity in vitro against Herpesvi-
ruses and Coxsackieviruses using Vero, HEp-2 and LLC-
MK2 cell lines.
In addition, Lee et al. (23) suggest that alleferon has sig-
nificant anti-inflammatory effects on the UVB-induced
response on the cutaneous inflammation. In our prelimin-
ary investigation, we found that alloferon inhibits the repli-
cation of Human Herpesvirus 1 and fails to affect the
Coxsackievirus replication in vitro (16,24). Furthermore,
our results show that this peptide suppresses the devel-
opment of plant pathogens (25). We also studied alloferon
analogues modified in position 1 of the peptide chain
such as: [des-His1]-, [Lys1]-, [Arg1]- and [Ala1]-alloferon
(24). The most active compound in this series was pep-
tide with lysine residue in position 1. This analogue was
very active against reference and clinical strains of the
Experimental
Materials
The Wang resins preloaded with Fmoc-Gly, Fmoc-Val and
Fmoc-Gln(Trt), Fmoc-amino acids, HOBt, HBTU and TFA
were purchased from IRIS Biotech (Marktredwitz, Ger-
many). Boc-Gly-OH, Boc-Val-OH, Boc-His(p-Bom)-OH,
Boc-Gln-OH, Merrifield resin and DCC were obtained from
Bachem (Heidelberg, Germany). N-ethylmorpholine (NEM)
was purchased from Fluka (as Sigma-Aldrich, St. Louis,
MO, USA). HPLC-grade solvents were purchased from
Fisher Scientific (Gliwice, Poland). All other reagents were
purchased from Sigma-Aldrich (St. Louis, MO, USA). All sol-
vents and reagents used for solid-phase synthesis were of
analytical quality and were used without further purification.
Human Herpesvirus
1 (HHV-1) in Vero cells (IC50 =
147.09 and 9.19 lg ⁄ mL) and Coxackievirus B2 (CVB-2) in
HEp-2 cells (IC50 = 107.04 and 74.0 lg ⁄ mL), respectively
(24).
This result inspired us to perform further studies on allofer-
on analogues. In this study, on structure ⁄ activity relation-
ship in alloferon, we present the synthesis and antiviral
activity of two new series of analogues of alloferon
(Table 1) such as: analogues with a shortened sequence
and analogues modified in position 1.
All the chemicals and reagents used for antimicrobial stud-
ies were of bacteriological grade. The viruses used for the
antiviral studies came from the collection of the Depart-
ment of Medical Microbiology, Medical University of War-
saw (Warsaw, Poland). The African green monkey kidney
Table 1: The primary structures
of alloferon and its synthesized
Peptide
Amino acid sequence
analogues
Alloferon
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
1
2
3
4
H-His-Gly-Gln-His-Gly-Val-His-Gly-OH
5
H-Gly-Gln-His-Gly-Val-His-Gly-OH
6
H-Gln-His-Gly-Val-His-Gly-OH
7
H-His-Gly-Val-His-Gly-OH
8
H-Gly-Val-His-Gly-OH
9
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-OH
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-OH
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-OH
10
11
12
13
14
15
16
17
18
H-His-Gly-Val-Ser-Gly-His-Gly-OH
H-Phe-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Tyr-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Trp-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Phg-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Phe(p-Cl)-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
H-Phe(p-OMe)-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
Chem Biol Drug Des 2013; 81: 302–309
303

Kuczer et al.
cells (Vero), rhesus monkey kidney cells (LLC-MK2) and
human larynx carcinoma cells (HEp-2) were obtained from
the American Type Culture Collection (Manassas, VA,
USA).
deprotection with 4.75 mL of TFA in the presence of
0.125 mL of EDT and 0.125 mL of water for 2 h at room
temperature according to the standard procedure. The
peptide was purified by preparative HPLC. The main frac-
tions were combined and lyophilized. Finally, the peptide
was redissolved in 50% acetic acid in water and then re-
lyophilized. The purity of all final products was checked
by HPLC, TLC, optical activity and molecular weight
determinations.
Eagle’s medium was purchased from Biomed (Lublin,
Poland). FBS was obtained from Gibco (Paisley, UK). Pep-
tides were purified by preparative high-performance liquid
chromatography on a Varian ProStar HPLC system, col-
umn: Tosoh Biosciences ODS-120T C18 (ODS 300 ·
21.5 mm) (Tokyo, Japan) with UV detection at 210 nm.
Peptides 2–3 and 9–18 were obtained and purified in the
same manner as peptide 1. The purity of free peptides
was checked by HPLC and found to exceed 95% in each
case. Their analytical data are presented in Table 2.
Analytical HPLC was performed on a Thermo Separation
Products HPLC system with a Vydac C18 column (ODS
250 · 4.6 mm) (Grace, Deerfield, IL, USA) with UV absorp-
tion determined at 210 nm. The molecular weight of the
peptides was confirmed with a Bruker Daltonics micrO-
TOF-Q mass spectrometer (Bremen, Germany). The opti-
cal activity of the chiral compounds was measured with a
Jasco DIP-1000 polarimeter (Jasco, Japan). TLC was per-
formed on aluminium sheets precoated with silica gel 60
from Merck (Darmstadt, Germany).
Synthesis of H-His-Gly-Gln-His-Gly-Val-His-Gly-OH
(4)
The peptide was obtained by a stepwise elongation of the
peptide chain by the method outlined below. The C-termi-
nal amino acids were bound to the Merrifield resin by the
caesium salt procedure to the substitution level of
0.68 mmol glycine per gram.
Cytotoxic activity of peptides was assessed by light
microscopy Olympus CK2 (Olympus Corp., Hamburg,
Germany). The plate readings were recorded spectropho-
tometrically on a reader (Reader 230, Organon Teknika
Turnhout, Belgium).
One gram of Boc-Gly-resin was suspended in solution of
30% TFA in CH2Cl2. The mixture was stirred for 30 min at
room temperature. Then, it was filtered and washed for
10 min with CH2Cl2 and EtOH, three times each. The resin
was neutralized with 10% TEA in CH2Cl2 for 10 min and
washed with EtOH and CH2Cl2, three times each. The
next amino acid, Boc-His(p-Bom)-OH (0.50 g, three equiv),
was dissolved in CH2Cl2 and coupled to the resin in the
presence of three equivalents of DCC ⁄ HOBt for 2 h. The
end of the reaction was determined by the Kaiser test.
The following amino acids (three equivalents) were coupled
to the resin by the DCC method: Boc-Gly-OH, Boc-His(p-
Bom)-OH. Boc-Val-OH (0.80 g, six equiv) was introduced
to the peptide chain by the symmetrical anhydride
method. Boc-Gln-OH (1.0 g, three equiv) was coupled to
the resin in the presence of HBTU (0.75 g, three equiv),
HOBt (0.30 g, three equiv), NEM (0.9 mL, six equiv). The
peptide-resin was washed with CH2Cl2, MeOH:CH2Cl2
(1:1, v ⁄ v), MeOH and then dried overnight over KOH under
reduced pressure.
Synthesis
Synthesis of H-Val-Ser-Gly-His-Gly-Gln-His-Gly-
Val-His-Gly-OH (1)
The peptide was obtained by a stepwise elongation of the
peptide chain by the method outlined below.
0.5 g of the Fmoc-Gly-resin (capacity 0.84 mmol ⁄ g) was
suspended in 20% solution of piperidine in dimethylfor-
mamide (DMF). The mixture was stirred for 20 min at
room temperature. Then, it was filtered and washed with
DMF. The next amino acid, Fmoc-His(Trt)-OH (0.826 g,
three equiv), was dissolved in DMF and coupled to the
resin in the presence of HBTU (1.352 g, three equiv),
HOBt (0.180 g, three equiv) and NEM (293 lL, six equiv)
for 2 h. The end of the reaction was determined by the
Kaiser test. Other Fmoc-amino acid derivatives: Fmoc-
Val-OH (0.453 g, three equiv), Fmoc-Gly-OH (0.397 g,
three equiv), Fmoc-His(Trt)-OH (0.826 g, three equiv),
Fmoc-Gln(Trt)-OH (0.815 g, three equiv), Fmoc-Gly-OH
(0.397 g, three equiv), Fmoc-His(Trt)-OH (0.826 g, three
equiv), Fmoc-Gly-OH (0.397, three equiv), Fmoc-Ser(But)-
OH (0.512 g, three equiv), Fmoc-Val-OH (0.453 g, three
equiv) were connected to the peptide-resin in the same
way. After the final removal of the Na-Fmoc group, the
peptide-resin was washed with DMF, MeOH:DMF (1:1,
v ⁄ v), MeOH and then dried overnight over KOH under
reduced pressure. The free peptide was obtained by
The free peptide was obtained according to the following
procedure: the peptide-resin was mixed with 0.9 mL of
anisole, 0.45 mL of EDT, 7.5 mL of TFA and 1.2 mL of
TFMSA. The mixture was kept at room temperature for
2 h. The resin was filtered off and the filtrate was triturated
with diethyl ether (200 mL). The above reaction mixture
gave a precipitate, which was separated, washed with
diethyl ether, dried in vacuo over KOH and then dissolved
in water and lyophilized. The peptide was purified by pre-
parative HPLC. Finally, the peptide was redissolved in
50% acetic acid in water and then relyophilized. The purity
of all final products was checked by HPLC, TLC, optical
activity and molecular weight determinations.
304
Chem Biol Drug Des 2013; 81: 302–309

New Alloferon Analogues
Table 2: Physicochemical data of alloferon analogues
Rf (TLC)b
[a]20 (c = 1.0,
MW
[M+H]+ m ⁄ z
D
Peptide
Yield (%)a
methanol)
Rt (HPLC)
calculated
found
X
Y
Z
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
78
74
82
74
75
78
86
83
76
84
85
78
75
79
75
84
81
79
)23.5
)27.3
)31.4
)20.8
)30.4
)22.0
)30.0
)19.3
)25.6
)24.5
)18.4
)39.6
)47.6
)12.0
)17.5
)23.6
)18.5
)17.3
14.2
13.2
12.8
12.9
12.5
12.3
12.6
11.8
15.6
14.9
14.1
13.4
15.0
13.5
16.2
14.5
15.1
15.6
1070.5
971.7
884.4
827.7
690.6
633.5
505.4
368.2
1070.5
971.4
777.4
649.3
1274.6
1290.6
1313.6
1260.6
1309.6
1305.6
1071.5
972.4
886.4
828.4
691.3
634.3
506.2
369.2
1071.5
972.3
778.4
650.3
1275.3
1291.3
1314.3
1261.3
1310.3
1306.3
0.35
0.34
0.35
0.35
0.33
0.30
0.32
0.32
0.41
0.36
0.34
0.37
0.28
0.23
0.22
0.20
0.32
0.28
0.27
0.29
0.28
0.18
0.30
0.18
0.29
0.30
0.22
0.19
0.19
0.22
0.30
0.19
0.30
0.17
0.22
0.31
0.17
0.21
0.19
0.22
0.19
0.33
0.22
0.22
0.18
0.23
0.28
0.26
0.15
0.29
0.26
0.23
0.20
0.19
aCrude yield: yield after cleavage from the resin. The purity of crude product was analysed according to HPLC peak integrals at k 210 nm
on analytical HPLC. The crude peptide had a purity of >80%.
bT.L.C. on silica gel plates; eluents: X = n-butanol ⁄ acetic acid ⁄ methanol (4:1:1), Y = chloroform ⁄ methanol ⁄ acetic acid (5:3:1), Z = n-buta-
nol ⁄ pyridine ⁄ acetic acid ⁄ water (30:20:6:24).
Peptides 5–8 were obtained and purified in the same man-
ner as peptide 4. The purity of free peptides were checked
by HPLC and found to exceed 95% in each case. Their
analytical data are presented in Table 2.
Herpesvirus 1 McIntire (HHV-1MC) or 971 PT Coxsackievirus
B2 (971 PT, CVB-2), and the clinical strain of Human Her-
pesvirus 1 (HHV-1) and Coxsackievirus B2 (CVB-2). The her-
pesviruses stock was propagated in Vero or HEp-2 cells.
The CVB-2 strains were grown in LLC-MK2 or HEp-2 cells.
Molecular modelling study
After the cytopathic effect was evident, the cells were fro-
zen-thawed three times. The cell debris was removed by
centrifugation. The supernatant was aliquoted, titrated and
kept at )70 ꢀC. In the antiviral assay, the medium was
supplemented with 2% FBS and the above-mentioned
antibiotics.
Theoretical calculations were performed using the
HYPERCHEM version 7.01 (Hypercube Inc., Gainsville, FL,
USA). The peptide structures were built using the Database
Amino Acids option. First geometric parameters of the pep-
tides in the zwitterionic form were optimized by molecular
mechanic computations with Amber force field. Subse-
quently, the peptide was placed in a box containing 200
water molecules and its geometry was optimized again.
Cytotoxicity assay
Cytotoxic activity of peptides was assessed by a light
microscopy and quantified by the MTT assay in vitro using
LLC-MK2, HEp-2 and Vero cell lines. The absorbance was
read in a reader at 405 nm. Cells were inoculated in 96-mi-
crowell plate. After incubation for 24 h, the peptides in the
serial twofold dilutions from 1:2 to 1:64 were added to the
culture medium and cultured for further 24 or 48 h. The
control was prepared without any sample. All experiments
were performed in triplicate. The toxicity was expressed as
maximum cytotoxic concentration to cause a microscopi-
cally detectable alteration of normal cell morphology.
Biology
Cells
Three types of cell lines were used in this study: Vero,
LLC-MK2, HEp-2. Cells were grown and maintained at
37 ꢀC in Eagle’s medium 1959 supplemented with 10%
FBS and 1% of antibiotic antimycotic solution (100 ·):
penicillin, streptomycin, amphotericin B.
Viruses
Two viruses, representing DNA and RNA viruses pathogenic
for humans, were used in these experiments. The viral strains
used in this study were the standard strains of Human
Antiviral assay
Antiviral activity was assessed in vitro using Vero, LLC-
MK2 or HEp-2 cell lines infected with 0.01 TCID50 ⁄ cell
Chem Biol Drug Des 2013; 81: 302–309
305

Kuczer et al.
Table 3: Cytotoxicity of alloferon analogues
(Tissue Culture Infectious Doses) of respective virus. The
cells after virus isolation were incubated for 2 days at
37 ꢀC with various concentrations of the respective com-
pounds, ranging from 1:2 to 1:64.
Peptide
CPEa (lM)
1
165
2
3
4
>420
>448
468
The antiviral activity of the tested peptides was determined
using a cytopathic effect. The inhibition of the viral CPE
was assessed by a light microscopy. Virus titres were
determined according to the Reed–Muench formula (26)
and expressed in TCID50 ⁄ mL at particular stages of the
experiments. The antiviral activity of the tested peptides
was finally expressed as the compound concentration that
reduces virus yield by 50% (IC50).
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Ribavirin
Acyclovir
>775
380
>826
>1174
260b
350b
447b
504b
290b
>315b
282b
Results and Discussion
350b
The 18 new analogues of alloferon were prepared by the
manual solid-phase techniques. The synthesis of peptides
1–3 and 9–18 were performed using the standard Fmoc
procedure on Wang resin. As a coupling reagent, HBTU in
the presence of HOBT was used. The N-Fmoc group was
removed with 20% piperidine in DMF. The peptide-resin
was cleaved with TFA in the presence of EDT and water.
Other peptides (4–8) were synthesized by the classical
solid-phase method according to the Boc-procedure.
DCC in the presence of HOBt was used as a coupling
reagent. The Boc-protecting group was removed with
30% TFA in CH2Cl2. Peptides were released from the resin
using TFA, TFMSA and EDT. All peptides were purified by
preparative HPLC. In each case, the free peptides had a
purity of >95%. Their analytical data are presented in
Table 2.
255b
312b
>300c, >100d
>250c,d
aMaximal non-cytotoxic concentration for target cells (Vero, HEp-
2, LLC-MK2).
bMaximal non-cytotoxic concentration for Vero cells.
cVero cells; the 50% cytotoxic concentration for target cells in
lg ⁄ mL.
dHEp-2 cells, the 50% cytotoxic concentration for target cells in
lg ⁄ mL.
Cytotoxic activity of new analogues of alloferon was exam-
ined against the Vero, LLC-MK2 and HEp-2 cell lines. The
cells were incubated in the presence of various doses of
tested peptides. As shown in Table 3, the investigated
peptides did not show any cytotoxic activity against exam-
ined cell lines and did not affect the growth or morphology
of the tested cells. The MTT assay also proved that they
have no effect on cell proliferation.
Figure 1: Antiviral activity of alloferon and its analogues against
HHV-1MC and HHV-1 in Vero cells. : standard strain of HHV-
1MC
;
: clinical strain of HHV-1, X without effect (concentration of
The antiviral activity of investigated peptides was tested in
vitro with respect to: DNA viruses (HHV-1MC and the clini-
cal strain of HHV-1 in a Vero or HEp-2 cells), and RNA
viruses (971 PT Coxsackievirus B2 and the clinical strain of
Coxsackievirus B2 using HEp-2 and LLC-MK2 cells).
tested peptide >> 500 lM), acyclovir IC50 = 4 lM.
complete loss of antiviral activity (analogue 3, [5-13]-allofer-
on) (Figure 1). A similar activity was observed for these pep-
tides against the clinical strain of HHV-1 in Vero cells with
IC50 values 173, 117 and 168 lM, respectively (Figure 1).
The antiviral bioassay showed that most of investigated
peptides inhibit in vitro the replication of viruses in Vero,
LLC-MK2 or HEp-2 cells (Figures 1–5). We found that
among the truncated analogues the most active against
HHV-1MC in Vero cells were these without three (com-
pound 2, [4-13]-alloferon, IC50 = 186 lM), five (compound
4, [6-13]-alloferon, IC50 = 179 lM) and six N-terminal
As shown in Figure 2, only the C-terminal truncated ana-
logue of alloferon without the fragment His-Gly ([1-11]-allo-
feron, analogue 9) displays the antiviral activity against
HHV-1MC at lower concentrations (IC50 = 178 lM) than
alloferon.
During the investigation of the influence of alloferon deriva-
tives on the replication of the standard strain HHV-1MC in
amino acids (compound 5, [7-13]-alloferon, IC50
215 lM), whereas the deletion of four residues causes
=
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Chem Biol Drug Des 2013; 81: 302–309

New Alloferon Analogues
Figure 5: Antiviral activity of alloferon and its analogues against
971 PT CVB-2 and CVB-2 in HEp-2 cells. : standard strain of
971PT CVB-2; : clinical strain of CVB-2, X without effect (con-
centration of tested peptide >> 500 lM), ribavirin IC50 = 820 lM.
Figure 2: Antiviral activity of alloferon and its analogues against
HHV-1MC in Vero cells. Acyclovir IC50 = 4 lM.
(peptide 7) and nine amino acid residues (peptide 8) at the
N-terminal region were completely inactive.
The antiviral bioassay against RNA viruses demonstrated
that alloferon did not show inhibitory effect on the replica-
tion of examined CVB-2 in LLC-MK2 cells, whereas all
truncated analogues were active against the reference
strain of CVB-2 (Figure 4). The most active analogue was
peptide 1 ([3-13]-alloferon), without the N-terminal dipep-
tide His-Gly (IC50 = 93 lM). Moreover, only four N-trun-
cated analogues of alloferon showed an inhibitory effect
against the clinical strain of CVB -2 in LLC-MK2 (Figure 4).
Figure 3: Antiviral activity of alloferon and its analogues against
HHV-1MC and HHV-1 in HEp-2 cells. : standard strain of HHV-
1MC
;
: clinical strain of HHV-1, X without effect (concentration of
Additionally, as shown in Figure 5, the N-truncated ana-
logues weakly inhibited the replication of the standard
strain of 971 PT Coxsackievirus B 2 in HEp-2 cells. The
antiviral test against the clinical strain of CVB-2 in HEp-2
showed that the majority of the peptides have the antiviral
activity. The most active compounds were peptides with-
out N-terminal fragments: His-Gly (analogue 1, [3-13]-allo-
feron, IC50 = 38 lM) or His-Gly-Val-Ser-Gly (analogue 4,
[6-13]-alloferon, (IC50 = 78 lM) (Figure 5).
tested peptide >> 500 lM), acyclovir IC50 = 8 lM.
These results indicated that the C-terminal amino acid res-
idues of alloferon are more important for antiviral activity
than the N-terminal ones. From the analysis of analogues
modified in position 1, we found that the substitution of
the N-terminal histidine by aromatic amino acids such as
Phe, Trp, Phg, Tyr, Phe(p-OMe) or Phe(p-Cl) gives ana-
logues with antiviral activity. All analogues modified in posi-
tion 1 inhibit the replication of the virus HHV-1MC in Vero
cells as it was also observed by us (24) for the native pep-
tide (Figure 2). Moreover, we discovered that the alloferon
analogue modified in position 1 by Ala is inactive (24).
These results suggest that for optimal biological function
an aromatic structure is required at N-terminal position of
alloferon.
Figure 4: Antiviral activity of alloferon and its analogues against
971 PT CVB-2 and CVB-2 in LLC-MK2 cells. : standard strain of
971PT CVB-2; : clinical strain of CVB-2, X without effect (con-
centration of tested peptide >> 500 lM), ribavirin IC50 = 820 lM.
HEp-2 cells, we found that most of the N-truncated ana-
logues did not inhibit the replication of this virus (Figure 3).
However, alloferon and most of truncated analogues of al-
loferon inhibited the replication of the clinical strain of
HHV-1 in HEp-2 cells. The highest antiviral effect was
observed for compounds
1
([3-13]-alloferon, IC50
=
The biological effects of alloferon and its analogues modi-
fied in position 1 presented here and in our earlier report
(24), prompted us to perform the preliminary conformational
studies by molecular modelling. Theoretical calculations
117 lM) and 4 ([6-13]-alloferon, IC50 = 186 lM). Other ana-
logues such as [4-13]- (2) and [5-13]- alloferon (3) show
weak inhibitory activity, but the analogues without eight
Chem Biol Drug Des 2013; 81: 302–309
307

Kuczer et al.
A
B
D
C
E
Figure 6: Superposition of mini-
mum energy conformations of
[Ala1]-alloferon (A), [Lys1]-alloferon
(B), [Phe1]-alloferon (C), [Tyr1]-allo-
feron (D) and [Trp1]-alloferon (E)
(blue) upon alloferon (red).
were performed for alloferon and its selected analogues
modified in position 1 such as [Phe1]-, [Tyr1]-, [Trp1]-,
[Lys1]- and [Ala1]-alloferon (Figure 6).
compared with the native peptide. However, the activity of
the tested peptides depends on the virus and the cell line
used. The analysis of biological effects shows that the ref-
erence CVB-2 is less sensitive to investigated peptides.
The conformations of the peptides were optimized using
Amber force field (HYPERCHEM 7.01) in a two-step proce-
dure. First, the conformation of molecules was optimized.
Subsequently, the peptide was placed in a box containing
200 water molecules and its geometry was optimized again.
Furthermore, both our previous (24) and the present work
with the analogues of alloferon suggest that the presence
of the aromatic ring in position 1 of the peptide chain can
play a role in the expression of antiviral properties.
The obtained structures of analogues of alloferon were
superimposed to compare their conformation with confor-
mation of alloferon. The similarities between the conforma-
tions of alloferon with peptides 13–15 were found
(Figure 6). The differences between alloferon and [Lys1]- or
[Ala1]-alloferon were observed for the relative spatial
arrangement of the side chains of lysine, alanine and histi-
dine in position 1 (Figure 6). These results are in good
agreement with the biological data presented above and
our earlier investigations (24) and suggest that the posi-
tively charged side chain of Lys, which is conformationally
flexible, can create the optimal conditions for biological
activity of such analogue.
It is worth noting that the tested peptides show no cyto-
toxic activity against Vero, LLC-MK2 and HEp-2 cells. Our
results indicate that among the analogues of alloferon
could be new non-toxic antiviral agents.
Acknowledgments
This study was supported by the Polish Ministry of Science
and Higher Education (Grant No. N N204085638), the Uni-
versity of Wroclaw (Grant No. 2237 ⁄ W ⁄ WCh ⁄ 09) and by
the Medical University of Warsaw grants AM20 ⁄ W1 and
W2 ⁄ 2008–2009.
However, the mechanism of antiviral activity of alloferon is
still unknown; therefore, only the hypotheses resulting from
structure ⁄ activity studies of alloferon analogues could be
used in design of potential new antiviral compounds.
Conflict of Interest
The authors declare no financial or commercial conflict of
interest.
Conclusion
References
We found that in a series of new analogues of alloferon
most of the evaluated peptides inhibit the replication of
DNA and RNA viruses in vitro at lower or equal dose as
1. Kitazato K., Wang Y., Kobayashi N. (2007) Viral infec-
tious disease and natural products with antiviral activ-
ity. Drug Discov Ther;1:14–22.
308
Chem Biol Drug Des 2013; 81: 302–309

New Alloferon Analogues
2. Kormelinka R., Garcia M.L., Goodin M., Sasaya T., Ha-
enni A.L. (2011) The plant-infecting counterparts. Virus
Res;162:184–202.
insect peptides alloferon, any-GS and their analogues.
Synthesis and antiherpes activity. J Pept Sci;16:186–
192.
3. De Clercq E. (2008) Antivirals: current state of the art.
Future Virol;3:393–405.
4. Lembo D., Cavalli R. (2010) Nanoparticulate delivery
systems for antiviral drugs. Antivir Chem Chemo-
ther;21:53–70.
5. Schang L.M. (2006) Herpes simplex viruses in antiviral
drug discovery. Curr Pharm Des;12:1357–1370.
6. Brown T.J., McCrary M., Tyring S.K. (2002) Antiviral
agents: nonantiviral drugs. J Am Acad Dermatol;47:
581–599.
7. Field H.J., Biswas S. (2011) Antiviral drug resistance
and helicase – primase inhibitors of herpes simplex
virus. Drug Resist Updat;14:45–51.
8. Abonyi D.O., Adikwu M.U., Esimone C.O., Ibezim E.C.
(2009) Plants as sources of antiviral agents. Afr J Bio-
technol;8:3989–3994.
9. Krawczyk E., Łuczak M. (2005) Antiviral activity of natu-
ral compounds and their derivatives. Post Mikrobi-
ol;44:239–252.
17. Donald D.O. (2010) Susceptibility in vitro of Epstein–Barr
virus to myristoylated-peptide. Peptides;31:1409–1411.
18. Albiol Matanic V.C., Castilla V. (2004) Antiviral activity
of antimicrobial cationic peptides against Junin virus
and herpes simplex virus. Int J Antimicrob Agents;
23:382–389.
19. Wang G., Watson K.M., Peterkofsky A., Buckheit R.W.
Jr (2010) Identification of novel human immunodefi-
ciency virus type 1-inhibitory peptides based on the
antimicrobial peptide database. Antimicrob Agents
Chemother;54:1343–1346.
20. Ryu M.J., Anikin V., Hong S.H., Jeon H., Yu Y.G., Yu
M.H., Chernysh S., Lee C. (2008) Activation of NF-jB
by alloferon through down-regulation of antioxidant
proteins and IjBa. Mol Cell Biochem;313:91–102.
21. Chernysh S., Irina K., Irina A. (2012) Anti-tumor activity
of immunomodulatory peptide alloferon-1 in mouse
tumor transplantation model. Int Immunopharmacol;
12:312–314.
10. Bhuwan B.M., Vinod K.T. (2011) Natural products: an
evolving role in future drug discovery. Eur J Med
Chem;46:4769–4807.
11. Ratcliffe N.A., Mello C.B., Garcia E.S., Butt T.M., Aza-
mbuja P. (2011) Insect natural products and pro-
cesses: new treatments for human disease. Insect
Biochem Mol Biol;41:747–769.
12. Bullet P., Sto¨ cklin R. (2005) Insect antimicrobial pep-
tides: structures, properties and gene regulation. Pro-
tein Pept Lett;12:3–11.
22. Lee N., Bae S., Kim H., Kong J.M., Kim H.R., Cho
B.J., Kim S.J., Seok S.H., Hwang Y., Kim S., Kang
J.S., Lee W.J. (2011) Inhibition of lytic reactivation of
Kaposi’s sarcoma-associated herpesvirus by alloferon.
Antivir Ther;16:17–26.
23. Lee S.K., Kang J.S., Kim J.E., Jung D.J., Bae S., Lee
N.E., Kong J.M., Lee S., Shin D.H., Hwang Y., Lee
W.J. (2008) The anti-inflammatory effect of alloferon on
UVB irradiated human skin keratinocyte cell line, Ha-
CaT via the regulation of pro-inflammatory cytokine
production. FASEB J;22:1072.15.
´
´
13. Słocinska M., Marciniak P., Rosinski G. (2008) Insects
antiviral and anticancer peptides: new leads for the
future? Protein Pept Lett;15:578–585.
24. Kuczer M., Midak-Siewirska A., Zahorska R., Łuczak
´
M., Konopinska D. (2011) Further studies on the antivi-
14. Chernysh S., Kim S.I., Bekker G., Pleskach V.A., Filat-
ova N.A., Anikin V.B., Platonov V.G., Bullet P. (2002)
Antiviral and antitumor peptides from insects. PNAS;
99:12628–12632.
ral activity of alloferon and its analogues. J Pept
Sci;17:715–719.
25. Kuczer M., Dziubasik K., Łuczak M., Majewska A., Ka-
´
mysz W., Saniewska A., Konopinska D. (2008) The
15. Ourth D.D. (2004) Antiviral activity against human
immunodeficiency virus-1 in vitro by myristoylated-pep-
tide from Heliothis virescens. Biochem Biophys Res
Commun;320:190–196.
search for new biological activities for selected insect
peptides. Pestycydy ⁄ Pesticides;1–2:5–11.
26. Reed L.J., Muench H.A. (1938) A simple method of esti-
mating fifty per cent endpoint. Am J Hyg;27:493–497.
16. Kuczer M., Dziubasik K., Midak-Siewirska A., Zahorska
´
R., Łuczak M., Konopinska D. (2010) Studies of
Chem Biol Drug Des 2013; 81: 302–309
309