Screening Of Inhibitory Peptides To Hepatitis C Virus NS5B RNA Polymerase

[Objective and background]There is about 185 millions of people infected hepatitis C virus (HCV) in the world, accounting for 3% of the total and the number is still growing rapidly each year. About twenty percent of HCV infected can be healed, but most persons (70%-80%) will grow into patients with chronic hepatitis C (CHC). Without timely treatment, 25% patients with CHC will gradually deteriorate as liver cirrhosis or even primary liver cancer.In the process of HCV replication, non-structural protein 5B (NS5B) polymerase not only plays an important catalytic role, but also has the activity of adjusting helices of HCV. In the different genotypes of HCV, NS5B polymerases are highly conserved. Enzymes close to the function of HCV NS5B polymerase have not yet been found in mammalian cells, which make the inhibitors to NS5B polymerase more specific. Based on the reasons above, study of inhibitors to HCV NS5B polymerase is becoming a hot spot in the researches of anti-HCV drugs.According to the active sites of the targets or the earliest known structural features of the active compounds, we could select reactive molecules from the compound library, which called virtual screening. The rapid development of virtual screening technology will push the new drug researches deeply.Molecular docking is one of the methods of drug design. It researches mainly about the interactions between ligand and receptor. Free energy is a very important concept in the process of binding between receptor and ligand. Now molecular docking technology has been used widely in the field of anti-tumor drug researches.The rapid development of computer aided design technology makes the study of micro combined of proteins and small molecule compounds more detailed and in-depth. Computer simulation of drug screening makes not only the intensity of selection and artificial screening reducing greatly, but also the development cycle shortens. At the same time, chance of success has been improved greatly. Great economic benefits would be achieved.This research designed inhibitory peptides to HCV NS5B RNA polymerase with mature molecular docking method, which provided a convenient way to subsequent screening.Part 1 Virtual Screening of Inhibitory Peptides to HCV NS5B RNA Polymerase1. ObjectiveTo design inhibitory peptides to HCV NS5B RNA polymerases by molecular docking software with the aids of virtual screening technology.2. MethodsMolecular docking software SYBYL-X2.0 was used. Active pocket including key amino acid sites to the NS5B RNA polymerases catalytic activity were designed. Peptides combinating with HCV NS5B RNA polymerases best were designed. Calculate van der Waals force, electrostatic interaction, hydrogen between the surface of NS5B RNA polymerase/peptides.3. Results(1) The active pocket completely covered the key active sites of HCV NS5B RNA polymerase.(2) The van der Waals force was a very important force; Electrostatic interactions in areas of Asp220, Thr221, Asn291, Asp318, Asp319, Gly317 were relatively strong. Electrostatic interaction was formed when polypeptides combined with HCV NS5B RNA polymerase. The affinity of hydrogen was huge between the ligands and the target proteins.4. ConclusionTwelve amino acids which could combine with the HCV NS5B RNA polymerase best were acquired.Part 2 the expression and purification of HCV NS5B RNA polymerase in vitro1. ObjectiveTo build over HCV NS5B proteins (NF), proteins of 21 amino acids missing (N-21) and proteins of 51 amino acids missing (N-51) in the C-terminal, which were then expressed in Escherichia coli.2. MethodsThe three plasmids were amplified with PCR, and then were cut off by BamH I and Xho I. System optimization and nickel column purification were performed to the target protein.3. Results(1) The three plasmids and fragments were the right size as expected.(2) The size of enzyme plasmids was 5360 bp and the purpose fragments were 1773 bp,1720 bp and 1620 bp respectively. When IPTG concentration was 1.1 mM,3 h, target protein was the best.(3) The purity of target proteins was greatly improved after Purification.4. ConclusionThe expression vectors meet the requirements of the experimental and the three kinds of HCV NS5B proteins could be expressed.Part 3 Identification to the activity of HCV RNA polymerase NS5B protein expressed in vitro1. ObjectiveTo identify the activity of HCV RNA polymerase NS5B protein expressed in Vitro.2. MethodsWestern Blot was performed to analysis the expressed proteins. NS5B proteins were reacted with peptides in ELISA system. Identify the activity of HCV RNA polymerase NS5B proteins expressed in vitro.3. Results(1) Three proteins could react with HCV positive serum. There was a clear reactive stripe at 55-70 kda.(2) All peptides could combine with expression protein. There was correlation between N-21 fusion protein and virtual docking results.(3) The activity of the expressed NS5B RNA polymerases was best when the reaction system was added 10 μl (10 μg/ml) NS5B proteins and the reaction time was 30 min.4. Conclusion The HCV NS5B fusion proteins of different length had the same protein activity and enzyme activity to normal NS5B.Part 4 screening and identification the inhibitory peptides of HCV NS5B RNA polymerase1. ObjectiveTo Screen and identify the inhibitory peptides of HCV NS5B RNA polymerase.2. MethodsEvaluation system in vitro was built to evaluate the inhibitory ability of the peptides to NS5B polymerase.3. ResultsP72 and P84 could inhibit the enzyme activity of three kinds of proteins significantly. IC50 of P72 was 9.85 μ M and P84 was 13.57 μ M, respectively.4. ConclusionThe peptides of HCV NS5B RNA polymerase by virtual screening could inhibit the biological activity of HCV NS5B protein.