影响禽流感病毒(H3N2/H4N6)感染机制的生物信息学分析

目的通过生物信息学分析禽流感病毒WDK/ST/27/03(H3N2)和Dk/ST/472/03(H4N6)HA、NA氨基酸序列,进一步探讨影响禽流感病毒感染血液肿瘤细胞株K-562和HL-60的机制。方法 RT-PCR扩增WDK/ST/27/03(H3N2)和Dk/ST/472/03(H4N6)HA、NA基因,经1%琼脂糖凝胶电泳鉴定,对纯化的PCR产物进行测序。在GenBank中选取登录的H3N2和H4N6序列,利用在线生物信息工具ClustalW2(http://www.ebi.ac.uk/Tools/msa/clustalw2/)对这些序列和WDK/ST/27/03(H3N2)和Dk/ST/472/03(H4N6)HA、NA氨基酸序列进行比对分析。结果 RT-PCR扩增产物的大小与预期相符。生物信息学分析显示,WDK/ST/27/03(H3N2)HA的裂解位点是QNR*GLFG,Dk/ST/472/03(H4N6)HA的裂解位点是KAAR*GLFG,此位置的氨基酸无变化。HA的226位氨基酸为Gln(Q),228位氨基酸为Gly(G),具有禽流感病毒HA受体结合位点的特征,影响受体结合的位点98Y、136S、152W、183H、190E、194L、222W、225G、227S未见变异。NA活性位点及影响酶活的位点均无变异,但WDK/ST/27/03(H3N2)NA在接近N-末端的茎部61⁷9位缺失19个氨基酸,致使NA茎部变短,且丢失2个潜在的糖基化位点。结论 WDK/ST/27/03(H3N2)的NA茎部缺失突变可能是造成其吸附、繁殖和复制能力低于Dk/ST/472/03(H4N6)的一个因素。     

Antiviral Effects of ABMA and DABMA against Influenza Virus In Vitro and In Vivo via Regulating the Endolysosomal Pathway and Autophagy

Influenza virus is an acute and highly contagious respiratory pathogen that causes great concern to public health and for which there is a need for extensive drug discovery. The small chemical compound ABMA and its analog DABMA, containing an adamantane or a dimethyl-adamantane group, respectively, have been demonstrated to inhibit multiple toxins (diphtheria toxin, Clostridium difficile toxin B, Clostridium sordellii lethal toxin) and viruses (Ebola, rabies virus, HSV-2) by acting on the host’s vesicle trafficking. Here, we showed that ABMA and DABMA have antiviral effects against both amantadine-sensitive influenza virus subtypes (H1N1 and H3N2), amantadine-resistant subtypes (H3N2), and influenza B virus with EC₅₀ values ranging from 2.83 to 7.36 µM (ABMA) and 1.82 to 6.73 µM (DABMA), respectively. ABMA and DABMA inhibited the replication of influenza virus genomic RNA and protein synthesis by interfering with the entry stage of the virus. Molecular docking evaluation together with activity against amantadine-resistant influenza virus strains suggested that ABMA and DABMA were not acting as M2 ion channel blockers. Subsequently, we found that early internalized H1N1 virions were retained in accumulated late endosome compartments after ABMA treatment. Additionally, ABMA disrupted the early stages of the H1N1 life cycle or viral RNA synthesis by interfering with autophagy. ABMA and DABMA protected mice from an intranasal H1N1 challenge with an improved survival rate of 67%. The present study suggests that ABMA and DABMA are potential antiviral leads for the development of a host-directed treatment against influenza virus infection.     

新型抗氧化剂4,4′-亚糠基双酚合成的研究

采用了H2 SO4,H3PO4,KOH ,N(C2 H5 ) 3四种催化剂催化苯酚与糠醛反应合成 4,4′ 亚糠基双酚。实验结果表明 ,N(C2 H5 ) 3的催化效果最好 ,当以异丁醇为溶剂 ,n(苯酚 )∶n[N(C2 H5 ) 3]=1 0 0∶0 .0 5 ,反应时间为 3.0h ,反应温度为 80℃时 ,合成产物 4,4′ 亚糠基双酚的收率最高 ,可达到 5 7.9%。并经实验测试其抗氧化性能优于BHT和双酚A ,在同等条件下用于猪油 ,加入BHT的样品在 15天后开始变质 ,而加入 4,4′ 亚糠基双酚的样品在 2 0d后还保持稳定 ;用于从废弃煤焦油中提取的燃料油中 ,加入BHT和双酚A的样品分别在 5d和 7d后开始变质 ,而加入 4,4′ 亚糠基双酚的油样在 10d后才开始有变质现象     

Molecular Docking Studies and Biological Evaluation of Berberine–Benzothiazole Derivatives as an Anti-Influenza Agent via Blocking of Neuraminidase

In this study, we have introduced newly synthesized substituted benzothiazole based berberine derivatives that have been analyzed for their in vitro and in silico biological properties. The activity towards various kinds of influenza virus strains by employing the cytopathic effect (CPE) and sulforhodamine B (SRB) assay. Several berberine–benzothiazole derivatives (BBDs), such as BBD1, BBD3, BBD4, BBD5, BBD7, and BBD11, demonstrated interesting anti-influenza virus activity on influenza A viruses (A/PR/8/34, A/Vic/3/75) and influenza B viral (B/Lee/40, and B/Maryland/1/59) strain, respectively. Furthermore, by testing neuraminidase activity (NA) with the neuraminidase assay kit, it was identified that BBD7 has potent neuraminidase activity. The molecular docking analysis further suggests that the BBD1–BBD14 compounds’ antiviral activity may be because of interaction with residues of NA, and the same as in oseltamivir.     

Recent Advances in Neuraminidase Inhibitor Development as Anti-influenza Drugs

The recent emergence of the highly pathogenic H5N1 subtype of avian influenza virus (AIV) and of the new type of human influenza A (H1N1) have emphasized the need for the development of effective anti‐influenza drugs. Presently, neuraminidase (NA) inhibitors are widely used in the treatment and prophylaxis of human influenza virus infection, and tremendous efforts have been made to develop more potent NA inhibitors to combat resistance and new influenza viruses. In this review, we discuss the structural characteristics of NA catalytic domains and the recent developments of new NA inhibitors using structure‐based drug design strategies. These drugs include analogues of zanamivir, analogues of oseltamivir, analogues of peramivir, and analogues of aromatic carboxylic acid and present promising options for therapeutics or leads for further development of NA inhibitors that may be useful in the event of a future influenza pandemic.     

流感病毒受体结合特性鉴别方法的建立

目的建立一种快速鉴别流感病毒受体结合特性的方法。方法应用流式细胞仪技术,通过地高辛标记的植物凝集素DIG-SNA和DIG-MAA,检测正常鸡红细胞、绵羊红细胞和经α-2,3唾液酸酶处理的鸡红细胞表面受体。分别应用两种受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性,并检测醛化红细胞对其受体结合特性的影响。结果流式细胞检测结果表明,鸡红细胞表面既有SAα2,6Gal受体,也有SAα2,3Gal受体,绵羊红细胞表面只有SAα2,3Gal受体,处理后的鸡红细胞表面只有SAα2,6Gal受体。经分别检测人禽流感病毒毒株,证实该方法能快速准确地鉴别流感病毒的受体结合特性,醛化红细胞未影响其受体结合特性。结论已成功建立了流感病毒受体结合特性的鉴别方法。     

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from β-Amyloid Toxicity

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an “open gate” mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from β-amyloid (Aβ) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aβ degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.     

Development of a Rapid Fluorescent Diagnostic System for Early Detection of the Highly Pathogenic Avian Influenza H5 Clade 2.3.4.4 Viruses in Chicken Stool

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 10⁴ RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.     

丙烯二聚合成四甲基乙烯均相催化剂的研究

研究了在均相催化丙烯二聚合成四甲基乙烯 (DMB - 2 )反应中 ,Ziegler型均相催化剂的组成对反应活性与选择性的影响。实验确定了均相催化剂组成为氯化镍、三乙基铝、三异丙基膦、五氯苯酚和异戊二烯 (isoprene) ,并在氯苯为溶剂 4～ 6℃反应温度和干燥N2 气氛下 ,制得该均相催化剂。当它的配比为n(NiCl2 )∶n(AlEt3 )∶n[P(i Pr) 3 ]∶n(C6Cl5OH)∶n(isoprene) =1∶30∶2∶72∶16 0时 ,制得的催化剂用于均相催化丙烯二聚合成四甲基乙烯有较好的反应活性与选择性 ,丙烯转化率为 5 5 %～ 5 7% ,四甲基乙烯选择性为 44 %～ 46 %。     

Reaction of ethyl N-(6-ethoxycarbonyl-2-methylthiothieno[2,3-d]-pyrimidin-5-yl) formimidate with alkyl- and arylhydrazines: Formation of modified thieno[2,3-d]pyrimidines and thieno[2,3-d:4,5-d]dipyrimidin-4-ones

The reaction of ethyl N-(6-ethoxycarbonyl-2-methyl-thiothieno[2,3-d]pyrimidin-5-yl)formimidate ( 1 ) with methyl-hydrazine, N,N′-dimethyl- and N,N′-diphenylhydrazines separately in refluxing ethanol resulted in hydrolysis of formimidate group to give ethyl 5-amino-2-methylthiothieno[2,3-d] pyrimidine-6-carboxylate ( 2 ). The reaction on 1 with methyl-hydrazine without solvent afforded ethyl 5-amino-2-(1-methyl-hydrazino)thieno[2,3-d]pyrimidine-6-carboxylate ( 3 ), which underwent hydrazone formation with p-nitrobenzaldehyde to give 4 . Treatment of 1 with N,N-dimethylhydrazine afforded a mixture of 3-dimethylamino-7-methylthiothieno[2,3-d:4,5-d']dipyrimidin-4(3H)-one ( 5 ) and 2 . Displacement of methylthio group in 5 with morpholine, piperidine and 4-methylpiperazine gave the corresponding 7-substituted derivatives 6a-c . The reaction of 1 with para-substituted phenylhydrazines resulted in the formation of 3-(p-substituted phenylamino)7-methylthiothieno[2,3-d:4,5-d']dipyrimidin-4 (3H)ones ( 7a-c ).     

Combining Molecular Docking and Molecular Dynamics to Predict the Binding Modes of Flavonoid Derivatives with the Neuraminidase of the 2009 H1N1 Influenza A Virus

Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R(2) = 0.75.     

Aryl and Arylalkyl Substituted 3-Hydroxypyridin-2(1H)-ones: Synthesis and Evaluation as Inhibitors of Influenza A Endonuclease

Seasonal influenza infections are associated with an estimated 250–500 000 deaths annually. Resistance to the antiviral M2 ion-channel inhibitors has largely invalidated their clinical utility. Resistance to neuraminidase inhibitors has also been observed in several influenza A virus (IAV) strains. These data have prompted research on inhibitors that target the cap-snatching endonuclease activity of the polymerase acidic protein (PA). Baloxavir marboxil (Xofluza®), recently approved for clinical use, inhibits cap-snatching endonuclease. Resistance to Xofluza® has been reported in both in vitro systems and in the clinic. An X-ray crystallographic screening campaign of a fragment library targeting IAV endonuclease identified 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating agent at the active site. We have reported the structure–activity relationships for 3-hydroxypyridin-2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones as endonuclease inhibitors. These studies identified two distinct binding modes associated with inhibition of this enzyme that are influenced by the presence of substituents at the 5- and 6-positions of 3-hydroxypyridin-2(1H)-ones. Herein we report the structure–activity relationships associated with various para-substituted 5-phenyl derivatives of 6-(p-fluorophenyl)-3-hydroxypyridin-2(1H)-ones and the effect of using naphthyl, benzyl, and naphthylmethyl groups as alternatives to the p-fluorophenyl substituent on their activity as endonuclease inhibitors.     

两性表面活性剂吡啶烷氧基磺酸盐的合成

初步合成出2种具有C5H5N^ -R-SO3(其中R为烷氧基和芳基)结构特征的吡啶衍生物类两性表面活性剂。对合成路线和方法进行了探讨,将产物应用于酸性电镀镍溶液中表现出良好的增光效果。     

Chemistry and Pharmacology of Nicotinic Ligands Based on 6‐[5‐(Azetidin‐2‐ylmethoxy)pyridin‐3‐yl]hex‐5‐yn‐1‐ol (AMOP‐H‐OH) for Possible Use in Depression

AMOP‐H‐OH (sazetidine‐A; 6‐[5‐(azetidin‐2‐ylmethoxy)pyridin‐3‐yl]hex‐5‐yn‐1‐ol) and some sulfur‐bearing analogues were tested for their activities in vitro against human α4β2‐, α4β4‐, α3β4*‐ and α1*‐nicotinic acetylcholine receptors (nAChRs). AMOP‐H‐OH was also assessed in an antidepressant efficacy model. AMOP‐H‐OH and some of its analogues have high potency and selectivity for α4β2‐nAChRs over other nAChR subtypes. Effects are manifested as partial agonism, perhaps reflecting selectivity for high sensitivity (α4)₃(β2)₂‐nAChRs. More prolonged exposure to AMOP‐H‐OH and its analogues produces inhibition of subsequent responses to acute challenges with full nicotinic agonists, again selectively for α4β2‐nAChRs over other nAChR subtypes. The inhibition is mediated either via antagonism or desensitization of nAChR function, but the degree of inhibition of α4β2‐nAChRs is limited by the partial agonist activity of the drugs. Certain aspects of the in vitro pharmacology suggest that AMOP‐H‐OH and some of its analogues have a set of binding sites on α4β2‐nAChRs that are distinct from those for full agonists. The in vitro pharmacological profile suggests that peripheral side effects of AMOP‐H‐OH or its analogues would be minimal and that their behavioral effects would be dominated by central nAChR actions. AMOP‐H‐OH also has profound and high potency antidepressant‐like effects in the forced swim test. The net action of prolonged exposure to AMOP‐H‐OH or its analogues, as for nicotine, seems to be a selective decrease in α4β2‐nAChR function. Inactivation of nAChRs may be a common neurochemical endpoint for nicotine dependence, its treatment, and some of its manifestations, including relief from depression.     

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3–H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC₅₀ = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC₅₀ = 12 µM by visual CPE score; EC₅₀ = 8.3 µM by TMS score; MCC > 100 µM, CC₅₀ = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC₅₀ in the range 14.5–97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at β-lactamase, thus protecting the antibiotic from undesirable biotransformation.     

新型抗氧化剂4，4＇-亚糠基双酚合成的研究

采用了H     

Toxicity of Certain Penta-Coordinated Organotin(IV) and Tetra-Coordinated Tin(II) Complexes of Heterocyclic beta-Diketones Against the Larvae of Aedes Aegypti (Liston)

Some organotin(IV) and tin(II) complexes of composition R(3)Sn[R'COC:CON(C(6)H(5))N:CCH(3)] (where R=C(4)H(9), R'=CH(3), C(3)H(5), p-ClC(6)H(4); R=C(6)H(5), R'=C(6)H(5) and p-ClC(6)H(4)) and Sn[p-ClC(6)H(4)COC:CON(C(6)H(5))N:CCH(3)](2) were screened for their toxicity against Aedes aegypti larvae. Organotin(IV) complexes were more active than tin(II) complexes.     

经Al掺杂改性的介孔CaZr4（PO4）6的酸催化活性

对溶胶-凝胶法合成的NZP族介孔CaZr4(PO4)6进行Al掺杂改性,获得了一系列含Al的新型酸性催化剂样品Al-CaZr4(PO4)6。利用X射线衍射、X射线荧光、N2吸附-脱附和NH3的程序升温脱附等技术,表征了样品的物相、Al掺杂量、孔结构和表面酸性;以α-蒎烯的异构化为模型反应表征了样品的酸催化活性。结果表明:经适量Al掺杂改性后的CaZr4(PO4)6仍保持了均匀分布的介孔结构特征,与未经掺杂的CaZr4(PO4)6相比,表面酸性有明显提高。当掺杂的Al与CZP中Zr的物质的量的比分别为0.15和0.20时,获得的催化剂样品Al-CaZr4(PO4)6-0.15和Al-CaZr4(PO4)6-0.20在150℃下对α-蒎烯异构化反应的转化率可以达到40%～47%,而未经掺杂改性的介孔CaZr4(PO4)6在相同反应条件下对α-蒎烯异构化反应的转化率仅为3%,因此,Al掺杂改性可以显著提高介孔CaZr4(PO4)6的酸催化活性。