Structure Modification Of Influenza Virus Hemagglutinin Inhibitory Peptides And The Biological Properties Evaluation

Influenza, commonly known as ’flu’, is a acute respiratory infection caused byinfluenza viruses, resulting in3-5million cases of severe illness and up to500,000deaths annually[1-2]. The influenza viruses are RNA viruses of the Orthomyxoviridaefamily, in which the viral genome is divided into multiple segments[3]. The total genomeof influenza A, which is considered to be a big threat to humans, consists of eightnegative sense RNA segments. Together, they code for10viral proteins: three subunitsof viral RNA-dependent RNA polymerase （PA、PB1、PB2）；major surface glycoproteins,neuraminidase （NA） and hemagglutinin （HA）; two matrix proteins （M1、M2）；nucleocapsid protein （NP）; and two nonstructural proteins, NS1and NS2[4]. The existingtwo anti-flu drugs M2ion channel inhibitors target M2protein and neuraminidaseinhibitors target viral NA, while the rest proteins can also be used to develop newanti-viral drugs as the target[5].Hemagglutinin （HA） is the receptor-binding and membrane fusion glycoprotein ofinfluenza virus, this step is the key to virus infection. Therefore, HA inhibitor has thedevelopment potential of antiviral drugs[6].Our lab found a12-amino-acid peptide called P19has a certain activity against theH1N1virus, in the process of using Phage display select HA-binding peptides. P19canprevent the H1N1virus infected host cells and cytopathic effect （CPE） was suppressedin high concentrations and its IC₅₀against H1N1was about100μmol/L. Because of P19specificity target HA with high affinity, we speculated that the mechanism of P19antiviral activity is combined with HA by high-affinity and occupied the active site. Dueto the weak antiviral activity of P19, we plan modify the amino acid residues of peptide sequence^[7]. Our study carried out a series of the modification research of P19sequence,and the biological properties of the modified peptides were preliminary evaluated.This research mainly focused on three aspects as follows.1) Established a quickand efficient synthesis, purification methods of P19modified peptides.2) Establishedthe peptides modified site and modification plan.3) Selection the antiviral activepeptides and evaluation the biological properties. The results are as following:1. To study the structure–activity relationship of P19, the amino acid in P19wasindividually replaced by alanine[8]. One peptide from9modified peptides which calledP19-8could suppress the CPE caused by H1N1, and IC₅₀of CPE was reduced to48.5μmol/L.2. To further modification for P19-8, the amino acid in P19-8was individuallyreplaced by D-amino acid, and the amino acid in active site of P19-8was replaced byanother18kinds of natural amino acids. The result showed that hydrophobic amino acidin the active site of P19-8has a huge impact on the inhibition of IV activity, and therewas no site Sensitive to D configuration.3. To improve solubility of P19-8Y, an RRKK tetramer was added at the aminoterminus. The result showed that carboxyl terminus hydrophobicity is very important tothe anti-viral activity of P19-8Y-EB. Its IC₅₀was8.9μmol/Ldetermined by CPE test.In conclusion, P19-8and P19-8Y were selected and preliminarily evaluated theantiviral activity of the two modified peptides in vitro and chick embryo. The solubilityoptimized peptide P19-8Y-EB was also proved to have good anti-H1N1activity.