Neuraminidase Inhibitors Screening From Compounds Of Traditional Chinese Medicinal Library And Their Anti-Influenza Virus Effects

Influenza caused by influenza virus is a highly contagious respiratory disease. Influenza virus is strongly pathogenic with strong infectivity and a short incubation period. It spreads with high speed, high morbidity and mortality.Influenza virus is a RNA virus belonged to orthomyxovirus, influenza virus genus. It has a single negative RNA strand in a coated structure. There are two significant membrane proteins on the surface of influenza virus: the hemagglutinin(HA) and neuraminidase(NA). NA has glycosidic enzyme activity, it can hydrolyze the glycosidic bond to separate the progeny virus from the infected cells. The NA plays an important role in influenza virus replication and facilitates the early process of influenza virus infection in human airway epithelium. Hence, as an ideal target for design of anti-influenza drugs, NA have attracted global attention. There are three drugs(zanamivir, peramivir and oseltamivir) belonged to NA inhibitor approved by globally for the clinical treatment. Due to lack of commercial drugs and the emergence of drug-resistant virus, new drugs are in urgent need to oppose new fatal influenza virus.Prevention and therapy of influenza have been practiced for thousand of years in China, but there is no systematic research to investigate which constituents in traditional Chinese medicines(TCMs) has an inhibitory effect against influenza virus.In this dissertation, we discussed 5 major aspects of TCMs regarding their anti-influenza activity:1. Screening NAI from the library of TCMsThe NA of influenza A has been divided into two different classes, known as group-1(comprising N1, N4, N5, N8) and group-2(comprising N2, N3, N6, N7, N9) according to there structural characteristics adjacent to the active site. We chose N1 and N2 from two different classes to represent group-1 and group-2 NAs, respectively. We screened 289 compounds from the library of TCMs to detect which one has NA inhibitory activities. We identified four effective compounds: rutin, hyperoside, jatrorrhizine hydrochloride and safflomin A.2. The anti-influenza virus activities in MDCK cells and in vivo using selected compoundsWe found these four compounds could prevent viral infection and protect MDCK cells from influenza virus. We selected rutin to test the weight, survival period and lung weight in ICR mouse infected FM1 virus. The results showed that rutin obviously increased the weight of ICR mouse infected virus, and the increase in a dose-dependent manner of rutin. In addition, rutin also reduced the mortality and decrease the lung index.3. Inhibitory mechanism of compounds against NAsTo study how these four compounds bind to NA, we firstly detected the inhibitory type of these four compounds with enzyme kinetics. The inhibitory were noncompetitive for rutin, hyperoside, and safflomin A toward N1, and mixed for jatrorrhizine hydrochloride toward N1. These inhibitory types were the same as these compounds interact with N2.In order to study the binding site for these compounds, we used the structure of N1 and N2 obtained by homologous modeling utilizing the MOEDLLER program in Discovery Studio. We analyzed the credibility of these simulative structures and then simulated the interaction between NA and the compounds with Autodock. The results were in agreement with the inhibitory types by the enzyme kinetics study. Finally, we also compared the simulated affinity and the IC50 values. It showed a corresponding relationship between the two indicators.4. The inhibition of NA for compounds against H7N9 influenza virus and their inhibitory mechanismIn 2013, a novel reassortant avian influenza A(H7N9) virus has been verified in China, accompanied with the high morbidity and morality. We measured inhibitory rate of N9 with the four compounds mentioned above. These compound showed inhibitory effect against N9. Using enzyme kinetics and molecular simulation, we found the inhibitory type of rutin, hyperoside and safflomin A against N9 is non-competitive. Jatrorrhizine hydrochloride is competitive toward N9. The simulated molecular affinity is in agreement with IC50 values.5. The combination of TCM compounds and oseltamivir carboxylate against NAsBy far, oseltamivir is the most effective drug to treat influenza, but resistant mutant has more frequently appeared along with the widespread using of the drug. In order to determinate the effect on the TCM compounds and oseltamivir combination, we tested the combination index for the above for compounds and oseltamivir carboxylate against influenza viruses. We found that the administrative sequence of the drug could affect the inhibitory activity toward NAs. We characterized the compounds that are synergistic in action with oseltamivir carboxylate against NAs.In summary, in this dissertation, we screened 289 compounds from the library of TCMs to detect which one has NA inhibitory activities. We identified four effective compounds: rutin, hyperoside, jatrorrhizine hydrochloride and safflomin A. These four compounds showed activities in protecting MDCK cells from influenza virus infection, and they acted synergistically with oseltamivir carboxylate against NAs. These researches elucidate the mechanism of the therapeutic effect of TCMs and give new insight in the field of NA inhibitor development.