In Vitro Anti-viral Activities and Structureactivity Relationship Studies of Flavones and Dihydroflavone Derivatives as Influenza Virus Potential Neuraminidase Inhibitors

From the bioassay tests, 14 neuraminidase inhibitors （NIs） flavones and dihydroflavones derivatives from natural plants displayed different degree of inhibitory activities. Further- more, compounds No. 8 and 14 showed good inhibitory activity against influenza A virus with IC50 = 835.4 and 860.6 μg/mL. Then, to investigate interactions between NIs and neuraminidase （NA）, molecular docking was performed. Docking results indicated that Arg118, Asp151, Arg292 and Arg371 were the key residues in the active pocket of 2ht8. Main influencing factors of interactions between NIs and NA were hydrogen bond and electrostatic, then hydrophobic factor. Moreover, experimental activities of NIs were consistent with total scores of the docking. In order to understand the chemical-biological interactions governing their activities toward NA, QSAR models of 14 NIs were developed. The obtained HQSAR （hologram quantitative structure activity relationship）, PLS （partial least squares） and SRA （stepwise regression analysis） models were robust and had good exterior predictive capabilities. Moreover, squared multiple correlation coefficients （R2） and squared cross-validated correlation coefficients （Q2） of HQSAR and PLS models based on descriptors by Gaussian and Sarchitect were 0.832 and 0.721, 0.925 and 0.688, 0.892 and 0.692, respectively. R2 and SE （standard error） of SRA model based on descriptors by Gaussian were 0.922 and 0.072. Therefore, these models may be further used to design and evaluate the bioactivity of new compounds.

QSAR Studies on Influenza Neuraminidase Inhibitors——Acylthiourea Analogue

The quantitative structure-activity relationship （QSAR） of 30 acylthiourea analogues was studied by using a three-dimensional holographic vector of atomic interaction field （3D-HoVAIF） to describe their chemical structures. The descriptors obtained were screened by stepwise multiple regression （SMR） and a partial least-squares （PLS） regression model was built. The correlation coefficient r^2 of the established model and Leave-One-Out （LOO） Cross-Validation （CV） correlation coefficient q^2 are 0.624 and 0.409, respectively. The model has favorable stability and good prediction capability, and further QSAR analysis showed that hydrophobic interaction has the most important effect on the activity of acylthiourea analogue and 3D-HoVAIF was applicable to the molecular structural characterization and biologicalactivity prediction.

Rapid identification and isolation of neuraminidase inhibitors from mockstrawberry(Duchesnea indica Andr.) based on ligand fishing combined with HR-ESI-Q-TOF-MS

Neuraminidase inhibitors(NAIs)are the mainstay antiviral drugs against influenza infection.In this study,a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads(NA-MB).After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors,the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr.Twenty-four NA binding compounds were identified from the normal butanol(n-BuOH)extract of D.indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)assisted with Compound Structure Identification(CSI):FingerID,including 12 ellagitannins,4 brevifolin derivatives,3 ellagic acid derivatives,and 4 flavonoids.Among them,9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens,and from oseltamivir sensitive and resistant influenza A virus strains.The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA,with half maximal inhibitory concentration(IC50)values of 197.9 and 125.4μmol/L,respectively.Moreover,B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin-Darby canine kidney(MDCK)cells at the concentrations of 40 and 200μmol/L.

Design, Synthesis, and Evaluation of 3-（（4-（t-Butyl）-2-（2- benzylidenehydrazinyl）thiazol-5-yl）methyl）quinolin- 2（1H）-ones as Neuraminidase Inhibitors

A series of novel 3-（（4-（t-buty-）-2-（2-benzylidenehydrazinyl）thiazol-5-yl）methyl）quinolin-2（1H）-ones （7a--7z） were designed, synthesized and evaluated for their ability of inhibiting neuraminidase （NA） of influenza H1N1 virus. Some compounds displayed moderate influenza NA inhibitory activity. Compound 71 with the scaffold of 2-（2-（2-methoxybenzylidene）hydrazinyl）thiazole was the best one, exhibiting moderate NA inhibitory activity with ICs0 of 44.66 ~tmol/L. Structure-activity relationship showed that compounds with methoxy or hydroxy groups at the ortho position, fluorine and nitro groups at the meta position and chlorine and bromine groups at the para posi- tion of phenyl ring were more active. Docking study indicated that compound 71 has important interactions with some key residues （including Asp151, Glu119, Arg292, Tyr406, and Asn347） and binds to 430-cavity adjacent to NA active site.

A nonsynonymous SNP in human cytosolic sialidase in a small Asian population results in reduced enzyme activity: potential link with severe adverse reactions to oseltamivir

The use of oseltamivir, widely stockpiled as one of the drugs for use in a possible avian influenza pandemic, has been reported to be associated with neuropsychiatric disorders and severe skin reactions, primarily in Japan. Here we identified a nonsynonymous SNP （single nucleotide polymorphism） in dbSNP database, R41Q, near the enzymatic active site of human cytosolic sialidase, a homologue of virus neuraminidase that is the target of oseltamivir. This SNP occurred in 9.29% of Asian population and none of European and African American population. Our structural analyses and Ki measurements using in vitro sialidase assays indicated that this SNP could increase the unintended binding affinity of human sialidase to oseltamivir carboxylate, the active form of oseltamivir, thus reducing sialidase activity. In addition, this SNP itself results in an enzyme with an intrinsically lower sialidase activity, as shown by its increased Km and decreased Vmax values. Theoretically administration of oseltamivir to people with this SNP might further reduce their sialidase activity. We note the similarity between the reported neuropsychiatric side effects ofoseltamivir and the known symptoms of human sialidase-related disorders. We propose that this Asian-enriched sialidase variation caused by the SNP, likely in homozygous form, may be associated with certain severe adverse reactions to oseltamivir.

Laninamivir-induced ischemic enterocolitis:A case report

BACKGROUND Neuraminidase inhibitor-associated acute hemorrhagic colitis is rare.We report a case of ischemic enterocolitis that was likely caused by laninamivir.CASE SUMMARY A 54-year-old female patient with influenza type A was administered 40 mg of laninamivir via inhalation once.On the same day,the patient experienced bloody stools and lower abdominal pain.A contrast-enhanced abdominal computed tomography showed edema-like changes from the descending colon to the sigmoid colon,which suggested ischemic enterocolitis.CONCLUSION We treated a patient with ischemic enterocolitis caused by laninamivir,a rare but similar symptom following the administration of oseltamivir.

Novel Dual-Site-Binding Neuraminidase Inhibitor from Virtual Screening by Pharmacophore and Molecular Dynamics Methods

Neuraminidase is a significant anti-influenza target that plays crucial role in virus replication cycle. The discov- ery of 150-cavity in Group-1 neuraminidase provides us a novel mentality of designing inhibitor which can bind with both conserved site and 150-cavity. In order to discover novel dual-site-binding inhibitors, a 3D chemi- cal-feature-based pharmacophore model was established to cover dual-site in neuraminidase. The dual-site-binding model was consistent in predicting the binding conformation of Group-1 neuraminidase inhibitor and applied for virtual screening of Specs database. Compound 4 （ZINC05790048） that aligned well to the model was selected after multiple filtrations for molecular dynamics simulations, indicating improved binding energy with neuraminidase. It can sever as the lead compound for a novel series of inhibitors.

Multivalent neuraminidase hydrolysis resistant triazole-sialoside protein conjugates as influenza-adsorbents

We report the synthesis of pseudo triazole-sialoside protein conjugates of various valency that are resistant to neuraminidase for the adsorption of influenza viruses. The glycotriazole monomer bearing an amine-functionalized linker was synthesized by click chemistry and grafted to the lysine residues of bovine serum albumin （BSA） or human serum albumin （HSA） via diethyl squarate and adipate-based strategy. The binding of hemagglutinin （HA） and neuraminidase （HA） on the virion surface by the synthetic neoglycoproteins were evaluated by hemagglutination and neuraminidase inhibition assay, respectively. The results demonstrated that these synthetic glycoproteins have significantly higher affinity with NA than HA. The interactions between these neoglycoproteins and intact influenza viruses were further investigated by Dynamic Light Scattering （DLS） technique. The pronounced agglutination indicated that these glycoconjugates can be used as adsorbents to prevent virus from invading host cells as well as the release of newly synthesized viral particles, which are crucial in the life cycle of the influenza virus. With the high binding affinity to intact influenza viruses, these neoglycoproteins can also be used as probe to elucidate the molecular mechanism of the sialic acid-influenza recognition and biosensors for influenza detection.