Isolation of antiviral proteins from natural product extracts.

从天然产物提取物中分离抗病毒蛋白。

• 批准号: 9153938
• 负责人: Barry Okeefe
• 金额: $ 37.37万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153938
• 关键词: Africa; Amino Acid Sequence; Animal Model; Animal Testing; Animals; Antiviral Agents; Avidity; Binding; Biochemical; Biological; Biological Assay; Biological Factors; Biological Models; CD209 gene; CD4 Positive T Lymphocytes; Carbohydrates; Categories; Cell Culture Techniques; Cells; Cervical; Clinical; Clinical Trials; Cloning; Collaborations; Communicable Diseases; Control Animal; Cyanovirin-N; Databases; Democratic Republic of the Congo; Dendritic Cells; Dependence; Development; Dose; Drug Formulations; Drug Kinetics; Ebola virus; Environment; Epithelial; Escherichia coli; Evaluation; Exhibits; Film; Filovirus; Frankfurt-Marburg Syndrome Virus; Funding; Gel; Glycoproteins; Grant; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Seropositivity; HIV-1; Hepatitis C virus; Herpesvirus 1; Human; Immune response; In Vitro; Induced Mutation; Infection; Infectious Agent; Injection of therapeutic agent; Institutes; Japan; Laboratories; Lectin; Literature; Liver; Modeling; Molecular Target; Monosaccharides; Mus; Names; National Institute of Allergy and Infectious Disease; Nipah Virus; Oligosaccharides; Patients; Peptide Fragments; Production; Proteins; Publications; Reporting; Research Personnel; Resistance; SCID Mice; Safety; Sequence Homology; Simplexvirus; Site; Source; Structure; Survival Rate; System; Tenofovir; Testing; Therapeutic; Tissues; Toxic effect; United States National Institutes of Health; Vaginal Douching; Viral; Viral Load result; Virus; Work; anti-HIV microbicide; aqueous; base; biodefense; cell killing; crosslink; dimer; expression vector; hepatocyte engraftment; immune activation; in vivo; in vivo Model; interest; irritation; large scale production; model development; molecular mass; monomer; mortality; mouse model; mutant; novel; polyclonal antibody; pre-clinical; prevent; research study; three dimensional structure; treatment duration

Structure-activity, biochemical and antiviral studies on griffithsin to elucidate its mechanism of action and its specific binding parameters to oligosaccharides present on viral envelope glycoproteins. These structural determinations, done in collaboration with Dr. Alex Wlodawer (MCL), have allowed us to modify the GRFT structure to create mutants that have demonstrated varying degrees of anti-HIV activity and helped define the GRFT mechanism of action. Taken together, these results indicate that GRFT likely exhibits its potent antiviral activity by cross-linking oligosaccharides on the HIV-I envelope glycoprotein gp120 and preventing the subsequent conformational changes necessary for viral entry. The loss of activity of the monomer may be due to its inability to cross-link as efficiently as the native obligate GRFT dimer. This novel mechanism, revealed by our biophysical and structural studies, is supported by recent literature concerning GRFT induced mutations in gp120 oligosaccharide attachment sites and the dependence of lectin avidity on multivalent interactions. Studies towards the pre-clinical and clinical development of griffithsin as a topical anti-HIV microbicide. Since the large-scale production of GRFT, we have pursued its pre-clinical development through collaborations largely funded by NIAID. We have reported on its stability and efficacy in the mucosal environment, its activity in human cervical explant models and its safety and lack of either immune activation or epithelial irritation in multiple in vitro and in vivo model systems. This has resulted in several publications by other researchers detailing GRFTs surprising resistance to proteolytic degradation, its synergistic activity in combination with ARVs such as tenofovir and other lectins, and its enhancement of immune response to HIV-1 gp120. GRFT is now considered a leading candidate for human clinical trials. The formulation of GRFT as both a gel and a film, its stability and release from those formulations, its activity against a broad spectrum of strains of HIV, and its in vivo activity against HSV have also elevated its status. Currently, we are continuing the clinical development of GRFT as an anti-HIV microbicide. Evaluation of griffithsin for activity against HIV & HCV.-Previously we and others had shown that the antiviral lectin cyanovirin-N had activity against other enveloped viruses in addition to HIV. As GRFT was more potent than CV-N and, as a monosaccharide-specific lectin more promiscuous in its carbohydrate binding motif we proceeded to collaborate with NIAID to determine the in vitro antiviral spectrum of activity for GRFT. Simultaneously, we began a collaboration with Dr. Lynn Morris at NICD in S. Africa to test GRFT against circulating strains of HIV. The evaluation of GRFTs activity across the various clades of HIV-1 resulted in several interesting findings. Initially, we showed that, as with laboratory strains, clinical strains of HIV-1 in Clade C were very susceptible to GRFT at low nM to pM levels. Furthermore, virus in cervical vaginal lavages from HIV positive patients was also shown to be susceptible to inhibition by GRFT. Finally, our work evaluated the ability of GRFT (and our other lectins) to inhibit HIV-1 binding to the dendritic cell-associated lectin DC-SIGN. DC-SIGN is known to be a positive effector of HIV infection via the transfer and presentation of infectious virus to CD4+ T-cells. In this study we determined that GRFT was able to inhibit HIV-1 binding to DC-SIGN and to inhibit subsequent transfer of virus to CD4+ cells. The culmination of these experiments was a better understanding of both the scope and mechanism of GRFT activity against HIV and its potential utility as a anti-HIV microbicide. GRFT and another MTL discovery, SVN, were tested for activity against hepatitis C virus (HCV). This was a collaboration initiated with Dr. Yutaka Takebe at the National Institute of Infectious Disease in Japan. When tested in the HCV cell culture assay system, both SVN and GRFT were potently active with GRFT displaying an EC50= 50 pM and little toxicity. I received funding from NIAID to support in vivo testing of GRFT against HCV in a Alb-uPA-SCID mouse model with human hepatocyte engraftment. Injections of 20 mg/kg/day given to study animals for 10 days revealed that GRFT was non-toxic and rapidly bioavalable following s.c injection. We then completed two efficacy studies in which animals were dosed for either 10 or 18 consecutive days with GRFT. The results showed that 10 day treatment with GRFT, post HCV challenge, reduced HCV titers 100 fold. The 18-day GRFT treatment again reduced viral load by >100-fold, but resulted in significant toxicity to test animals (6/14 animals died) though not to the human liver tissue therein. Evaluation of the antiviral proteins griffithsin and scytovirin for activity against ebola.- I have an ongoing collaboration with both USAMRIID and NIAID to investigate the proteins we have discovered for activity against pathogenic viruses of potential biodefense interest. Part of this work has been funded by a Trans-NIH grant that I received from NIAID to work on the use of the proteins SVN and GRFT against the ebola virus. Ebola virus is a category A infectious agent with no approved therapeutic options and has a mortality rate in humans of >50%. Initial in vitro pseudoparticle assays on SVN and GRFT against ebola Zaire and Marburg virus showed that these proteins had nanomolar activity against both filoviruses. Tolerability studies in mice indicated that GRFT and SVN were well tolerated at doses up to 40 mg/kg/day with GRFT being dosed either Q12 or Q24 and SVN dosed Q6. Efficacy studies in mice with both proteins in various dose ranges indicated that treatment with either SVN or GRFT for 10 days resulted in survival rates of 90% for treated animals (compared to 0% for control animals). Isolation, characterization and cloning of anti-HIV proteins isolated from natural products extracts. The aqueous extract of Synthecium sp. showed anti-HIV activity and yielded 3 novel anti-HIV proteins. The purified cnidarin proteins were named cnidarin 1-3 (CNID-1, CNID-2, CNID-3). The proteins, homogenous by SDS-PAGE, showed single peaks for each protein by ESI/MS, corresponding to exact molecular masses of 18,122 Da (CNID-1), 18,088 Da (CNID-2) and 17,963 Da (CNID-3). Amino acid sequences of the purified CNID proteins were established. The peptide fragments for CNID-1 and CNID-3 sufficiently overlapped to sequence both proteins. CNID-1 and CNID-3 share a 71% sequence similarity when aligned against each other, and when compared against known proteins using the NCBI database they showed no significant sequence homology to any known protein. All three CNID proteins elicited concentration-dependent inhibition of virus-induced cell killing with picomolar EC50 values. The CNIDs were remarkably potent with picomolar to low-nanomolar range activity against HIV, which is, on average, lower or comparable to the activity of other antiviral proteins isolated from natural sources, with the exception of griffithsin. Of the three CNID proteins, CNID-1 was the most potent at protecting against HIV-1RF-induced cytopathic effects in CEM-SS cells (EC50 = 85 pM). CNID-3 was most potent at inhibiting the HIV-1ROJO primary isolate (EC50 of 1.5 nM) while CNID-2 was the least potent. CNID-1 bound to gp41 and gp120 equivalently well which is distinct from our previous discoveries of CV-N, SVN and GRFT. I am also on the steering committee of two clinical trials that have been funded for the evaluation of griffithsin as an anti-HIV microbicide. Additional intramural studies on GRFT activity against Nipah virus, ebola and MERS have been initiated. We have also collaborted extramurally on activity against HSV-1 & 2 and Trichnomas vaginalis.

关于griffithsin的结构活性，生化和抗病毒研究，以阐明其作用机理及其与病毒包膜糖蛋白上存在的寡糖的特异性结合参数。与Alex Wlodawer博士（MCL）合作进行的这些结构确定使我们能够修改GRFT结构，以创建突变体，这些突变体显示出不同程度的抗HIV活性，并有助于定义了GRFT的作用机理。综上所述，这些结果表明，GRFT可能通过在HIV-I包膜糖蛋白GP120上交联寡糖来表现出其有效的抗病毒活性，并防止了病毒进入所需的随后构象变化。单体的活性损失可能是由于其无法像天然义务GRFT二聚体那样有效地交联。我们的生物物理和结构研究揭示了这种新型机制，它得到了最近的文献，该文献涉及GP120寡糖附着位点的GRFT诱导的突变以及凝集素亲和力对多价相互作用的依赖性。研究格里菲辛（Griffithsin）作为局部抗HIV微生物的研究。自GRFT大规模生产以来，我们通过NIAID在很大程度上资助的合作进行了临床前的发展。我们已经报告了其在粘膜环境中的稳定性和功效，其在人体宫颈外植体模型中的活性以及其安全性以及缺乏多种体外和体内模型系统中的免疫激活或上皮刺激。这导致了其他研究人员的出版物，详细介绍了对蛋白水解降解的抗药性，其协同活性与Tenofovir和其他凝集素的结合以及对HIV-1 GP120的免疫反应的增强。现在，GRFT被认为是人类临床试验的领先候选人。 GRFT作为凝胶和膜的表述，其稳定性和从这些配方中释放，其对广泛的HIV菌株的活性以及其对HSV的体内活性也提高了其状态。目前，我们正在继续将GRFT作为抗HIV杀菌剂的临床发展。格里菲辛（Griffithsin）针对HIV和HCV的活性评估我们和其他人表明，除HIV外，抗病毒凝集素Cyanovirin-N除了其他包膜病毒还具有活性。由于GRFT比CV-N更有效，并且在其碳水化合物结合基序中，单糖特异性凝集素更加混杂，我们开始与NIAID合作，以确定GRFT活性的体外抗病毒药谱。同时，我们与非洲的NICD的Lynn Morris博士开始了合作，以测试GRFT对艾滋病毒的循环菌株。 HIV-1各个进化枝的GRFT活动的评估导致了一些有趣的发现。最初，我们表明，与实验室菌株一样，进化枝C中HIV-1的临床菌株非常容易受到低NM至PM水平的GRFT。此外，HIV阳性患者的宫颈阴道灌洗病毒也被证明容易受到GRFT的抑制作用。最后，我们的工作评估了GRFT（和其他凝集素）抑制HIV-1与树突状细胞相关凝集素DC-SIGN的能力。已知DC-SIGN是通过转移和表现为CD4+ T细胞的转移和表现，是HIV感染的阳性效应。在这项研究中，我们确定GRFT能够抑制HIV-1与DC-SIGN的结合，并抑制病毒随后转移至CD4+细胞。这些实验的高潮是对针对HIV的GRFT活性的范围和机理的更好理解及其作为抗HIV杀菌剂的潜在效用。测试了针对乙型肝炎病毒（HCV）的GRFT和另一个MTL发现SVN。这是与日本国家传染病研究所的Yutaka Takebe博士发起的合作。当在HCV细胞培养测定系统中进行测试时，SVN和GRFT均在GRFT上都具有有效的活性，其EC50 = 50 pm且毒性很少。我从NIAID获得了基于人类肝细胞植入的Alba-scID小鼠模型中对HCV的体内测试。对研究动物进行10天的20 mg/kg/day注射10天表明，S.C注射后GRFT无毒，可快速生物助长。然后，我们完成了两项疗效研究，其中将动物连续10或18天加入GRFT。结果表明，使用GRFT，HCV挑战后的10天治疗，HCV滴度减少了100倍。 18天的GRFT治疗再次使病毒载量降低了> 100倍，但导致对动物测试（6/14只动物死亡）的毒性显着，尽管对其中的人肝组织没有。对埃博拉病毒活性的抗病毒蛋白的评估。-我与USAMRIID和NIAID进行了持续的合作，以研究我们发现的蛋白质，我们发现了我们发现的针对潜在生物探索的致病性病毒的活性。这项工作的一部分是由我从NIAID获得的Trans-NIH赠款资助的，用于使用蛋白质SVN和GRFT对埃博拉病毒的使用。埃博拉病毒是一种没有批准的治疗选择，人类死亡率> 50％的感染剂类别。针对埃博拉Zaire和Marburg病毒对SVN和GRFT的初始体外假颗粒测定表明，这些蛋白质对这两种丝状病毒均具有纳莫尔活性。在小鼠中的耐受性研究表明，GRFT和SVN的耐受性高达40 mg/kg/day，GRFT的剂量被Q12或Q24和SVN剂量do Q6。在各种剂量范围内使用两种蛋白质的小鼠的疗效研究表明，用SVN或GRFT进行10天的治疗导致治疗的动物的存活率为90％（对照动物为0％）。从天然产物提取物中分离出的抗HIV蛋白的分离，表征和克隆。 Synthecium sp。的水提取物。表现出抗HIV活性，并产生3种新的抗HIV蛋白。纯化的CNIDARIN蛋白称为CNIDARIN 1-3（CNID-1，CNID-2，CNID-3）。通过SDS-PAGE均匀的蛋白质显示了每种蛋白的单个峰通过ESI/MS，对应于18,122 DA（CNID-1），18,088 DA（CNID-2）和17,963 DA（CNID-3）的精确分子质量​​（CNID-1）。建立了纯化的CNID蛋白的氨基酸序列。 CNID-1和CNID-3的肽片段足够重叠以序列两种蛋白质。当相互对齐时，CNID-1和CNID-3具有71％的序列相似性，并且使用NCBI数据库与已知蛋白质进行比较时，它们与任何已知蛋白质都没有显着的序列同源性。所有三种CNID蛋白均引起浓度依赖性抑制病毒诱导的细胞杀死，并具有皮莫尔EC50值。与HIV相对于HIV的皮摩尔至低纳摩尔范围活性，CNID非常有效，除Griffithsin外，它的平均而言与其他从天然来源分离的其他抗病毒蛋白的活性较低或可比。在三种CNID蛋白中，CNID-1是防止CEM-SS细胞中HIV-1RF诱导的细胞病变作用（EC50 = 85 pm）最有效的。 CNID-3最有效地抑制HIV-1ROJO原发性分离株（EC50为1.5 nm），而CNID-2的有效性最低。 CNID-1与GP41和GP120结合，这与我们以前对CV-N，SVN和GRFT的发现不同。我也是指导委员会的两项临床试验委员会，这些临床试验已被资助用于评估格里菲辛作为抗HIV杀生型。对NIPAH病毒，埃博拉病毒和MER的GRFT活性进行了其他壁内研究。我们还在针对HSV-1和2和Trichnomas阴道的活动外面合作。