Perspective of Use of Antiviral Peptides against Influenza Virus
Abstract
The threat of a worldwide influenza pandemic has greatly increased over the past decade with the emergence of highly virulent avian influenza strains. The increased frequency of drug-resistant influenza strains against currently available antiviral drugs requires urgent development of new strategies for antiviral therapy, too. The research in the field of therapeutic peptides began to develop extensively in the second half of the 20th century. Since then, the mechanisms of action for several peptides and their antiviral prospect received large attention due to the global threat posed by viruses. Here, we discussed the therapeutic properties of peptides used in influenza treatment. Peptides with antiviral activity against influenza can be divided into three main groups. First, entry blocker peptides such as a Flupep that interact with influenza hemagglutinin, block its binding to host cells and prevent viral fusion. Second, several peptides display virucidal activity, disrupting viral envelopes, e.g., Melittin. Finally, a third set of peptides interacts with the viral polymerase complex and act as viral replication inhibitors such as PB1 derived peptides. Here, we present a review of the current literature describing the antiviral activity, mechanism and future therapeutic potential of these influenza antiviral peptides.
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Re iew
Pe spec i e o Use o An i i al Pep ides agains
In luenza Vi us
Syl ie Skalicko a 1, Zbynek Hege 1,2, Ludmila K ejco a 1, Vladimi Peka ik 1, Ka el Bas l 3,
Joze Janda 4, F an isek Kos olansky 5, E a Va ecko a 5, Ond ej Zi ka 1,2, Voj ech Adam 1,2 and
Rene Kizek 1,2,*
Recei ed: 21 July 2015 ; Accep ed: 30 Sep embe 2015 ; Published: 20 Oc obe 2015
Academic Edi o : Alexande Ploss
1Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, Zemedelska 1, B no CZ-613 00,
Czech Republic; [email p o ec ed] (S.S.); [email p o ec ed] (Z.H.);
[email p o ec ed] (L.K.); [email p o ec ed] (V.P.); [email p o ec ed] (O.Z.);
[email p o ec ed] (V.A.)
2Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Technicka 3058/10,
B no CZ-616 00, Czech Republic
3Wool and Kni ing Resea ch Ins i u e, B no, Sujano o names i 3, B no CZ-602 00, Czech Republic;
[email p o ec ed]
4Labo a o y o Tumou Biology, Ins i u e o Animal Physiology and Gene ics, Academy o Sciences o he
Czech Republic, Libecho CZ-277 21, Czech Republic; [email p o ec ed]
5Ins i u e o Vi ology, Slo ak Academy o Sciences, Dub a ska ces a 9, 84505 B a isla a, Slo ak Republic;
[email p o ec ed] (F.K.); [email p o ec ed] (E.V.)
*Co espondence: [email p o ec ed]; Tel.: +420-5-4513-3350; Fax: +420-5-4521-2044
Abs ac : The h ea o a wo ldwide in luenza pandemic has g ea ly inc eased o e he pas
decade wi h he eme gence o highly i ulen a ian in luenza s ains. The inc eased equency
o d ug- esis an in luenza s ains agains cu en ly a ailable an i i al d ugs equi es u gen
de elopmen o new s a egies o an i i al he apy, oo. The esea ch in he ield o he apeu ic
pep ides began o de elop ex ensi ely in he second hal o he 20 h cen u y. Since hen, he
mechanisms o ac ion o se e al pep ides and hei an i i al p ospec ecei ed la ge a en ion due o
he global h ea posed by i uses. He e, we discussed he he apeu ic p ope ies o pep ides used in
in luenza ea men . Pep ides wi h an i i al ac i i y agains in luenza can be di ided in o h ee main
g oups. Fi s , en y blocke pep ides such as a Flupep ha in e ac wi h in luenza hemagglu inin,
block i s binding o hos cells and p e en i al usion. Second, se e al pep ides display i ucidal
ac i i y, dis up ing i al en elopes, e.g., Meli in. Finally, a hi d se o pep ides in e ac s wi h he
i al polyme ase complex and ac as i al eplica ion inhibi o s such as PB1 de i ed pep ides. He e,
we p esen a e iew o he cu en li e a u e desc ibing he an i i al ac i i y, mechanism and u u e
he apeu ic po en ial o hese in luenza an i i al pep ides.
Keywo ds: ca ionic pep ides; hemagglu inin; in luenza i us; memb ane usion; neu aminidase;
i al eplica ion
1. In oduc ion
Gene ally, some biological ac i e pep ides ac in compliance wi h o he de ense mechanisms o
plan s o mammals [1–3] and can be conside ed as one o he i s o ms o “chemical” p o ec ion
o euka yo ic cells agains bac e ia, p o ozoa, ungi, and i uses de eloped h oughou he cou se
o e olu ion [4,5]. These e ec s o na u al pep ides ha e been s udied since 1970s and since hen,
a ious he apeu ic ac i i ies we e p oposed agains G am-nega i e and G am-posi i e bac e ia [6].
The mechanisms o pep ide ac ion depend on hei s uc u e and can be enhanced by modi ica ions
Vi uses 2015,7, 5428–5442; doi:10.3390/ 7102883 www.mdpi.com/jou nal/ i uses
Vi uses 2015,7, 5428–5442
o na i e pep ides o chemically syn hesized coun e pa s. In addi ion o sc eening o lib a ies o
na i e s uc u es, e icien pep ides can be selec ed wi h commonly used phage display o in silico
app oaches [7]. Pep ides can be designed o mimic o in e ac wi h conse ed su ace p o eins and
in he case o a a ie y o pa hogens wi h mu agenic shi he pep ide sequence could be modi ied
o p ese e he apeu ic e iciency. In ecen yea s, esea che s ha e been explo ing a ious me hods
o imp o e pep ide syn hesis echnology om solid/liquid phase syn hesis up o comme cial scale.
The economic and biological p ospec s ha e been well discussed in he s eng hs, weaknesses,
oppo uni ies and h ea s (SWOT) analysis by Fosge au [8]. The good e icacy, sa e, selec i i y, and
p edic able me abolism a e he s eng hs o pep ide d ugs p oduc ion. On o he hand, chemical
and physical s abili y, p one o hyd olysis, and endency o agg ega ion a e he weaknesses o
pep ide pha maceu ics.
In luenza is highly con agious, eb ile and in luenza i uses cause acu e espi a o y disease.
In luenza i uses cause illness wi h signi ican mo bidi y and mo ali y wo ldwide and hey a e
conside ed as po en ial pandemic agen s due o hei high mu a ion a e, which may esul in he
o ma ion o new sub ypes [9,10]. The eme ging h ea o no el pandemic in luenza s ains sp eading
in o he human popula ion, as well as inc easing esis ance agains con en ional an i i al d ug
encou aged esea ch e o s o de elop new he apies agains in luenza i uses [11–14]. In ou e iew,
we p esen a comp ehensi e o e iew o pep ides wi h he apeu ic po en ial agains speci ic a ge s
o in luenza i uses.
2. Design and Cha ac e is ics o An i i al Pep ides
Cu en ly, he pep ides a e he candida e he apeu ic agen s ha o e selec i i y and speci ici y,
low le els o side e ec s, and possibili y o scaling up he p oduc ion om mg o kg le els.
On he o he hand, hey a e p edisposed o p o eoly ic deg ada ion in i o and a e apidly clea ed
om he ci cula ion. In he case o in luenza i us, he pulmona y deli e y ou e is he simples
way o deli e he apeu ic pep ides o he a ge cells. The main ad an age o his d ug deli e y
me hod is o a oid enzymes o gas oin es inal ac and also o sus ain la ge su ace a ea o d ug
abso p ion [15]. Imp o ed he apeu ic a ge ing can be achie ed h ough s uc u al changes such as
chemical modi ica ions, cycliza ion o u iliza ion o s able D-amino acids iso o ms [16].
Conside ing he ac ha i al in ec ion is o en ollowed by seconda y bac e ial in ec ions [17],
i would be highly ad an ageous i pep ide he apy can a ge bo h he p ima y i al and a seconda y
bac e ial in ec ion. The e is a possibili y o complemen ing ea men o s anda d an i i al d ugs wi h
an ibac e ial d ugs, such as neu aminidase inhibi o s (o al osel ami i and inhaled zanami i ) o M2
ion channel blocke s (aman adine and iman adine). The syne gic e ec and immune-modula o y
ole o such d ug combina ions ha e no been s udied ye in p o ec ion agains po en ial seconda y
bac e ial in ec ion.
Pep ides can be di ided in o se e al g oups based on hei ne cha ge, hyd ophobici y, helici y
o s uc u e. The balance be ween hyd ophobici y and he cha ge is an impo an ma ke o possible
he apeu ic applica ion o pep ides as well as amphipa ici y and molecula mass [18,19]. Al hough
hese e ec s we e adequa ely in es iga ed in he case o an imic obial pep ides’ e ec s on bac e ia
( e iewed by Teixei a e al. [18]), in he case o i uses, he ela ion be ween pep ide hyd ophobici y
and cha ge has no be clea ly es ablished ye . Howe e , he oxici y e ec s on mammalian cells and
hemoly ic ac i i y ha e been pa ially explained by Yin e al. [20]. Pep ides wi h low hyd ophobici y,
displayed no hemolysis e en a high concen a ions (up o 320 µM). In s iking con as , pep ides
wi h high hyd ophobici y showed hemoly ic ac i i ies a all concen a ions es ed. This could be
explained by ela i ely highe hyd ophobici y ha unde goes a s uc u al ansi ion in con ac wi h
bac e ial- ype memb anes om α-helical- o β-s and- ype s uc u es compa ed o he co esponding
pep ides wi h lowe hyd ophobici y [20]. An i i al ac i i ies o pep ides ha e been s udied o ea
se e e i al disease like HIV [21,22], hepa i is [23], he pes simplex [24,25], and in luenza i us [26,27].
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The g ea ad an age o pep ides agains i uses consis s in he educed possibili y o de eloping
esis ance du ing he ea men [28].
In luenza Vi us Replica ion Cycle
In o de o unde s and he apeu ic po en ial o pep ides agains in luenza i us, i is necessa y
o unde s and he i al eplica ion cycle. The in luenza i us (Figu e 1A) is an en eloped i us o
he O homyxo i idae amily. The i al genome is composed o eigh segmen s o single-s anded
nega i e sense RNA c ea ing a ibonucleop o ein complex (RNP) wi h polyme ase p o eins PB1, PB2,
PA and nucleop o ein (NP) [29]. The li e cycle s a s a e he a achmen o i us o he hos cell
ia i al hemagglu inin (HA). Hemagglu inin is a ime ic su ace glycop o ein ecep o ecognizing
sialic acids on he su ace o hos cells [30]. A e i us en y h ough ecep o -media ed endocy osis,
he usion p ocess media ed by HA ime s is ac i a ed by low pH in la e endosomes, unde which
HA s uc u e is des abilized and con o ma ionally changed. Consequen ly, he N- e minus o HA2
glycop o ein is exposed and inse ed in o he endosomal memb ane esul ing in he usion o i al
and endosomal memb anes. Only HA molecules ha a e p e iously p o eoly ically clea ed in o he
HA1 and HA2 glycop o eins a e able o media e he usion. The in e io en i onmen o i al pa icle
is acidi ied h ough an ion channel o med by M2 p o ein, leading o he dissocia ion o i al ma ix
p o ein (M1) om i al ibonucleop o ein ( RNP) complex. The M1 p o ein is subsequen ly eleased
in o he hos cell cy oplasm and anspo ed o he nucleus, he si e o i al RNA eplica ion and
ansc ip ion [31]. The eplica ion and ansc ip ion o RNA is ca alyzed in he cell nucleus by i al
RNA polyme ase complex [32]. In luenza i al mRNA a e ansla ed by he hos cell ansla ion
machine y. The newly syn he ized i al p o eins HA, neu aminidase (NA) and M2 p o eins a e
anspo ed o he plasma memb ane [33]. I is gene ally assumed ha he in luenza en elope is
de i ed om he hos cell memb ane, which includes lipid a s ich in choles e ol and sphingolipids
(Figu e 1B). These lipid a s se ing as a pla o m o concen a ing HA and NA o e ec i e i al
usion and elease om he hos cell [34,35]. The M1 p o ein plays a ole in he assembly p ocess since
i in e ac s wi h lipid memb anes. The M2 p o ein, which is abundan in he in ec ed cells, egula es
he pH in endoplasmic e iculum and in anspo esicles du ing he HA syn hesis, ime iza ion
and i s anspo o he plasma memb ane. The M2 p o ein hus ensu es he co ec olding o HA
ime . Neu aminidase, he second main su ace glycop o ein is needed o elease new assembled
i on pa icles om he cell su ace. The M2 p o ein, which is ound in he a pe iphe y, appea s
o media e memb ane scission and pa icle elease om he in ec ed cells du ing he i us budding
p ocess [36].
3. Mode o Ac ion o Va ious An imic obial Pep ides wi h An i i al Ac i i y
The h ee main mechanisms o an i i al e ec s o an i i al pep ides a e: (i) pep ides ha inhibi
a achmen o i uses and i us-cell memb ane usion; (ii) pep ides ha dis up he i al en elope;
and (iii) pep ides ha inhibi eplica ion o in luenza i us by in e ac ing wi h i al polyme ase
(Table 1). In his ega d, he same mechanism ha is desc ibed o in luenza A, which is he commonly
epo ed ype o in luenza in publica ions, ha e also been epo ed in he case o in luenza B ype.
3.1. The Pep ides Inhibi ing Vi us A achmen and Vi us-Cell Memb ane Fusion
Two mechanisms o inhibi ion o i us en y by pep ides ha e been p oposed. In he i s case,
he pep ides compe e wi h sialic acid (SA) binding by blocking ecep o si e o HA (Figu e 2, S ep I.).
The second mechanism in ol es he in e e ence wi h HA con o ma ion change necessa y o i al
usion (Figu e 2, S ep II.). Thus, he usion o i al and endosomal memb anes is blocked and elease
o RNA o he hos cell is p e en ed. The i al eplica ion and mechanisms o pep ide ac ion a e
shown in Figu e 2.
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Vi uses 2015, 7, page–page
3
In luenza Vi us Replica ion Cycle
In o de o unde s and he apeu ic po en ial o pep ides agains in luenza i us, i is necessa y
o unde s and he i al eplica ion cycle. The in luenza i us (Figu e 1A) is an en eloped i us o
he O homyxo i idae amily. The i al genome is composed o eigh segmen s o single-s anded
nega i e sense RNA c ea ing a ibonucleop o ein complex (RNP) wi h polyme ase p o eins PB1,
PB2, PA and nucleop o ein (NP) [29]. The li e cycle s a s a e he a achmen o i us o he hos
cell ia i al hemagglu inin (HA). Hemagglu inin is a ime ic su ace glycop o ein ecep o
ecognizing sialic acids on he su ace o hos cells [30]. A e i us en y h ough ecep o -media ed
endocy osis, he usion p ocess media ed by HA ime s is ac i a ed by low pH in la e endosomes,
unde which HA s uc u e is des abilized and con o ma ionally changed. Consequen ly, he N- e minus
o HA2 glycop o ein is exposed and inse ed in o he endosomal memb ane esul ing in he usion o
i al and endosomal memb anes. Only HA molecules ha a e p e iously p o eoly ically clea ed
in o he HA1 and HA2 glycop o eins a e able o media e he usion. The in e io en i onmen o
i al pa icle is acidi ied h ough an ion channel o med by M2 p o ein, leading o he dissocia ion
o i al ma ix p o ein (M1) om i al ibonucleop o ein ( RNP) complex. The M1 p o ein is
subsequen ly eleased in o he hos cell cy oplasm and anspo ed o he nucleus, he si e o i al
RNA eplica ion and ansc ip ion [31]. The eplica ion and ansc ip ion o RNA is ca alyzed in
he cell nucleus by i al RNA polyme ase complex [32]. In luenza i al mRNA a e ansla ed by he
hos cell ansla ion machine y. The newly syn he ized i al p o eins HA, neu aminidase (NA) and
M2 p o eins a e anspo ed o he plasma memb ane [33]. I is gene ally assumed ha he in luenza
en elope is de i ed om he hos cell memb ane, which includes lipid a s ich in choles e ol and
sphingolipids (Figu e 1B). These lipid a s se ing as a pla o m o concen a ing HA and NA o
e ec i e i al usion and elease om he hos cell [34,35]. The M1 p o ein plays a ole in he
assembly p ocess since i in e ac s wi h lipid memb anes. The M2 p o ein, which is abundan in he
in ec ed cells, egula es he pH in endoplasmic e iculum and in anspo esicles du ing he HA
syn hesis, ime iza ion and i s anspo o he plasma memb ane. The M2 p o ein hus ensu es he
co ec olding o HA ime . Neu aminidase, he second main su ace glycop o ein is needed o
elease new assembled i on pa icles om he cell su ace. The M2 p o ein, which is ound in he
a pe iphe y, appea s o media e memb ane scission and pa icle elease om he in ec ed cells
du ing he i us budding p ocess [36].
Figu e 1. (A) The s uc u e o in luenza i us pa icle; (B) S uc u e o he lipid a
localized in he in luenza lipid bilaye . The lipid a s a e composed mainly om
glycolipids (GPLs), choles e ol and sphingolipids. These mic odomains a e esponsible
o he e ec i e i al usion.
Figu e 1. (A) The s uc u e o in luenza i us pa icle; (B) S uc u e o he lipid a localized in he
in luenza lipid bilaye . The lipid a s a e composed mainly om glycolipids (GPLs), choles e ol and
sphingolipids. These mic odomains a e esponsible o he e ec i e i al usion.
Vi uses 2015, 7, page–page
6
3. Mode o Ac ion o Va ious An imic obial Pep ides wi h An i i al Ac i i y
The h ee main mechanisms o an i i al e ec s o an i i al pep ides a e: (i) pep ides ha inhibi
a achmen o i uses and i us-cell memb ane usion; (ii) pep ides ha dis up he i al en elope;
and (iii) pep ides ha inhibi eplica ion o in luenza i us by in e ac ing wi h i al polyme ase
(Table 1). In his ega d, he same mechanism ha is desc ibed o in luenza A, which is he
commonly epo ed ype o in luenza in publica ions, ha e also been epo ed in he case o
in luenza B ype.
3.1. The Pep ides Inhibi ing Vi us A achmen and Vi us-Cell Memb ane Fusion
Two mechanisms o inhibi ion o i us en y by pep ides ha e been p oposed. In he i s case,
he pep ides compe e wi h sialic acid (SA) binding by blocking ecep o si e o HA (Figu e 2, S ep I.).
The second mechanism in ol es he in e e ence wi h HA con o ma ion change necessa y o i al
usion (Figu e 2, S ep II.). Thus, he usion o i al and endosomal memb anes is blocked and
elease o RNA o he hos cell is p e en ed. The i al eplica ion and mechanisms o pep ide ac ion
a e shown in Figu e 2.
Figu e 2. Mechanisms o inhibi ion o i us en y by pep ides. Vi al en y can be blocked ia
in e ac ion o pep ide wi h hemagglu inin (HA), commonly in e ac ing wi h esidue o sialic acid.
This phenomenon esul s in he al e a ion o HA unc ions, and hus in luenza i ion canno be
a ached o he memb ane o a hos cell. The second an i i al ac ion o pep ides may be ca ied ou
in acellula ly due o blocking o HA con o ma ion change ha commonly leads o open o
endosome and dissemina ion o i al genome.
Figu e 2. Mechanisms o inhibi ion o i us en y by pep ides. Vi al en y can be blocked ia
in e ac ion o pep ide wi h hemagglu inin (HA), commonly in e ac ing wi h esidue o sialic acid.
This phenomenon esul s in he al e a ion o HA unc ions, and hus in luenza i ion canno be
a ached o he memb ane o a hos cell. The second an i i al ac ion o pep ides may be ca ied ou
in acellula ly due o blocking o HA con o ma ion change ha commonly leads o open o endosome
and dissemina ion o i al genome.
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Table 1. Lis o an i i al pep ides.
The Pep ides Inhibi ing Vi us A achmen and Vi us-Cell Memb ane Fusion
Pep ide In luenza Se o ype Sequence Con o ma ion Ne Cha ge * Hyd ophobic Residue * IC50 Re e ence
EB pep ide B oad spec um RRKKAAVALLPAVLLALLAP linea 4 70 3 o 20 µM [37]
De i ed EB pep ide B oad spec um RRKKLAVLLALLA linea 4 69 3.5 µM [38]
P1 H9N2 NDFRSKT linea 1 14 48 µM [39]
P1 cyclic H9N3 CNDFRSKTC cyclic 1 33 71 µM [39]
FluPep 1 H1N1 WLVFFVIFYFFR α-helix 1 83 0.093 µM [40]
FluPep 2 H1N1 WLVFFVIAYFAR α-helix 1 83 0.0009 µM [40]
FluPep 3 H1N1 WLVFFVIFYFFRRRKK α-helix 5 62 0.00003 µM [40]
FluPep 4 H1N1 RRKKWLVFFVIFYFFR α-helix 5 62 0.00004 µM [40]
FluPep 7 H1N1 RRKKIFYFFR α-helix 5 40 0.15 µM [40]
FluPep 8 H1N1 WLVFFVRRKK α-helix 4 60 0.63 µM [40]
FluPep 9 H1N1 FFVIFYRRKK α-helix 4 50 1.48 µM [40]
C18-s2 H1N1, H3N2 C17H35CO-ARLPRTMVHPKPAQP-NH2 - 3 33 11–15 µM [41]
Pal L1 H5N1 C16-ARLPRTMVHPKPAQP micelle 3 33 - [42]
Pal M1 H5N1 C16-ARLPRTMV micelle 2 50 - [42]
Pal S1 H5N1 C16-ARLPR micelle 2 40 - [42]
Flu i i ide B oad spec um - - - - - [43]
PEP 19-2.5 H7N7, H3N2, H1N1 GCKKYRRFRWKFKGKFWFWG α-helix 8 40 - [44]
PEP 19-4 H7N7, H3N2, H1N1 GKKYRRFRWKFKGKWFWFG α-helix 8 36 - [44]
PEP 19-8D H7N7, H3N2, H1N1 GFWFKGKWRFKKYRGGRYKKFRWKGKFWFG α-helix 12 33 - [44]
PEP 19-CP H7N7, H3N2, H1N1 SSNKSTTGSGETTTA α-helix 0 6 - [44]
De ensins H1N1, H3N2 ACYCRIPACIAGERRYGTCIYQGRLWAFCC β-shee 3 53 - [45]
The Pep ides Dis up ing Vi al En elope
Pep ide In luenza Se o ype Sequence Con o ma ion Ne Cha ge * Hyd ophobic Residue * IC50 Re e ence
LF C-lobe pep ide 1 H1H1, H3N2 SKHSSLDCVLRP α-helix 1 33 4–6 pM [46]
LF C-lobe pep ide 2 H1H1, H3N2 AGDDQGLDKCVPNSKEK α-helix ´1 23 4–7 pM [46]
LF C-lobe pep ide 3 H1H1, H3N2 NGESSADWAKN α-helix ´1 27 22–225 pM [46]
Muc opo in-M1 H5N1, H1N1 LFRLIKSLIKRLVSAFK α-helix 5 58 1.03 µM [47]
LL-37 H1N1, H3N2 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES α-helix 6 35 - [48]
The Pep ides Inhibi ing Vi al Replica ion
Pep ide In luenza Se o ype Sequence Con o ma ion Ne Cha ge* Hyd ophobic Residue* IC50 Re e ence
PB11´25 B oad spec um MDVNPTLLFLKVPAQNAISTTFPYT α-helix 0 44 - [49]
PB21´37 H1N1, H5N1 MERIKELRDLMSWSRTREILTKTTVDHMAIIKKYTSG α-helix 3 35 375 nM [50]
PB1731´´757 H5N1 ESGRIKKEEFAEIMKICSTIEELGRQK α-helix 0 33 - [51]
PB11´´25AT6Y H1N1, H5N1 MDVNPYLLFLKVPAQ α-helix 0 53 22–107 nM [52]
Kille pep ide H7N1 AKVTMTCSAS α-helix 1 50 2.6 µM [53]
HNP-1 H3N2 CYCRIPACIAGERRYGTCIYQGRLWAFCC β-shee 3 51 - [54]
Pep id 6 H1N1, H3N2 CATCEQIADSQHRSHRQMV Zn- inge 0 36 0.7 nM [55,56]
* Calcula ed by APD2: An imic obial Pep ide Calcula o and P edic o .
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Vi uses 2015,7, 5428–5442
The en y blocke pep ides a e e y p omising p ospec i e candida es o i al he apy
applica ions. Jones e al. [37] demons a ed he use o a 20 amino acid pep ide de i ed om
signal sequence o ib oblas g ow h ac o 4. This s udy con i med he b oad-spec um ac i i y o
pep ides agains human, swine, and a ian in luenza A H1N1, H2N2, H3N2, H5N1, H5N9, and
H7N3 s ains and in luenza B i uses. P e ea men o mice wi h pep ide shows 100% p o ec ion
agains in luenza i us, demons a ed by a dec ease in i al i e s in he lungs o in ec ed animals.
Al hough pos in ec ion ea men wi h pep ide was no as e ec i e as p e ea men , i should be
no ed ha he pep ide was as e ec i e as iman adine in p o ec ing mice om H5N1 in ec ion.
The pep ide inhibi ed chicken ed blood cells agglu ina ion wi h IC50 alues anging om 3 o 20 µM.
These esul s con i med he abili y o pep ide inhibi i al a achmen . Fu he mo e, he analysis
showed low cy o oxici y o he pep ide o he Madin-Da by canine kidney (MDCK) cells anging
in concen a ions exceeding 50 µM in medium con aining 1% BSA [37]. In a subsequen s udy, he
minimal and op imal sequence, RRKKLAVLLALLA, con e s an i i al ac i i y simila o ha o EB.
In addi ion, a newly iden i ied pep ide, RRKKVALLAVLLALLA, possessing signi ican ly enhanced
an i i al and po en ially i ucidal ac i i y agains in luenza A was explo ed. The N- e minus o
hese pep ides wi h cha ac e is ic sequence RRKK in luenced hei solubili y. The esul s o his s udy
showed ha up o ou amino acids om C- e minus and up o se en amino acids om N- e minus
could be dele ed while p ese ing he an i i al ac i i y [37,38]. O he esul s indica e ha pep ide P1
(NDFRSKT) has he abili y o in e ac wi h HA and exhibi s a s ong an i i al e ec s and negligible
hemoly ic ac i i y.
FluPep is a mix o p edominan ly hyd ophobic α-helical pep ides capable o in e ac ion wi h
HA blocking he i al usion. These pep ides a e de i ed om Tkip pep ide, which is a mime ic
o he supp esso o cy okine signaling p o ein, known o be ac i e in modula ing in lamma o y
cy okine esponses and known as an e ec i e an i i al d ug agains Pox i uses [57]. A a ie y
o in luenza sub ypes we e inhibi ed by FluPep in nanomola concen a ions in MDCK cells [40].
O he inhibi o y pep ides we e iden i ied using he Phage display lib a y and he no el alkyla ed
pep ide wi h he sequence C17H35CO-ARLPRTMVHPKPAQP was e ie ed. By docking simula ion
i was p o en ha he pep ide was mimicking sialic acid and was ecognized he by ecep o -binding
si e in HA [41]. I seems ha RLxRxMxxxK mo i is c ucial o he inhibi o y ac i i y, as i is
homologous wi h highly conse ed sequence wi hin HA in many in luenza s ains. The amino
e minal alkyl chain can play an impo an ole in di ec ing pep ides in o sel -assembling micelle,
s abilizing he pep ide and allowing in e ac ion wi h mul iple binding pa ne s. Hu l e al.
desc ibed N-modi ied pep ides wi h palmi ic acid (C16-ARLPRTMVHPKPAQP, C16-ARLPRTMV,
and C16-ARLPR) [42]. Due o he micelle s uc u e o he pep ides, hei en opy is educed [58,59]
and a ini y o HA is inc eased in compa ison wi h unmodi ied linea pep ides. Al hough he
mechanism o he binding o he micelle pep ide o he HA emains unclea , he concep has he
po en ial o u u e exploi s. Ano he pep ide blocking binding o HA o sialic acid is Flu i i ide,
which is cu en ly es ing in clinical ials. Besides in e e ing wi h he i us en y, i modula es
he immune sys em by ac i a ion o p oduc ion o an i-in lamma o y cy okines and chemokines,
inc easing he ac i i y o neu ophilic cells, and imp o ing phagocy osis o mac ophages [43].
A special g oup o pep ides agains in luenza i us a e cyclic del a de ensins ( e ocyclins), o med
by coupling o N- and C- e minal domains. Thei occu ence has been desc ibed in p ima es [43,45].
Meanwhile, p e ious s udies ha e shown hei abili y o inhibi HIV i us by hei abili y o bind o
HIV su ace p o ein and he simila mechanism is supposed o in luenza i us [60,61]. Ano he class
o an i i al pep ides a e an i-lipopolysaccha ide pep ides (SALPs). The SALPs a e o iginally based on
he LPS-binding domain o Limulus an i-lipopolysaccha ide- ac o (LALF) and ha e been disco e ed
by Gu smann and colleagues as pep ides wi h an imic obial ac i i y agains G am-posi i e and
G am-nega i e bac e ia [62]. Recen ly, SALPs, which show an i i al ac i i y agains some en eloped
i uses (HIV, HCV and HBV), ha e been in es iga ed [63]. Ho man and cowo ke s epo ed ha
SALPs a e able o inhibi in luenza i us eplica ion o a ious in luenza i us sub ypes (H7, H3 and
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Vi uses 2015,7, 5428–5442
H1) by p e en ing i us a achmen o hos cells in i o and in i o by binding o N-Ace ylneu aminic
acids as majo componen s o he in luenza i us ecep o [44].
3.2. The Pep ides Dis up ing Vi al En elope
The i al en elope is de i ed om hos cell memb anes con aining lipid a s and is ich in
sphingolipids and choles e ol [34,35], as shown in Figu e 1B. These compounds p o ide amphipa hic
cha ac e and nega i e cha ge [64], which is esponsible o elec os a ic in e ac ions wi h he
posi i ely cha ged ca ionic pep ides [65,66]. Gene ally, pep ide-memb ane in e ac ions a e media ed
by elec os a ic in e ac ions, while memb ane dis up ion can be accomplished by di e en means.
In he ield o an imic obial pep ides, he mechanism o ac ion is qui e well unde s ood ( he opic
is desc ibed in de ailed in [67]). The i al en elope is adop ed om hos cell and hus does no
exhibi a s ong nega i e cha ge as bac e ial memb ane. I may seem ha an i i al pep ides esul
in less selec i i y, howe e , se e al an i i al pep ides ha can cause he i al en elope dis up ion
ha e been epo ed. The mechanism o an i i al ac ion agains i uses can ei he a ge he i al
memb ane in gene al o he lipid a s ich in choles e ol. The la e case esul s in des abiliza ion
o i al su ace p o eins ha a e al eady en iched in he lipid a s domains [68]. The common
mechanisms o ac ion a e summa ized in Figu e 3A. Ca helicidins a e human an i i al pep ides
ha a e able o dis up i al en elope and we e shown o elici a numbe o hos p o ec i e
mechanisms such as p omo ion o ba ie epai s, chemokine and cy okine p oduc ion, modula ion
o dend i ic cell di e en ia ion, and T-cell pola iza ion, as well as demons a e po en an i-sepsis
and an i-in lamma o y p ope ies [69]. One ca helicidin, namely human LL-37, is p oduced as a
p ecu so o hCAP-18 ha accumula es in neu ophil g anules, bu i may also be p oduced in
epi helial cells as an acu e esponse o pa hogens [70]. Thei mechanism o ac ion is ela ed o he
in e ac ions be ween he pep ide and i al en elope by he ca pe model cha ac e ized by o ma ion
o con inuous laye on lipid bilaye su ace esul ing in memb ane des abiliza ion [71]. The po ency
o LL-37 agains in luenza i us seems o be simila o human de ensins, in ol ing di ec in e ac ions
wi h he i us wi hou a ec ing i al agg ega ion o inhibi ion o binding o up ake o i us by cells.
LL-37 may be an impo an con ibu o o he ini ial inna e de ense agains in luenza i us [48].
Lac o e in, widely p esen in a ious sec e o y luids, is able o in e ac no only wi h he i al
en elope, bu also wi h ecep o s on he cell memb ane o he hos cells by in e ac ion wi h i al
hemagglu inin [72]. The e o e, i is no su p ising ha bo ine lac o e in was ound as a possible
agen wi h an abili y o dis up he i us en elope [73]. Th ee sequences de i ed om lac o e in,
SKHSSLDCVLRP, AGDDQGLDKCVPNSKEK, and NGESSADWAKN, inhibi in luenza i us (H1H1,
H3N2) ac i i y a em omola concen a ions [46]. To imp o e s abili y and ci cula ion ime,
Balco e al. showed ha lac o e in could be encapsula ed in liposomes wi hou loss o ac i i y [74].
Ligh , ionic s eng h o pH change s imuli can s imula e ca go elease [75]. Thus, he liposomes could
be used o p ese e pep ide d ugs h ough issues anspo . De elopmen o his app oach dese es
de ailed a en ion in he u u e. P obably he bes -known memb ane dis up ing pep ide is meli in,
a 26-amino acid pep ide ha o ms he majo componen o Eu opean honeybee (Apis melli e a)
enom [76,77]. Meli in, wi h a p ima y s uc u e GIGAVLKVLTTGLPALISWIKRKRQQ, exhibi s a
a ie y o e ec s on lipid bilaye memb anes, such as de o ma ion o esicles, o ma ion o a i icial
po es, dis up ion, and lysis [78,79]. Cu en ly, pep ide-induced dis up ion o i ion en elopes is
aguely unde s ood. Two o he mos discussed models o memb ane dis up ion ha a e in oked
o explaining he elease o lipid con en o he bilaye a e: (i) o ming o up u es [80,81], mos ly
in he o m o o oidal po es cha ac e ized by pep ide agg ega ion on he lipid bilaye su ace and
subsequen pe pendicula pe mea ion h ough lipid bilaye by ansmemb ane po en ial change;
and/o (ii) lipid bilaye des uc ion/solubiliza ion [82,83] by he ca pe mechanism, men ioned
abo e. Lu e al. used eal- ime qua z c ys al mic obalance o acing he dynamic beha io o
lipid bilaye s in e ac ing wi h meli in. These esul s showed ha eaching a h eshold pep ide
concen a ion ( ypical o ca pe model) ollowed by mass emo al includes he elease o lipids,
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Vi uses 2015,7, 5428–5442
p obably as lipid-meli in complex, and he leakage o esicle componen s [84], by dis up ing he
bilaye cu a u e leading o micelliza ion o eleased lipids, is c ucial [82]. Finally, he i ion is
des oyed by ansien openings in he memb ane enabling he passage o low molecula mass
molecules p io o comple e memb ane lysis. Li and co-wo ke s es ed muc opo in and i s op imized
pep ide a ian muc opo in-M1 LFRLIKSLIKRLVSAFK and employed hese pep ides o an i i al
ac ion agains measles, SARS-CoV and in luenza H5N1 i uses [85]. Mucopo in M-1 design was
based on he p o ein sequence o muc opo in o enhance he ne posi i e cha ge o he hyd ophilic
side by eplacing glycine and p oline esidues wi h lysine and a ginine. I was ound ha he i ucidal
ac i i y o muc opo in-M1 was no ably inc eased, whe eas he o iginal muc opo in showed no
i ucidal ac i i y wi h EC50 o 2.10 µg/mL (1.03 µM) agains in luenza s ain H5N1. The inhibi ion
model could be explained by di ec in e ac ion wi h he i us en elope, he eby dec easing he
in ec i i y o i us. Due o his ac muc opo in-M1 analogues ep esen s a p ac ical ool o
de eloping b oad-spec um an i i al agen s, especially agains RNA i uses [85].
Vi uses 2015, 7, page–page
9
op imized pep ide a ian muc opo in-M1 LFRLIKSLIKRLVSAFK and employed hese pep ides o
an i i al ac ion agains measles, SARS-CoV and in luenza H5N1 i uses [85]. Mucopo in M-1
design was based on he p o ein sequence o muc opo in o enhance he ne posi i e cha ge o he
hyd ophilic side by eplacing glycine and p oline esidues wi h lysine and a ginine. I was ound
ha he i ucidal ac i i y o muc opo in-M1 was no ably inc eased, whe eas he o iginal
muc opo in showed no i ucidal ac i i y wi h EC50 o 2.10 μg/mL (1.03 μM) agains in luenza s ain
H5N1. The inhibi ion model could be explained by di ec in e ac ion wi h he i us en elope,
he eby dec easing he in ec i i y o i us. Due o his ac muc opo in-M1 analogues ep esen s a
p ac ical ool o de eloping b oad-spec um an i i al agen s, especially agains RNA i uses [85].
Figu e 3. (A) O e all scheme o he mos common in e ac ions be ween an i i al pep ides wi h an
in luenza i us lipid bilaye . Due o elec os a ic in e ac ions posi i ely cha ged pep ides a e a ac ed
by lipid bilaye wi h nega i e cha ge. The pep ides inse in o lipid bilaye (I.). The c i ical
concen a ion o pep ides igge s he lipid bilaye dis up ion. These phenomenon esul s in
o ma ion o a i icial po es (II.) h ough which he low mass molecules pene a e in o he capsid
and con ibu e o he lipid bilaye des uc ion and leakage o i al componen s (III.) as well as
dis up ion o NA and HA unc ions (IV.); (B) Scheme o unc ion o polyme ase assembly in i us
eplica ion cycle. An i i al pep ides may bind o PB2 subuni (pep ides de i ed om PB1 subuni )
and hus p e en he assembly o in luenza polyme ase complex ia blocking o ac i e binding si e
o PB2 subuni .
3.3. The Pep ides Inhibi ing Vi al Replica ion
Vi al RNA-dependen RNA polyme ase (RdRp), one o he a e-limi ing enzymes o influenza
i us ansc ip ion and eplica ion [86,87], is composed o h ee polyme ase subuni s (PB1, PB2 and
PA). The PB1 subuni is esponsible o polyme iza ion eac ion and endonuclease clea age [88,89],
while PB2 is esponsible o ecognizing and binding he cap s uc u e o hos mRNAs [90,91].
The exac ole o PA was ecen ly cla i ied: he N- e minus PA subuni o ming he domain wi h he
endonuclease ac i i y and PA endonuclease is esponsible o clea age o hos p e-mRNA [92,93].
The RdRp is held oge he h ough nonco alen in e ac ions. Dis up ion o RdRp assembly ep esen s
Figu e 3. (A) O e all scheme o he mos common in e ac ions be ween an i i al pep ides wi h
an in luenza i us lipid bilaye . Due o elec os a ic in e ac ions posi i ely cha ged pep ides a e
a ac ed by lipid bilaye wi h nega i e cha ge. The pep ides inse in o lipid bilaye (I.). The c i ical
concen a ion o pep ides igge s he lipid bilaye dis up ion. These phenomenon esul s in o ma ion
o a i icial po es (II.) h ough which he low mass molecules pene a e in o he capsid and con ibu e
o he lipid bilaye des uc ion and leakage o i al componen s (III.) as well as dis up ion o NA and
HA unc ions (IV.); (B) Scheme o unc ion o polyme ase assembly in i us eplica ion cycle. An i i al
pep ides may bind o PB2 subuni (pep ides de i ed om PB1 subuni ) and hus p e en he assembly
o in luenza polyme ase complex ia blocking o ac i e binding si e o PB2 subuni .
3.3. The Pep ides Inhibi ing Vi al Replica ion
Vi al RNA-dependen RNA polyme ase (RdRp), one o he a e-limi ing enzymes o in luenza
i us ansc ip ion and eplica ion [86,87], is composed o h ee polyme ase subuni s (PB1, PB2 and
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Vi uses 2015,7, 5428–5442
PA). The PB1 subuni is esponsible o polyme iza ion eac ion and endonuclease clea age [88,89],
while PB2 is esponsible o ecognizing and binding he cap s uc u e o hos mRNAs [90,91].
The exac ole o PA was ecen ly cla i ied: he N- e minus PA subuni o ming he domain wi h
he endonuclease ac i i y and PA endonuclease is esponsible o clea age o hos p e-mRNA [92,93].
The RdRp is held oge he h ough nonco alen in e ac ions. Dis up ion o RdRp assembly ep esen s
a ema kable oppo uni y o inhibi he enzyme unc ion and i us eplica ion (Figu e 2, S ep III.).
Fo his eason, he in e ac ion be ween PB1 and PA/PB2 is a p omising a ge o design o new
an i-in luenza d ugs (Figu e 3B). Nume ous au ho s ha e used PB1-de i ed pep ides in o de o
in e e e wi h polyme ase unc ion o he enzyme. Ghanem and cowo ke s es ed kine ics o i al
polyme ase subuni in e ac ions by immunop ecipi a ion me hod. PB11´25 and PB1715´740 pep ides
could bind PA subuni and inhibi in luenza eplica ion cycle by in e e ing wi h he i al polyme ase
ac i i y. P e e ably, he PB1715´740 pep ide binds o conse ed si e o in luenza PA subuni , his
app oach ep esen s p omise ool o block mos o in luenza A i us s ains [49]. Chase e al.
desc ibed an ELISA-based assay o in es iga e pep ides PB11´25 and PB21´37 capable o impai ing
polyme ase complex o ma ion [50]. The p esen ed sys em does no include o he ac o s, which
could play a ole in p o ein-p o ein in e ac ion such as o he binding domains, binding kine ics, and
s abiliza ion h ough ime o ma ion. This me hod enables o es lib a ies o a ian small pep ides.
In ano he s udy, Li and cowo ke s used PB1731´757 pep ide de i ed om in luenza i us s ain
H5N1. The au ho s showed ha PB1731´757 is capable o inhibi ing i al polyme ase ac i i y and
i al eplica ion [51]. PB1 de i ed pep ide can dis up he in e ac ion be ween he C- e minal pa o
PB1 (co esponding o PB1676´757) and he N- e minal pa o PB2 (co esponding o PB21´40) [51,94].
Wunde lich e al. in es iga ed a pep ide de i ed om he PA-binding domain o PB1 and ound
ha he pep ide blocked bo h he polyme ase ac i i y and i al sp ead. This wo k p o ides
oppo uni y o de eloping new an i i als ha speci ically in e e e wi h he polyme ase complex
assembly o bo h in luenza A and B i uses [41,52]. Con i and cowo ke s desc ibed an i in luenza
e ec o Kille pep ide (KP); a oxin isola ed om yeas wi h p o en an imic obial and an i-human
immunode iciency i us ype 1 (HIV-1) ac i i ies [53,95]. T ea men wi h KP demons a ed a
signi ican inhibi o y ac i i y on he eplica ion o wo in luenza A i us s ains, as e alua ed by
hemagglu ina ion, hemadso p ion, and plaque assays. In addi ion, KP demons a ed he comple e
inhibi ion o i us pa icle p oduc ion and a ma ked educ ion o he syn hesis o i al p o eins a a
KP concen a ion o 4 µg/mL [53].
3.4. O he Possible Mechanisms o In luenza Vi us Inhibi ion
Nume ous s udies ha e shown ha a g oup o an imic obial pep ides called de ensins
can posi i ely o nega i ely modula e in ec ion caused by bo h en eloped and non-en eloped
i uses [96,97]. De ensins play di ec ole in hos agains mic obial in ec ions as inna e immune
molecules [98] and a e able o inc ease he ac i i y o mucosal epi helia and inhibi he syn hesis
o i al RNA and p o eins [54]. Sal a o e and cowo ke s also showed ha human α-de ensin-1
(“human neu ophil pep ide–1” (HNP-1)) e ec i ely inhibi s eplica ion o in luenza i us and
syn hesis o i al p o eins when applied soon a e in ec ion. Fu he in es iga ion indica es ha i al
inhibi ion could be caused by he modula ion o p o ein kinase C ac i i y in in ec ed cells, sugges ing
he in ol emen o he PKC pa hway [54]. The p oposed s a egy in ol es pep ides de i ed om
in luenza ma ix p o ein (M1). Pep ide 6 was designed co esponding o a zinc inge egion o he M1
sequence o in luenza i us s ain A/PR/8/34 (H1N1), cen e ed a ound amino acids 148 o 166 [56].
The polyme ase inhibi o y p ope ies o pep ide 6 we e e alua ed on in ec ions induced in mice by
in luenza A/PR/8/34 and A/Vic o ia/3/75 (H3N2) i uses [56]. To a oid he enzyma ic b eakdown
o he pep ide, he d ug was adminis a ed by in anasal ou e and was well ole a ed up o a dose o
60 mg/kg/day. Based on sugges ed esul s, zinc inge pep ides may p o ide a new class o an i i als
e ec i e agains in luenza i us [55].
5436