| Virus-induced diseases greatly harm human health, but the research development of the antiviral drugs is very slow, therefore it has an important practical significance to develop antiviral drugs vigorously.Part1Preparation, screening and evaluation of antiviral activity of the compounds.Antiviral activity of preparative compounds from liquid fermentation of strains offered by our laboratory were detected. EV71、ADV-7and H1N1were used as screening targets. Base on the cytopathogenic effect (CPE) and MTT assay, we had detected the antiviral activity of618natural products isolated from the113microbial fermentation broth. The results showed that57extracts exhibited anti-EV71activity,82extracts exhibited anti-ADV-7activity and32extracts could inhibit H1N1virus. Among of them,20compounds were over85%pure and one compound was novel. We also detected antiviral activity of72agents (above95%purity) from chemical structural modification based on natural products (Dehydroepiandrosterone and its synthetic derivatives named W-Series; Gramine and its synthetic derivatives named I-Series, and cycloalkylthiophene-imine derivatives named L-series). The results demonstrated that some of three series compounds exhibited the anti-EV71and anti-ADV-7activity in various degrees, only one compound (W-16) of W series had anti-influenza virus effect.The EC50s (Median effective concentrations) of the pure compounds, with good antiviral activity, against EV71, CVB3, ADV-7and H1N1were measured. The CC50s (Median cyctoxic concentrations) in the corresponding cells were also determinated. The SI (Selectivity therapeutic indexes) were calculated.These results enrich the antiviral source to a certain extent, also fully prove feasibility and huge potential that seeking the antiviral agents from microorganism and chemistry structural modification compounds.Part2Antiviral effects against EV71of pimprinine, WS-30581A and WS-30581B.We choosed a type of indole alkaloids with good antiviral activity isolated from microbial fermentation for further study. These compounds were identified as pimprinethine, and its derivatives WS-30581A and WS-30581B. Their ability in inhibiting viral infections has not yet been reported.Pharmacodynamic study showed that pimprinethine,WS-30581A and WS-30581B could preferably inhibit virus-induced CPE, reduced progeny EV71yields, as well as prevented EV71-induced apoptosis in RD cells.Preliminary studies on the mechanisms of action of pimprinethine, WS-30581A and WS-30581B were performed. The virucidal activity analysis, preventive effect analysis, inhibition of adsorption analysis, therapeutic effect analysis and release analysis were conducted. The results showed that these compounds mainly blocked the post-attachment stage of the viral infection, rather than inactivating virus directly, preventing virus infection, blocking the viral entry or affecting the viral release from the cells. A time-of-addition experiment was performed to further understand the mechanism of pimprinethine, WS-30581A and WS-30581B against EV71propagation in cells. The results indicated that these compounds mainly acted on the early stage of the viral replication post infection.To investigate the effects of pimprinethine, WS-30581A and WS-30581B on EV71replication further, the inhibitory efficacy of the compounds at the different time points after virus inoculation in RD cells was analyzed. These compounds showed strong activity against progeny viral yields and viral RNA synthesis, but not completely inhibited viral protein synthesis. These results implied that these compounds targeted viral replication in RD cells. There is a direct effect on viral RNA synthesis, and the inhibition of viral protein synthesis by the compounds is the result of their cumulative inhibitory action on viral RNA synthesis.The data described herein demonstrate that the pimprinine family of compounds are inhibitors effective against the replication of EV71, so they might be feasible therapeutic agents for the treatment of EV71infection. Part3Antiviral activity of Gramine and its derivatives against EV71.The antiviral activity of Gramine and21Gramine derivatives against EV71was investigated in cell-based assays. The results showed that18derivatives displayed some degree of inhibitory effects against EV71, but the anti-EV71activity of the lead compound Gramine was not observed.Based on antiviral activity and structure specificity, the compounds4a,4i,4r and4s were chosen for further investigation on modes of action against EV71. The results showed that these compounds functioned by targeting the early stage of the EV71lifecycle after viral entry, rather than inactivating the virus directly, inhibiting virus adsorption or affecting viral release from the cells. Among these derivatives, compound4s showed the most potency against EV71. Further studies of inhibitory effect on viral replication in cells demonstrated that compound4s could profoundly inhibit viral RNA replication, protein synthesis, and virus-induced apoptosis in RD cells.These results indicate that derivative4s might be a feasible therapeutic agent against EV71infection and that these Gramine derivatives may provide valuable inductor for the further design and synthesis of potential antiviral agents.Part4Antiviral activity of DHEA and its derivatives (W-3and W-12) against EV71.DHEA synthetic derivatives, W-3and W-12, with the most powerful antiviral activity and the highest SI, were chosen for further study on preliminary mechanisms against EV71. DHEA showed a lower anti-EV71efficacy, but DHEA derivatives, W-3and W-12could inhibit completely virus-induced CPE in Vero and RD cells. The cytotoxicity of W-3was similar with that of DHEA, however, W-12reduced significantly the cytotoxic effect of DHEA, with a superhigh SI. W-3and W-12could also inhibit completely progeny virus yield, but DHEA showed a slight inhibitory effect. The assays of dependence of the EC50on EV71inoculation amount were performed, the results suggested that W-3and W-12could be utilized over a wide range of viral burden.The antiviral mechanisms against EV71of W-3and W-12were investigated. W-3and W-12appeared not to be virucidal with EV71. They could not only have obvious effects on cellular function to prevent virus infection, but also affect virus adsorption and virus replication in cells. In comparison, treatment with tested compounds after EV71infection showed the strongest inhibitory efficiency, indicating that these compounds targeted mainly the replication of EV71in cells after virus adsorption. Time of addition assays further confirmed the results above. W-3and W-12could inhibit strongly EV71replication in RD cells, including RNA and protein synthesis, and virus-induced apoptosis.The metabolism of DHEA and W-3in RD cells by the HPLC (High performance liquid chromatography) assay was investigated. The results showed that these compounds could exist stably after interaction for24h in RD cells, indicating that W-3played a strong antiviral activity by the chemical structure itself, rather than by the interaction of DHEA and the other substances, or by new degradation products.We inferred that W-3and W-12mainly targeted on the cells to produce the immunologic factors, so that the virus could not be adsorbed and internalized into the cells or biosynthesis in the cells. Derivative W-3and W-12, with the novel structure and powerful antiviral activity, were expected to develop into new anti-EV71drugs.Part5Antiviral activity of DHEA derivative (W-16) against Influenza A virus (H1N1) in vitro and in vivo.W-16was chosen to investigate the antiviral activity against H1N1in vitro and in vivo. The results in vitro showed that W-16had no direct virucidal activity, and exhibited the strongest inhibitory activity when treatment after virus inoculation. W-16also inhibited H1N1-induced plages formation.The inhibitory effect of W-16against H1N1in Balb/c mice was investigated. The results indicated that W-16could improve the general physical conditions of the mice, increase the survival rate and prolong life, also reduce the lung lesions and lung index, decrease the lung viral titer.These results indicate the anti-H1N1activity of W-16in vitro and in vivo. We believe that W-16has an optimistic research prospect considering its new structure and good antiviral activity. |
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