.1. Based On The Structure Of The Target Enzyme Of The Hcv Ns3 Helicase Inhibitors, Virtual Screening And Molecular Biological Activity Evaluation. Fungi 03-9007 Extraction And Separation Of Metabolites Of Anti-atherosclerosis Active Ingredient

HCV is an enveloped, positive-stranded RNA virus. Chronic hepatitis C virus(HCV) infections can be cured only in a fraction of patients treated with alphainterferon (IFN-a) and ribavirin combination therapy, which could not completelyremove the virus followed by significant side effects. HCV NS3 helicase unravels theRNA double-strand to facilitate the virus replication, representing as a key enzymealong the virus replication cycle. To accelerate the discovery of anti-HCV agents,virtual screening targeting HCV NS3 helicase was performed in our work. Virtualscreening relies on the 3D structure of specific receptors or other macromoleculartargets simulated by computer, and evaluate the affinity between the receptors andappropriate ligands. It could significantly reduce the scale of screening samplestogether with the time and resources, which could optimize the process of new drugdiscovery. We adopted the Syby17.1 software package developed by Tripos DiscoveryInformatics, and completed virtual screening towards HCV NS3 helicase mainlyusing the module FlexX. The data pool targeted by virtual screening was fromMicrobial Natural Products Database containing about 8,000 effective structures,from which we got about 50 structural formula of compounds with higher scores.Three authentic compounds were purchased from different companies.To further evaluate the results of virtual screening, we completed activityvalidation for these three true compounds via a cell-based model. The cell line used inthe validation was GS4.3 cells, which was a Huh-7 cell line transfected with HCVsubgenomic replicon. The virus RNA expressed in this cell line was about 300bp.When activity of non-structural proteins such as HCV NS3 was inhibited, the copiesof virus RNA would be lowered. Using alpha interferon as the positive control, weevaluated 5 compounds' activity on the cell level (including two other syntheticcompounds for primary validation). The result proved by quantitive RT-PCR showedthat copiamycin possessed HCV NS3 helicase inhibitory effect. Cardiovascular diseases such as coronary heart disease (CHD) remain to be adisastrous syndrome that devastates human health. It is commonly known thatatherosclerosis is the key pathological inducer for these diseases. Former researchesconcentrated on the contribution of LDL to this pathological change. Lately the HDLwas thought to be a positive factor that ameliorates atherosclerosis, and the discoveryof several receptors which could balance serum lipids like SR-BI, CD36 and ABCA1updates our knowledge about lipid regulation. All receptors involving in this processcould be potential targets for novel antiatherogenic agents.The antiatherosclerotic effect of HDL was mainly based on its role in reversecholesterol transport (RCT). Scavenger receptor class B typeⅠ(SR-BI) is thehigh-affinity HDL receptor at the molecular level, and the CLA-1 (CD36 andLysosomal integral membrane protein-ⅡAnalogous-1) is the human HDL receptor;ABCA1 might be an important factor that regulated the efflux of intracellularphospholipids and cholesterol to the unloaded HDL (including pre-βHDL andαHDL)which contained apoA-Ⅰand thus constructed the mature HDL. Therefore, bothcharacterize as significant receptors which could modulate serum HDL andintracellular cholesterol level. Their contribution to the reverse cholesterol transportendows them with the specificity of antiatherosclerotic effect.To search for antiatherogenic compounds, we established two cell-based highthroughput screening models using the same method, CLAP-LUC HepG2 cells forCLA-1 up-regulators and ABCA1-LUC HepG2 for ABCA1 up-regulators. Wescreened microbial secondary metabolite crude extracts using CLAP-LUC HepG2cells, resulting in eumycete 03-9007, one of the several positive strains which couldenhance the expression of luciferase. However, the assay during the isolation processtowards 03-9007 demonstrated that the components herein had more stable andspecific activity to ABCA1-LUC HepG2 cells. Therefore we decided to employ thelatter cells to steer the isolation work, which led to the purification of 03-9007A.Primary work regarding the identification of structure and activity was complete.