Identification of novel virus inhibitors by influenza A virus specific reporter cell based screening

As influenza viruses have developed resistance towards current drugs, it is urgent to find potential novel antiviral inhibitors. Here we generated an influenza virus reporter cell line in which the luciferase gene was driven by the influenza virus promoter and screened a small compound library (NCI Diversity Set II). Ten compounds were identified to have inhibitory activity against influenza A virus H1N1. Among them, four compounds blocked influenza virus replication through inhibiting the activity of vRNP. The compound NSC 335506 inhibited HA-mediated membrane fusion. It showed the inhibitory activity against H1N1, H9N2 and H5N1 subtype but not H3N2. Our results demonstrated that influenza virus reporter cell is a very useful tool to identify novel inhibitors against influenza A virus.     

Neuraminidase inhibitors for influenza B virus infection: Efficacy and resistance

Many aspects of the biology and epidemiology of influenza B viruses are far less studied than for influenza A viruses, and one of these aspects is efficacy and resistance to the clinically available antiviral drugs, the neuraminidase (NA) inhibitors (NAIs). Acute respiratory infections are one of the leading causes of death in children and adults, and influenza is among the few respiratory infections that can be prevented and treated by vaccination and antiviral treatment. Recent data has suggested that influenza B virus infections are of specific concern to pediatric patients because of the increased risk of severe disease. Treatment of influenza B is a challenging task for the following reasons:

1.

NAIs (e.g., oseltamivir and zanamivir) are the only FDA-approved class of antivirals available for prophylaxis and treatment of influenza.     

Inhibition of influenza hemagglutinin with the antiviral inhibitor arbidol using a proteomics based approach and mass spectrometry

A proteomics gel electrophoresis based approach has been applied to study the effect of arbidol on the proliferation of influenza virus in vitro through quantitation of hemagglutinin levels. An arbidol concentration of 20 μg/ml was required to achieve a 50% reduction in virus proliferation and hemagglutinin levels. The use of a MALDI mass spectrometry approach to study the binding of arbidol to influenza hemagglutinin revealed it bound solely to residues 104–120 of the HA2 subunit, a region known to contain an arbidol resistance mutation. Parallel molecular docking results revealed that this binding site was favoured in which the arbidol molecule binds in two possible orientations approximately 180° to one another at HA2 residues 118–123. The combined studies support the recognized potential of arbidol as an effective and targeted antiviral agent against the influenza virus.     

Plant-derived antiviral drugs as novel hepatitis B virus inhibitors: Cell culture and molecular docking study

Despite high anti-HBV efficacies, while the nucleoside analogs (e.g., lamivudine) lead to the emergence of drug-resistance, interferons (e.g., IFN-α causes adverse side-effects. Comparatively, various natural or plant products have shown similar or even better efficacy. Hence, new antiviral strategies must focus not only on synthetic molecules but also on potential natural compounds. In this report, we have combined the in vitro cell culture and in silico molecular docking methods to assess the novel anti-HBV activity and delineate the inhibitory mechanism of selected plant-derived pure compounds of different classes. Of the tested (2.5-50?μg/ml) twelve non-cytotoxic compounds, ten (10?μg/ml) were found to maximally inhibit HBsAg production at day 5. Compared to quercetin (73%), baccatin III (71%), psoralen (67%), embelin (65%), menisdaurin (64%) and azadirachtin (62%) that showed high inhibition of HBeAg synthesis, lupeol (52%), rutin (47%), β-sitosterol (43%) and hesperidin (41%) had moderate efficacies against HBV replication. Further assessment of quercetin in combination with the highly active compounds, enhanced its anti-HBV activity up to 10%. Being the most important drug target, a 3-D structure of HBV polymerase (Pol/RT) was modeled and docked with the active compounds, including lamivudine as standard. Docking of lamivudine indicated strong interaction with the modeled HBV Pol active-site residues that formed stable complex (?G?=??5.2?kcal/mol). Similarly, all the docked antiviral compounds formed very stable complexes with HBV Pol (?G?=??6.1 to ?9.3?kcal/mol). Taken together, our data suggest the anti-HBV potential of the tested natural compounds as novel viral Pol/RT inhibitors.     

Packaging signals in the 5'-ends of influenza virus PA, PB1, and PB2 genes as potential targets to develop nucleic-acid based antiviral molecules

In a previous study a 15-mer phosphorothioate oligonucleotide (S-ON) derived from the packaging signal in the 5′ end of segment 1 (PB2) of influenza A virus (designated 5–15b) proved markedly inhibitory to virus replication. Here we investigated whether analogous inhibitory S-ONs targeting the 5′ end of segments 2 (PB1) and 3 (PA) could be identified and whether viral resistance to S-ONs can be developed. Similar to our earlier result, 20-mer S-ONs reproducing the 5′ ends of segments 2 or 3 (complementary to the 3′-coding regions of PB1 and PA, respectively) exerted a powerful antiviral activity against a variety of influenza A virus subtypes in MDCK cells. Serial passage of the A/Taiwan/1/86 H1N1 strain in the presence of S-ON 5–15b or its antisense as5–15b analogue showed that mutant viruses with reduced susceptibility to the S-ON could indeed be generated, although the resistant viruses displayed reduced replicative fitness. Sequencing the resistant viruses identified mutations in the PB1, PB2, PA and M1 genes. Introduction of these changes into the A/PR/8/34 H1N1 strain by reverse genetics, suggested that alterations to RNA function in the packaging regions of segments 2 and 3 were important in developing resistance to S-ON inhibition. However, many of the other sequence changes induced by S-ON treatment were markedly deleterious to virus fitness. We conclude that packaging signals in the influenza A virus polymerase segments provide feasible targets for nucleic acid-based antivirals that may be difficult for the virus to evade through resistance mutations.     

Edible bird's nest extract inhibits influenza virus infection

Edible bird's nest (EBN) is the nest of the swift that is made from its saliva. Although EBN has been widely used for enhancing immunocompetence, its antiviral efficacy has not been studied in detail. We found that EBN extract could strongly inhibit infection with influenza viruses in a host range-independent manner when it was hydrolyzed with Pancreatin F. Western blotting assay showed that the EBN extract bound to influenza virus. Furthermore, EBN extract could neutralize the infection of MDCK cells with influenza viruses and inhibit hemagglutination of influenza viruses to erythrocytes, but it could not inhibit the activity of influenza virus sialidase. Fluorometric HPLC indicated that the major molecular species of sialic acid in EBN is N-acetylneuraminic acid. The results suggest that EBN is a safe and valid natural source for the prevention of influenza viruses.     

复方抗病毒制剂体外抗流感病毒和呼吸道合胞病毒作用及毒性研究

目的 测定复方抗病毒制剂体外对流感病毒和呼吸道合胞病毒的抗病毒作用. 方法 用鸡红细胞血凝素滴定法测定不同浓度复方抗病毒制剂在狗肾传代细胞（MDCK）中对流感病毒的抑制作用;用中和实验测定复方抗病毒制剂在Hep 2细胞中对呼吸道合胞病毒的活性. 结果 复方抗病毒制剂浓度＜1.5 mg•mL^-1时对MDCK细胞及Hep 2细胞无明显毒性. 浓度＞0.15 mg•mL^-1能抑制流感病毒的血凝活性和呼吸道合胞病毒对靶细胞的攻击作用. 结论 复方抗病毒制剂体外对流感病毒和呼吸道合胞病毒具有较强的抑制作用,且有较大的安全性.     

Synthetic anti-lipopolysaccharide peptides and hepatitis C virus infection

Introduction: Hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma. Although antiviral therapy has been markedly improved by the licensing of direct-acting antivirals, safety, resistance, high costs and difficult-to-treat patients remain important challenges.

Areas covered: This article focuses and comments on the recent development of synthetic anti-lipopolysaccharide peptides (SALPs) which bind to highly sulfated glycosaminoglycan/heparan sulfate (HS) on cell surface. HS serves as a primary docking site for several viruses to their respective host cells before the viruses interact with their cell surface receptor(s). In vitro studies have shown that SALPs inhibit entry of HCV without cell toxicity.

Expert opinion: SALPs prevent viral infection in cell culture model systems. Treatment studies of established HCV infection in cell culture models as well as proof-of-concept and safety studies in animal models are needed to evaluate their potential for drug development. The mechanism of action of SALPs as entry inhibitors suggests a potential application for HCV-infected patients to prevent reinfection of the liver graft in liver transplantation. Potential limitations may include high doses to obtain an antiviral effect and a target which is widely expressed and has a key function in cell physiology.     

Development of a broad-spectrum antiviral with activity against Ebola virus

We report herein the identification of a small molecule therapeutic, FGI-106, which displays potent and broad-spectrum inhibition of lethal viral hemorrhagic fevers pathogens, including Ebola, Rift Valley and Dengue Fever viruses, in cell-based assays. Using mouse models of Ebola virus, we further demonstrate that FGI-106 can protect animals from an otherwise lethal infection when used either in a prophylactic or therapeutic setting. A single treatment, administered 1 day after infection, is sufficient to protect animals from lethal Ebola virus challenge. Cell-based assays also identified inhibitory activity against divergent virus families, which supports a hypothesis that FGI-106 interferes with a common pathway utilized by different viruses. These findings suggest FGI-106 may provide an opportunity for targeting viral diseases.     

Repurposing of cyclophilin A inhibitors as broad-spectrum antiviral agents

Cyclophilin A (CypA) is linked to diverse human diseases including viral infections. With the worldwide emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2), drug repurposing has been highlighted as a strategy with the potential to speed up antiviral development. Because CypA acts as a proviral component in hepatitis C virus, coronavirus and HIV, its inhibitors have been suggested as potential treatments for these infections. Here, we review the structure of cyclosporin A and sanglifehrin A analogs as well as synthetic micromolecules inhibiting CypA; and we discuss their broad-spectrum antiviral efficacy in the context of the virus lifecycle.     

知母宁抗流感病毒作用研究

目的:考察知母宁抗流感病毒的作用.方法:采用人流感病毒A型(H1N1)滴鼻感染小鼠,以利巴韦林作为阳性对照药,对知母宁抗病毒活性进行评价.结果:与生理盐水对照组相比,知母宁可改善感染小鼠的临床症状,减少14 d内动物的死亡数,并能延长其平均存活时间.组织病理学检查表明25,50及75 mg/kg的知母宁对小鼠肺部感染均有一定的疗效,随剂量的降低疗效减弱.其中75 mg·kg-1的知母宁治疗作用与利巴韦林(15 mg·kg-1)相似.结论:知母宁具有较强的抗流感病毒A型(H1N1)的活性.     

Synergic effect of curcumin and its structural analogue (Monoacetylcurcumin) on anti-influenza virus infection

Curcumin (Cur), a polyphenolic compound extracted from spice and common food colourant turmeric, contains versatile bio-activities. Monoacetylcurcumin (MAC), a structural analogue of Cur, differs from Cur by acetyl modification, but retains enone groups. Comparative analysis revealed MAC effectively inhibited influenza virus infection (IAV) to a similar extent as, if not superior to, curcumin. Both compounds mildly reduced viral NA activity. Surprisingly, unlike Cur, the MAC inhibition of IAV did not occur through the blocking of HA activity. However, MAC strongly dampened Akt phosphorylation, the prerequisite signalling for efficient IAV propagation. A much stronger inhibition effect on IAV infection was observed when MAC treatment was in combination with Cur. Collectively, MAC demonstrated clear antiviral activity, and likely inhibited IAV via multiple mechanisms that were not identical to Cur. Importantly, Cur and MAC in combination synergistically inhibited IAV infection.     

抗病毒抗生素17997体外联合用药研究

目的：研究抗病毒抗生素17997与临床应用的抗病毒药物－阿昔洛韦、病毒唑及入干扰素α的联合用药抗单纯疱疹病毒I型的作用。方法：应用VERO细胞培养法及MTT染色法进行实验及观察结果,以Calcusyn软件计算,分析结果。结果：实验结果显示抗生素17997与阿昔洛韦、病毒唑及人干扰素α在ED50、ED70（或ED75）、ED90条件下均有协同抗单纯疱疹病毒I型的作用,表现在降低ED50值及联合指数＜1。仅抗生素17997与人干扰素α联合组,在ED90条件下,表现为相加或拮抗作用。结论：抗生素17997抗疱疹病毒活性强,在多次单纯疱疹病毒I型实验感染的兔多膜炎模型上显示抗病毒疗效。本研究结果为抗生素17997临床联合用药提供实验依据。     

Synthesis and antiviral activity of novel methylene cyclopropyl nucleosides

Novel exomethylene cyclopropyl nucleosides were synthesized as potential antiviral agents. The key intermediate 5 was synthesized in 4 steps, from Feists acid 1 and was condensed with purine derivatives by the SN2 type reaction to give some cyclopropyl nucleosides. The synthesized nucleosides did not showed any significant antiviral activity against HSV-1, HSV-2, HCMV, HIV-1, HIV-2, and HBV up to 100 microM.     

广谱抗病毒抗生素17997体内外抗病毒活性研究

从我国云南省土壤中分离到１株放线菌，基们生的抗生素１７９９７具有广谱抗病毒作用在细菌培养内对单纯疱疹病毒１型，单纯疱疹病毒２型，人免疫缺陷病毒１型，水疱性口炎病毒及柯萨奇病毒Ｂ３均有显著抑制活性，ＩＣ５０在μｍｏｌ／Ｌ水平。其在小鼠体内对单纯疱疹病毒１型及２型所致疱疹性脑炎有确切治疗效果，对死亡保护率平均生存日与对照组相比有统计意义的显著差别。     

Potent antiviral activity of brequinar against the emerging Cantagalo virus in cell culture

In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5 μM BQR reduced virus progeny production by >90%, revealing an EC₅₀ (drug concentration required to inhibit 50% of virus replication) of 0.017 μM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures.     

Synthetic Tyr-phospho and non-hydrolyzable phosphonopeptides as PTKs and TC-PTP inhibitors*

Tyrosine-specific protein kinases and phosphatases are important signal transducing enzymes in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. In order to develop agents which inhibit the function of these two classes of enzymes by interfering with the binding of their substrates, we synthesized analogs derived from the peptide EDNEYTA. This sequence reproduces the main autophosphorylation site of Src tyrosine kinases. In this work we report the synthesis, by classical solution methods, of the phosphotyrosyl peptide ED-NEYpTA as well as of three analogs in which the phosphotyrosine is replaced by a phosphinotyrosine and by two unnatural, non-hydrolyzable amino acids 4-phosphonomethyl-l -phenylalanine and 4-phosphono-l -phenylalanine. The Src peptide and its derivatives were tested as inhibitors of three non-receptor tyrosine kinases (Lyn, belonging to the Src family, CSK and PTK-IIB) and a non-receptor protein tyrosine phosphatase obtained from human T-cell (TC-PTP). The biomimetic analogues, which do not significantly affect the activity of CSK, PTK-IIB and TC-PTP, act:is efficient inhibitors on Lyn, influencing both the exogenous phosphorylation and, especially, its autophosphorylation. In particular, the Pphe derivative may provide a basis for the design of a class of inhibitors specific for Lyn and possibly Src tyrosine kinases, capable of being used in vivo and in vitro conditions. © Munksgaard 1995.     

A heterogeneous collection of novel antiviral pyrimidines

A number of patents dealing with a variety of antiviral pyrimidine analogues and granted to various companies and institutions are discussed. For convenience, the claimed pyrimidine structures are separated into a ‘nucleoside’-section and a ‘non-nucleoside’-section.     

新型冠状病毒肺炎抗病毒药物的安全应用

国家卫生健康委员会和国家中医药管理局联合印发的《新型冠状病毒肺炎诊疗方案(试行第五版修正版)》推荐了3种抗病毒药物,包括干扰素α(雾化吸入)、洛匹那韦/利托那韦(口服)和利巴韦林(静脉输注)。另外,一种尚未上市的抗病毒药物瑞德西韦也在我国展开了临床试验。本文主要就干扰素、洛匹那韦/利托那韦和利巴韦林在SARS冠状病毒、中东呼吸综合征冠状病毒、人类免疫缺陷病毒感染等治疗中的安全性数据以及瑞德西韦在动物实验、Ⅰ期临床试验、治疗埃博拉病毒感染临床试验中的安全性数据及用于1例新型冠状病毒肺炎治疗的初步数据进行简要综述,为临床安全用药提供参考。     

抗病毒抗生素17997不同时间给药抗单纯疱疹病毒Ⅰ型的效果

目的：广谱抗病毒抗生素17997，对单纯疱疹病毒Ⅰ型（HSV－Ⅰ）有较好的疗效，本实验通过观察不同给时间的抗病毒效果。方法：以HSV－Ⅰ感染Vero细胞，在感染前4h，感染后0、3、6h给予抗生素17997（10μmol/L），以透射电镜观察Vero细胞内的病毒，比较不同给药时间的抗病毒效果。结果：在病毒感染前4h给药无效，细胞内外有大量病毒颗粒；在感染早期（0.3h）给药效果好，细胞内外均极少见病毒颗粒，胞浆内可见病毒空壳聚集；感染晚期（6h）给药在细胞核内见病毒核壳体及有囊膜病毒颗粒，但细胞浆及细胞外均未见病毒颗粒。结论：抗病毒抗生素17997在病毒复制早期抑制单纯疱疹病毒DNA的合成，晚期可抑制有囊膜单疱疹病毒颗粒从核释放至胞浆。