The Role And Mechanism Of Asn-145 Site In The Design Of HIV Membrane Fusion Inhibitors

HIV fusion inhibitors are a kind of polypeptide drugs that can block the invasion of virus and cut off the spread of HIV at the stage of initial infection by specifically targeting to the region of HIV gp41 NHR to interrupt the formation of six helix bundle.T20(Enfuvirtide),the only inhibitor targeting to the HIV gp41,is the first fusion inhibitor clinically approved by FDA,U.S.A.It has slightly side effects and shows good therapeutic effects on patients who resist transcriptase inhibitors,protease inhibitors and other drugs.However,there exist problems such as relatively low activity,short half-life and low resistant gene barrier.The ideal fusion inhibitors should have good solubility,high potency,strong stability,as well as low costs.So it is necessary to design a new kind of inhibitor with high antiviral efficiency,broad-spectrum activity,high resistant gene barrier and short segments.Naito et al designed the SC29EK,a peptide combining EK(X-EE-XX-KK)motifs with the core residues of C34,which had efficient antiviral activity and could inhibit both T20-resistant HIV-1 strains and wild-type HIV-1 strains.In order to further investigate the mechanism of action of SC29EK,we truncated the SC29EK from the N terminal of peptides one by one.We found that there was a remarkable difference on the antiviral activity between SC29EK and SC28EK.We assumed that the amino acids Asn(gp41-145 loci in HIV)at the N terminal of SC29EK was vital for the binding between peptides and virus.A series of experiments were conducted to investigate this phenomenon.This study investigated the role of gp41 Asn-145 site.First of all,single-cycle infection assay was performed to determine the antiviral activities of SC29EK and series of truncated peptides.Subsequently,the representative peptides,T20 and C34,were selected to replace the Asn-145 site with Ala,and the potency of these peptides were detected.The stability of HIV 6-HB was evaluated by circular dichroism spectrum.The differences of heat variation during the interaction between C peptide and NHR were monitored by isothermal titration calorimetry.At the same time,the Asn-145 site located on the virus was also quite important,and we explored that the invasion and fusion ability of wild type strain and mutant strain.The MT-hook was a special structure of the N-terminal peptide,while the Asn-145 site was located at the C terminal.We explored the relationship bewteen MT-hook and Asn-145.Lu et al designed IDL,a peptide containing 29 amino acids,which could form a special hook-like structure at C-teriminal of peptides and interact with the special hydrophobic domain of NHR.We compared the antiviral activities and secondary structures among the peptides with 29 amino acids.To further study the conformation of Asn-145 in the 6-HB,we determined the crystal structure of complexes containing SC29EK peptide.Based on the comprehensive analysis,we can draw the following conclusions.(1)The Asn-145 site was critical for peptides' antiviral activity and potency to inhibit cell fusion,and also played an important role in the invasion and fusion of virus.(2)The Asn-145 site at the C-terminal of peptide regulated the MT-hook at the N-terminal of peptide.(3)We further illuminated the antiviral mechanism of SC29EK,the mechanism of drug resistance and the conformation and interaction mechanism of Asn-145 through analyzing the structure of the six-helix-bundle complex containing SC29EK peptide.Based on the structure,we analyze the antiviral mechanism of SC29EK and the mechanism of drug resistance strains.In this study,we determine the interaction mechanism of the peptides and virus by analyzing the differences of antiviral activities of peptides,which provides a new idea for the research on the mechanism of the virus entry and fusion.In addition,previous studies pay more attention to the interaction mechanism of the N-terminal of peptide,such as MT-hook,while this study focuses on the role of the C-terminal of peptide.What's more,we further analyze the relationship between amino acids at two ends.It provides new ideas and strategies for the modification of peptide sequences and the designation of peptides.