| Influenza virus is a serious pathogen which causes acute respiratory disease and results in high morbidity and mortality around the world. The widespread of influenza pandemics and seasonal flu has led to great panic and economic losses. The highly pathogenic avian influenza H5N1and H7N7virus which has the possibility to generate the next pandemic has also raised great concern. The threat of influenza thus has emphasized the urgent need of effective therapeutic strategies.Influenza virus neuraminidase (NA), which facilitates release of newly-formed virus and migration of virus in the respiratory tract, has been proved to be the most successful influenza drug target. Influenza A virus NA is classified into two Groups: Group1(N1,N4,N5,N8) and Group2(N2,N3,N6,N7,N9), based on its primary sequence. The Group1NA has an extra150-cavity in their active site. A novel N10is identified recently which is very different from N1-N9. The design and development of two approved drugs against the influenza virus NA:Zanamivir (Relenza(?)) and Oseltamivir (Tamiflu(?)), which has relied tremendously on the structural and catalytic mechanism study of NA, is an excellent example of structure-based drug design.However, the Zanamivir exhibits low oral bioavailability and is inconvenient for administration. Moreover, the broad use of Oseltamivir has caused frequent emergence and global spreading of Oseltamivir-resistant influenza virus. For these reasons, development of novel effective drugs is vital to the prevention and control of emerging highly pathogenic influenza strains.To direct the novel anti-influenza drugs design, we carried out our research on three aspects by means of biochemical and structural approaches (X-ray crystallography):1) characterize the structures of newly emerged influenza virus NA;2) comprehensively evaluate the effectiveness of novel influenza virus NA inhibitors and investigate the structural basis of binding mode;3) clarify the molecular mechanisms of NA drug-resistance. First of all, the head domain of2009pandemic H1N1influenza virus NA (09N1) was expressed with the baculovirus system, of which the crystal structure was then successfully solved soon after the outbreak of pandemic. We found that09N1has no150-cavity in its active site although it belongs to Group1members. This result has challenged the NA classification rule and thus we classify the09N1as an atypical Group1member. Further structural analysis showed that09N1could bind to the Group1specific inhibitor IG-173and the150-loop was broken to form a damaged150-cavity. Then we mutated the residue1491(usually seen in Group2NAs) of09N1to V (most in Group1NAs) and solved the09N1I149V as well as09N1I149V/IG-173complex structures. Accompanying the simulation result, we speculated that the09N1149V mutation will increase the flexibility of the150-loop.Moreover, we comprehensively evaluated the effectiveness and provided the structural basis of a novel influenza NA inhibitor Laninamivir (Inavir(?)) as well as its prodrug CS-8958. CS-8958was recently approved in Japan and it has long-lasting anti-influenza virus activity. We ultilized the recombinant NA proteins from different Groups with variant150-cavity properties:N5(typical Group1),09N1(atypical Group1) and N2(typical Group2). Our NA activity inhibition assay indicated that Laninamivir is more effective against Group1NA than to Group2NA and this result is highly correlated with the structural basis. Furthermore, we have found out that the binding mode of CS-8958/09N1is different from that of CS-8958/57N2, but with some similarities to NA/Oseltamivir binding, which provided a novel insight into Group specific differences of Oseltamivir binding and resistance.Meanwhile, we also analyzed the efficiency and structural basis of MS-257which is a novel influenza virus NA inhibitor with the Oseltamivir carboxylate core and Zanamivir4-guanidino group. MS-257could inhibit different Group NAs with high efficiency at nanomolar level. Inhibition of09N1H274Y by MS-257was about8fold higher than that of Oseltamivir. The interaction of MS-2574-guanidino group with NA is stronger than that of Oseltamivir4-amino group, which may explain why MS-257works better than Oseltamivir against09N1H274Y. This study has provided favarable evidence for the further development of MS-257. In addition, to comprehensively analyze the structural and functional characteristics of all the influenza A virus and prevent the potential pandemics, we also did some research on the swine origin N3, a high pathogenic avian influenza virus N7and the novel N10. The sequence and structure analysis of N3showed that N3had a Group1specific Y252residue while the other Group2NA had a T252, which was previously believed to be primary factor accounting for why N1was prone to Oseltamivir-resistance with H274Y mutation. However, our N3H274Y mutation showed no Oseltamivir-resistance although with the Y252residue. By solving the N3H274Y structure, we found novel Y274and W295conformations different from that of N1. The hydrogen bond network among the three loops(240,270and290-loop) and the π-π interaction between W295and adjacent296residue may contribute to these novel conformations. Our work has provided further insights into the Oseltamivir-resistant mechanisms. Up to now, we have obtained purified N7and N10proteins and achieved the crystals. The N10protein showed no sialidase activity.In conclusion, we have characterized several novel NA structures including the2009pandemic influenza virus NA (09N1). We found that09N1lacks the150-cavity in its active site. We have comprehensively evaluated the effectiveness of several novel influenza virus NA inhibitors such as Laninamivir and MS-257, and investigated the structural basis of binding mode. We provided further insights into the Oseltamivir-resistant mechanism. The comprehensive study of these key questions has provided considerable novel insights into the design and development of next generation anti-influenza drug which will benefit for the prevention and control of influenza. |
PDF Full Text Request