Study On Anti - HCV Pharmacodynamics Of IMB - DM122

HCV is a positive single-stranded RNA virus belonging to the Flaviviridae family and is a causative agent for hepatitis C around world. Given that the effect of the current standard of pegylated interferon a and ribavirin is limited, and there is no protective vaccine special for HCV. Thus developing new antivirals against HCV is imminent. In recent years, the arrival of the direct-acting antiviral (DAA) era has resulted in the development of well-tolerated and highly effective anti-HCV regimens and brought new hope to cure hepatitis C. However, long-term use of DAAs leads to drug-resisitance. So in order to overcome this problem, scientists turn to study drug combinations and develop anti-viral drugs targeted on host proteins. Our team have found an effective compound named IMB-DM122, which is a new anti-HCV candidate targeted on host proteins with low toxicity. We have identified the anti-HCV effect of IMB-DM122 that acts on HSC70 mRNA 3’UTR region and reduces its stability, down-regulates host HSC70 expression, and then reduces the level of HSC70 protein which is packaged into HCV particles. This will result in decreased infectivity of the HCV particle, and finally make an effect on resisting the HCV replication. But, the effect and characteristics of IMB-DM122 needs more researches, and this is also the content of the paper.In this paper, we optimized semi-quantitative western blot (WB) firstly, and then focus on the discussion of the IMB-DM122’ pharmacodynamics effects and advantages.Total proteins were extracted from the Huh7.5 cells infected with hepatitis C virus (HCV) firstly and quantified with BCA kit, HCV Core protein was chose as target protein, and protein Tubulin or Gapdh, which was encoded by housekeeping gene, as the internal control. By controlling the experimental factors, such as loading sample amount, concentration and the incubation time of first antibodies, and dilutions of chemiluminescent fluid as well, we analyzed those factors how to impact the semi-quantitative results. The semi-quantitative results showed that there is a linear relationship between relative intensity of target protein and the amount of total protein at must protein concentration range, beyond which, the rangeability of relative intensity of target protein reduced, even no changes. However, sample volume loaded, or protein selected as internal reference has little influence on the result of semi-quantitative WB. In the context of obtaining high-qualityband, concentration and incubation time of antibodies or dilution of Chemiluminescent fluid also has no influence on it. Yet, the semi-quantitative WB has certain defects and our results show that the permissible error of semi-quantitative result should be controlled within 15%. Therefore, the key impact factor on the result of semi-quantitative WB is the target protein amount of loading. On the premise of obtaining clear and high-quality bands, appropriately selecting loading volume of samples, concentration and incubation time of first antibodies, and dilutions of chemiluminescent fluid is conducive to accomplish experiments without affecting the final results of semi-quantitative WB.On an occasional basis, with the progress and optimization of the semi-quantitative western blot (WB), we will further explore the anti-HCV effects and characteristics of the IMB-DM122. The result showed that IMB-DM122 not only can resist the acute HCV infection, but have the activity of anti-chronic infection. It works in a dose-dependent manner and has the timeliness. Through the experiment of the combination of IMB-DM122 with the IFN and VX-950, we found that the group which combines IMB-DM122 with the IFN or VX-950 has an antiviral advantage over the single ones. Then, we analyzed the antiviral efficacy of IMB-DM122 against the HCV resistant variant. We utilize semipervir and sofosbuvir to induce drug resistantance through acting on Huh7.5 cells infected HCV, and obtain the drug-resistant strains. Then, we analyze the activity to drug-resistant strains of IMB-DM122. And the result show that IMB-DM122 has the same antiviral activity. Finally, we explore the IMB-DM122 specificity on its target family. And it appears to be highly efficient in inhibiting HSC70-related HCV replication, but the effect to other members of the HSP70 family such as HSP70, GRP75, GRP78 and other heat shock protein such as HSP90, HSP60 is not obvious. And this result indicates that the target of IMB-DM122 is specific.Conclusions:IMB-DM122 has advantages on resisiting not only the HCV acute infection, but also the HCV chronic infection in the cell culture. And the combination of IMB-DM122 with the IFN or VX-950 can improve the antiviral activity. In addition, it also shows the same antiviral activity to the drug-resistant variants. Furthermore, its antival effect has the target specificity, and it does not have obvious effect on other heat shock protein. The results can provide the scientific secundum for searching novel anti-hepatitis drugs, and also offer an important mode for the discovery of new antiviral drugs.