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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010In Vitro Antiviral Properties of Alloferon, Any-GS andTheir Analogues against Human Herpes Virus andCoxsackie B2 VirusMariola Kuczer 1 , Anna Midak-Siewirska 2 , Renata Zahorska 2 ,Mirosław Łuczak 2 , and Danuta Konopińska 11 Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland; e-mail: km@wchuwr.pl2 Department of Microbiology, Medical University of Warsaw, Warsaw, PolandIntroductionThe present study was carried out to search for new antiviral activity among selectedpeptides originating from the insect such as alloferon (HGVSGHGQHGVHG), yamamarin(DILRGa) (Any-GS) and series of their analogues.Alloferon has been isolated from the blow fly Calliphora vicina [1]. In vitro alloferonstimulates natural killer lymphocytes and in vivo this peptide has antiviral and antitumorcapabilities. Pentapeptide Any-GS has been isolated from the wild silkmoth Antheraeayamamai [2]. This peptide suppresses the proliferation of the rat hepatoma cells [3]. Inpreliminary investigations we found that alloferon and Any-GS inhibit the replication ofHuman Herpes Virus type 1 (HHV-1) [4]. Continuing our studies on these peptides weperformed a further search for their other antiviral activity.We obtained the following peptides: 1/ alloferon and its analogues modified atposition 1 of the peptide chain - HGVSGHGQHGVHG (I), AGVSGHGQHGVHG (II),KGVSGHGQHGVHG (III), RGVSGHGQHGVHG (IV); 2/ Any-GS and its shortenedderivatives - DILRGa (V), ILRGNa (VI), DILRa (VII), DILa (VIII); 3/ analogues ofAny-GS modified at position 1 of the peptide chain - RILRGa (IX), QILRGa (X), GILRGa(XI), KILRGa (XII), AILRGa (XIII).Peptides I-IV were synthesized by the classical solid phase method according to theFmoc-procedure. Other peptides (V-XIII) were synthesized by classical solid-phase methodaccording to the Boc-procedure. All peptides were purified by preparative HPLC. Peptideswere tested in vitro for the antiviral activity in respect to DNA (Human Herpes Virus type 1and wild-type HHV), and RNA viruses (971 PT Coxsackie type B2 and wild-typeCoxsackie type B2) and cytotoxic activity using a Vero and HEp-2 cell lines. Theherpesviruses were propagated in Vero or HEp-2 cells. The coxsackieviruses werepropagated in HEp-2 cells. Antiviral and cytotoxic activities were assessed in vitroaccording to [4]. Antiviral activity of tested peptides was finally expressed as thecompound concentration that reduces virus yield by 50% (IC 50 ).Results and DiscussionMicroscopic observations showed that no changes occurred in Vero and HEp-2 cellsgrowth or morphology in the presence of tested peptides. The MTT assay also proved thatthey had no effect on cell proliferation. The antiviral bioassay showed that most ofinvestigated peptides inhibit in vitro the replication of viruses in Vero or HEp-2 cells(Table 1). We found that alloferon inhibits the replication of viruses HHV-1MC andwild-type HHV-1 in Vero cells with IC 50 values 305.50 μg/ml and 479.00 μg/ml,respectively. Among analogues of alloferon, only [Lys 1 ]-alloferon (III) was very activeagainst herpesviruses in Vero cells (IC 50 =147.09 μg/ml and 4.95 μg/ml) andcoxackieviruses in HEp-2 cells (IC50=74.0 μg/ml and 107.04 μg/ml). Any-GS (V) inhibitsalso the replication of HHV-1MC and wild-type HHV-1 in Vero cells. The IC 50 valueswere 235.30 μg/ml and 216.6 μg/ml respectively. A similar activity was observed for fourpeptides X-XIII. However, [Arg 1 ]-Any-GS (IX) was very active against HHV-1MC andwild-type HHV-1 in Vero cells. Moreover, the shortened derivatives (peptides VI and VIII)were very active only against the wild-type HHV-1. During the investigation of theinfluence of Any-GS and its derivatives on the replication of viruses in HEp-2 cells, wefound that [Gly 1 ]-Any-GS (XI) was very active against HHV-1MC, wild-type HHV-1, 971PT Coxsackie and wild-type Coxsackie B2 with IC 50 values 191.62 μg/ml, 102.76 μg/ml,197.92 μg/ml and 158.92 μg/ml, respectively. A similar activity was observed for peptidesVII and XIII. However, these peptides show a weak inhibitory effect on the replication ofHHV-1MC and Coxsackie B2.604