Establishment Of H7N9 Influenza A Virus Neuraminidase Activity Model And Neuraminidase Inhibitors Screening

Neuraminidase (NA) is an important functional protein which anchors in the influenza virus surface as tetramer. It cleaves the sialic acid (SA) to release progeny virons which makes it an important target for antiviral agents. All current approved neuraminidase inhibitors (NAI) are the analogues of SA. They competitively bind to NA and subsequently block the virus release.The first human infected H7N9 strain A/Hangzhou/1/2013(H7N9) was isolated in 2013. Its neuraminidase was expressed and enzyme activity evaluation assay was established. Pharmacodynamics of zanamivir and oseltamivir were tested and compared of H7N9 with H1N1, H3N2 and H5N1. Drug resistant virus leads clinical therapy failure. Therefore, to investigate the susceptibility of possible mutant viruses to current drugs would provide information for clinical medication. Oseltamivir is the drug stockpile against influenza virus. Viruses carrying H274Y or R292K mutation on NA are the first two frequent appeared oseltamivir-resistant viruses on clinic. In this study NAH7N9-H271Y and NAH7N9-R289K were also expressed and the effects of zanamivir and oseltamivir on them were tested. For evaluation of zanamivir and oseltamivir on H7N9 neuraminidase activity, five more strains of influenza a viruses NA were used. The five strains are A/California/04/2009(H1N1), A/Puerto Rico/8/34/Mount Sinai (H1N1), A/WSN/1933 (H1N1), A/Wyoming/03/2003(H3N2) and A/Viet Nam/1203/2004(H5N1).The results show that IC50 values of zanamivir on NAs are:NAH7N9-WT:1.1 ±0.1 nmol·L-1; NAH7N9-H271Y:1.4±0.2 nmol·L-1; NAH7N9-R289K:38.0±5.9 nmol·L-1; NACA/04-WT: 0.3±0.1 nmol·L-1; NAPR/8-WT:0.3±0.1 nmol·L-1; NAWY/N2-WT:1.1±0.4 nmol·L-1; NAWS/33-WT:0.4±0.1 nmol·L-1; NAVN/04-WT:0.8±0.1 nmol·L-1; IC50 values of oseltamivir carboxylate on NAs are:NAH7N9-WT:1.6 ±0.6 nmol·L-1; NAH7N9-H271Y:15.1±2.6 nmol·L-1; NAH7N9-R289K:over 1000.0 nmol·L-1. The results indicated that: ①zanamivir has an equivalent effect on NAH7N9-WT comparing to other strains which implicates that it could be used in clinic; ②NAH7N9-R289K is resistant to both zanamivir and oseltamivir with 34 folds and over 625 folds respectively, which implicates that they should not be used when this mutation is tested; ③NAH7N9-H271Y is sensitive to both zanamivir and oseltamivir, implicating that this mutant strain has poor possibility to occur in clinic.NAIs of SA analogues are first-front antivirals against influenza virus. The risks of this kind drug are development of resistant viruses and neuropsychiatric adverse events (NPAEs). Influenza a virus is an RNA virus. Due to the poor fidelity during replication, the genes exhibit high genetic variability. Viruses harboring drug resistant mutations are selected by drugs and survive. Because all the current available NAIs are SA analogues, they have similar drug resistant mutant sites which lead to cross resistance, i.e. multi-drug resistance. Neuraminidase is widely distributed in nature and also exists in human. Thus zanamivir and oseltamivir may bind to endogenous NAs causing suppression of NA enzymatic activities and leading to NPAEs. The symptoms include delusion, abnormal behavior, convulsions and delirium, which reduce the patients’living quality. Therefore, novel non-substrate analogues NAIs are demanded in consideration to coping with the drug-resistant virus and NPAEs.In this study, drug library was screened to seek for novel NAIs. Firstly, the wild-type enzyme model of high pathogenic influenza a virus [A/Viet Nam/1203/2004(H5N1)] was applied to evaluate 1600 post-marketing drugs. When drug concentration at 10-4 mol·L-1, 40 active compounds were selected with inhibition rate over 60%. Then, we summarized the drug information including original indication, action target, mechanism, adverse effect and plasma concentration. After that 12 drugs were selected to assess their inhibitory effects on NAPR/8-WT. Finally, we obtained four better active drugs:rifampicin, phenazopyridine hydrochloride, sulfasalazine and protoporphyrin Ⅸ. The IC50 values of them on NAH7N9-WT are over 100μmol·L-1,90.6 μmol·L-1,77.6 μmol·L-1 and 38.6 μmol·L-1; on NAH7N9-H271Y are over 100 μmol·L-1, over 100 μmol·L-1,88.5 μmol·L-1 and over 100 μmol·L-1; on NAH7N9-R289K are 99.8 μmol·L-1,91.4 μmol·L-1,57.8 μmol·L-1 and 61.0 μmol·L-1;on NACA/04-WT are over 100 μmol·L-1,94.3 μmol·L-1,97.5 μmol·L-1 and 54.5 μmol·L-1; on NAPR/8-WT are over 100 μmol·L-1, over 100 μmol·L-1,90.8 μmol·L-1 and 43.0 μmol·L-1; on NAWY/N2-WT are over 100 μmol·L-1,69.7 μmol·L-1,85.6 μmol·L-1 and 69.9 μmol·L-1; on NAWS/33-WT are 98.7 μmol·L-1,94.4 μmol·L-1,75.6 μmol·L-1 and 51.2 μmol·L-1; on NAVN/04-WT are over 100 μmol·L-1,90.8 μmol·L-1,86.8 μmol·L-1 and 54.8 μmol·L-1.In this study,236 of natural sourced, chemical or biological synthesis compounds were also screened. One compound WB813 presented activity with the IC50 values of 15.6 μmol·L-1 (NACA/04-WT),58.0 μmol·L-1 (NAPR/8-WT),6.3 μmol·L-1 (NAWY/N2-WT), 66.9 μmol·L-1 (NAWS/33-WT) and 13.2 μmol·L-1 (NAVN/04-WT). There is no effect on NAH7N9-WT, NAH7N9-H271Y and NAH7N9-R289K at the final concentration of 100 μmol·L-1.In conclusion, in this study, the wild-type and oseltamivir-resistant NAs of H7N9 models were constructed. Enzymatic pharcodynamics of zanamivir and oseltamivir were tested by using these models. The results suggested that both zanamivir and oseltamivir could be used for patients infected by wild-type H7N9, but not by NAH7N9-R289K.The result could also help to explain the reason of the absence of NAH7N9-H271Y in clinic. Drug library which consists of 1600 drugs and 236 small molecule chemicals were screened for NAIs by using our models. As the results, five compounds presented weak inhibitory activities on neuraminidase. It’s notable that four of them are not SA structural analogues, which might offer clues for novel NAIs discovery.