Design and synthesis of peptides involved in the inhibition of influenza virus infection

Abstract

The main purpose of the present research project was the identification of peptide capable of exercising potent anti-influenza activity. We identified bovine lactoferrin (bLf) as drug target, a multifunctional glycoprotein that plays an important role in innate immunity against infections, including influenza. Previously, by protein-protein docking calculations, it was demonstrated that different loops of bLf C-lobe, corresponding to the sequences 418-429 (SKHSSLDCVLRP, 1), 506-522 (AGDDQGLDKCVPNSKEK, 2), 552-563 (NGESSADWAKN, 3), 441-454 (TNGESTADWAKN, 4), 478-500 (KANEGLTWNSLKDK, 5), 552-563 (TGSCAFDEFFSQSCAPGADPKSR, 6), 619-630 (GKNGKNCPDKFC, 7), 633-638 (KSETKN, 8) and 642-659 (NDNTECLAKLGGRPTYEE, 9) can contribute to the binding to HA. These peptides were synthesized and tested for their ability to inhibit hemagglutination and cell infection. The results showed that peptide 1 binds to HA and neutralizes hemagglutination and cell infection of different strains of Influenza virus with a very high potency. Therefore, we considered peptide 1 as lead compound for the development of novel compounds with improved anti-influenza activity. To identify the shortest amino acid sequence needed for the peptide activity, we designed a new small library of peptides through addition and deletion of four amino acid residues at both the N- and C-terminals of this fragment (peptide 10-17). Three tetrapeptides, 14 (VLRP), 15 (SLDC) and 17 (SKHS), retained the inhibitory potency of the fragment 418-429, inhibiting the Influenza virus hemagglutination and cell infection in a concentration range of femto- to picomolar. Therefore, focused on peptide 14, we evaluated the importance of the net positive charge (Arg428) for the biological activity (peptides 18-23). We then decided to apply to peptides 15 and 17 L-Alanine scanning approach, a classical chemical approach to check the relevance of side chains of each aminoacidic residue in the interaction with the target molecule (peptides 24-31). Finally, in order to improve the pharmacokinetic properties of biologically active tetrapeptides 15 and 17, we synthetized N-methyl peptides (peptides 32-41) and peptoid analogues (compound 42-51). We demonstrated that no compound was able to inhibit HA activity in a greater extent of peptides 15 and 17. NMR spectroscopy analysis performed on compounds 1 showed a global turn conformation for this peptide and hypothesized the preferred bioactive conformation of our tetrapeptides. Moreover, based on conformational analysis, we tried to stabilize 3D structure of peptide 1, S[KHSSLD]CVLRP, through cyclization of the peptide backbone. To synthesize this cyclic peptide, we used the allyl ester (OAll) as orthogonal protecting group for aspartic acid side-chain. Surprisingly, our approach to synthesize the peptide mentioned above, failed completely. In particular, after the deprotection of aspartic acid Fmoc group by piperidine (20% v/v in DMF) for 30 min at room temperature, the formation of unexpected side-products was observed. Therefore, we studied this phenomenon and tried to obtain the desidered sequence with different approaches. We hypothesized that the aspartimide and pyperidinil derivative formation is conformation-dependent. Finally, we synthetized the desidered peptide, using as β-protecting group of aspartic acid and lysine, β-2-phenylisopropyl ester and methoxytrityl, respectively. LC-MS analysis confirmed the presence of the desired cyclic peptide. [edited by author]