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: 8553216
• 负责人: Barry Okeefe
• 金额: $ 36.74万
• 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/8553216
• 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; 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; 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; glycosylation; 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. CNID-1 binding to gp120 was not glycosylation-dependant indicating that CNID-1, unlike CV-N, SVN and GRFT, is not a lectin.

对 griffithsin 进行结构活性、生化和抗病毒研究，以阐明其作用机制及其与病毒包膜糖蛋白上存在的寡糖的特异性结合参数。 这些结构测定是与 Alex Wlodawer 博士 (MCL) 合作完成的，使我们能够修改 GRFT 结构以创建突变体，这些突变体已表现出不同程度的抗 HIV 活性，并有助于确定 GRFT 的作用机制。总而言之，这些结果表明GRFT可能通过交联HIV-1包膜糖蛋白gp120上的寡糖并阻止病毒进入所必需的后续构象变化而表现出其有效的抗病毒活性。单体活性的丧失可能是由于其无法像天然专性 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一新机制，并得到了最近关于 GRFT 诱导的 gp120 寡糖附着位点突变以及凝集素亲和力对多价相互作用的依赖性的文献的支持。 Griffithsin 作为局部抗 HIV 杀菌剂的临床前和临床开发研究。 自从 GRFT 大规模生产以来，我们一直通过主要由 NIAID 资助的合作来进行其临床前开发。我们报道了其在粘膜环境中的稳定性和功效、其在人宫颈外植体模型中的活性以及其安全性以及在多个体外和体内模型系统中缺乏免疫激活或上皮刺激。这导致其他研究人员发表了几篇出版物，详细介绍了 GRFT 对蛋白水解降解的惊人抵抗力、其与抗逆转录病毒药物（如替诺福韦和其他凝集素）结合的协同活性，以及​​其对 HIV-1 gp120 免疫反应的增强。 GRFT 现在被认为是人体临床试验的主要候选者。 GRFT 的凝胶和薄膜配方、其稳定性和从这些配方中的释放、其针对广谱 HIV 病毒株的活性以及其针对 HSV 的体内活性也提高了其地位。目前，我们正在继续将 GRFT 作为抗 HIV 杀菌剂进行临床开发。评估 Griffithsin 的抗 HIV 和 HCV 活性。-之前我们和其他人已经证明，抗病毒凝集素氰基维林-N 对除 HIV 之外的其他包膜病毒也具有活性。由于 GRFT 比 CV-N 更有效，而且作为一种单糖特异性凝集素，其碳水化合物结合基序更加混杂，我们继续与 NIAID 合作，以确定 GRFT 的体外抗病毒活性谱。 与此同时，我们开始与南非 NICD 的 Lynn Morris 博士合作，测试 GRFT 对抗流行的 HIV 病毒株的效果。对不同 HIV-1 分支的 GRFT 活性的评估得出了一些有趣的发现。最初，我们表明，与实验室毒株一样，C 分支中的 HIV-1 临床毒株在低 nM 至 pM 水平下非常容易受到 GRFT 的影响。此外，HIV 阳性患者宫颈阴道灌洗液中的病毒也被证明容易受到 GRFT 的抑制。最后，我们的工作评估了 GRFT（以及我们的其他凝集素）抑制 HIV-1 与树突状细胞相关凝集素 DC-SIGN 结合的能力。已知 DC-SIGN 通过将感染性病毒转移和呈递给 CD4+ T 细胞而成为 HIV 感染的正效应子。在这项研究中，我们确定 GRFT 能够抑制 HIV-1 与 DC-SIGN 的结合，并抑制随后病毒向 CD4+ 细胞的转移。这些实验的最终成果是更好地了解 GRFT 抗 HIV 活性的范围和机制及其作为抗 HIV 杀菌剂的潜在用途。GRFT 和另一个 MTL 发现 SVN 进行了抗丙型肝炎病毒 (HCV) 活性测试。这是与日本国立传染病研究所的 Yutaka Takebe 博士发起的合作。在 HCV 细胞培养测定系统中进行测试时，SVN 和 GRFT 均具有有效活性，其中 GRFT 显示 EC50 = 50 pM，且毒性很小。我获得了 NIAID 的资助，用于支持在具有人肝细胞移植的 Alb-uPA-SCID 小鼠模型中进行 GRFT 抗 HCV 的体内测试。给研究动物注射 20 毫克/公斤/天，持续 10 天，结果表明 GRFT 无毒，并且皮下注射后可快速生物利用。然后我们完成了两项功效研究，其中动物连续 10 或 18 天接受 GRFT 给药。结果表明，HCV 攻击后 GRFT 治疗 10 天，HCV 滴度降低了 100 倍。 18 天的 GRFT 治疗再次将病毒载量降低了 100 倍以上，但对测试动物产生了显着的毒性（6/14 动物死亡），但对其中的人类肝组织没有影响。评估抗病毒蛋白 griffithsin 和 scytovirin 对抗埃博拉病毒的活性。-我与 USAMRIID 和 NIAID 持续合作，研究我们发现的具有潜在生物防御意义的抗病原病毒活性的蛋白质。这项工作的一部分是由我从 NIAID 获得的 Trans-NIH 拨款资助的，用于研究使用蛋白质 SVN 和 GRFT 对抗埃博拉病毒。埃博拉病毒是 A 类传染源，尚无批准的治疗方案，人类死亡率>50%。针对埃博拉扎伊尔病毒和马尔堡病毒的 SVN 和 GRFT 的初步体外伪粒子测定表明，这些蛋白质对两种丝状病毒均具有纳摩尔级活性。小鼠耐受性研究表明，GRFT 和 SVN 在高达 40 mg/kg/天的剂量下具有良好的耐受性，其中 GRFT 剂量为 Q12 或 Q24，SVN 剂量为 Q6。在不同剂量范围内使用两种蛋白的小鼠中进行的功效研究表明，用 SVN 或 GRFT 治疗 10 天，治疗动物的存活率为 90%（相比之下，对照动物的存活率为 0%）。从天然产物提取物中分离出的 HIV 蛋白。 Synthecium sp. 的水提取物。显示出抗 HIV 活性并产生了 3 种新型抗 HIV 蛋白。纯化的cnidarin蛋白被命名为cnidarin 1-3（CNID-1、CNID-2、CNID-3）。这些蛋白质通过 SDS-PAGE 分析为均质，通过 ESI/MS 显示每种蛋白质的单峰，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​​。建立了纯化的 CNID 蛋白的氨基酸序列。 CNID-1 和 CNID-3 的肽片段充分重叠，可对两种蛋白质进行测序。当相互比对时，CNID-1 和 CNID-3 具有 71% 的序列相似性，并且当使用 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没有显着的序列同源性。所有三种 CNID 蛋白质均引起浓度依赖性抑制病毒诱导的细胞杀伤具有皮摩尔 EC50 值。 CNID 具有非常有效的抗 HIV 活性，具有皮摩尔至低纳摩尔范围的活性，平均而言，其活性低于或与从天然来源分离的其他抗病毒蛋白的活性相当，但 griffithsin 除外。在三种 CNID 蛋白中，CNID-1 在防止 CEM-SS 细胞中 HIV-1RF 诱导的细胞病变效应方面最为有效 (EC50 = 85 pM)。 CNID-3 对 HIV-1ROJO 初级分离株的抑制作用最强（EC50 为 1.5 nM），而 CNID-2 的抑制作用最弱。 CNID-1与gp41和gp120的结合效果相当好，这与我们之前发现的CV-N、SVN和GRFT不同。 CNID-1 与 gp120 的结合不依赖于糖基化，表明 CNID-1 与 CV-N、SVN 和 GRFT 不同，不是凝集素。