Isolation of antiviral proteins from natural product extracts

通过对griffithin的结构活性、生化和抗病毒研究，阐明其作用机制及其与病毒包膜糖蛋白上低聚糖的特异性结合参数。与Alex Wlodawer博士（MCL）合作完成的这些结构测定使我们能够修改GRFT结构，以创建具有不同程度抗hiv活性的突变体，并帮助确定GRFT的作用机制。综上所述，这些结果表明GRFT可能通过在HIV-I包膜糖蛋白gp120上交联寡糖并阻止病毒进入所需的后续构象变化来显示其有效的抗病毒活性。单体活性的丧失可能是由于它不能像天然的专性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，并得到了近期关于GRFT诱导gp120寡糖附着位点突变和凝集素亲和力对多价相互作用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GRFT大规模生产以来，我们一直通过NIAID资助的合作进行临床前开发。我们已经报道了它在粘膜环境中的稳定性和有效性，它在人宫颈外植体模型中的活性，以及它在多种体外和体内模型系统中的安全性和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文章，详细介绍了GRFTs对蛋白水解降解的惊人抵抗力，它与抗逆转录病毒药物（如替诺福韦和其他凝集素）联合的协同活性，以及它增强对HIV-1 gp120的免疫反应。GRFT现在被认为是人体临床试验的主要候选者。GRFT作为凝胶和薄膜的配方，其稳定性和从这些配方中释放，其抗广谱HIV毒株的活性，以及其抗HSV的体内活性也提高了其地位。目前，我们正在继续进行GRFT作为抗hiv杀菌剂的临床开发。griffithsin抗HIV和HCV活性的评价。-之前我们和其他人已经证明抗病毒凝集素cyanovirin-N对除HIV外的其他包膜病毒也有活性。由于GRFT比CV-N更有效，并且作为单糖特异性凝集素，其碳水化合物结合基序更加混杂，我们继续与NIAID合作确定GRFT的体外抗病毒活性谱。同时，我们开始与南非NICD的林恩·莫里斯（Lynn Morris）博士合作，测试GRFT对艾滋病毒传播株的抑制作用。对HIV-1不同分支中GRFTs活性的评估产生了几个有趣的发现。最初，我们发现，与实验室菌株一样，C支的HIV-1临床菌株在低nM至pM水平下对GRFT非常敏感。此外，HIV阳性患者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（和我们的其他凝集素）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是HIV感染的阳性效应，通过感染性病毒向CD4+ t细胞的转移和呈递。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。这些实验的高潮是更好地了解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MTL的另一个发现SVN对丙型肝炎病毒（HCV）的活性进行了测试。这是与日本国立传染病研究所的Yutaka Takebe博士发起的一项合作。在HCV细胞培养实验系统中测试时，SVN和GRFT都具有强活性，GRFT显示EC50= 50 pM，毒性很小。我获得了NIAID的资助，支持在人肝细胞植入的Alb-uPA-SCID小鼠模型中进行GRFT抗HCV的体内试验。给研究动物注射20mg /kg/天，连续10天，结果显示GRFT在s.c注射后无毒且生物利用迅速。然后，我们完成了两项疗效研究，在这些研究中，动物连续服用GRFT 10天或18天。结果显示，GRFT治疗10天后，HCV攻击后，HCV滴度降低100倍。18天的GRFT治疗再次将病毒载量降低了100倍，但对试验动物产生了显著的毒性（6/14只动物死亡），但对人体肝脏组织没有毒性。抗病毒蛋白griffithsin和scytovirin对埃博拉病毒活性的评价。-我正在与USAMRIID和NIAID合作，研究我们发现的对潜在生物防御感兴趣的致病病毒有活性的蛋白质。这项工作的一部分是由我从NIAID获得的跨nih拨款资助的，用于研究使用SVN和GRFT蛋白对抗埃博拉病毒。埃博拉病毒是一种a类传染性病原体，没有批准的治疗方案，人类死亡率为50%。对SVN和GRFT抗埃博拉扎伊尔病毒和马尔堡病毒的初步体外假颗粒试验表明，这些蛋白对这两种丝状病毒具有纳摩尔活性。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，GRFT给药Q12或Q24， SVN给药Q6。对两种蛋白在不同剂量范围内的小鼠的疗效研究表明，用SVN或GRFT治疗10天，治疗动物的存活率为90%（相比之下，对照组动物的存活率为0%）。从天然产物提取物中分离的抗hiv蛋白的分离、鉴定和克隆。Synthecium sp.的水提物具有抗hiv活性，并产生了3个新的抗hiv蛋白。纯化得到的cnidarin蛋白命名为cnidarin 1-3 （cnid1, cnid2, cnid3）。SDS-PAGE均质，ESI/MS显示单峰，分别对应18122 Da （CNID-1）、18088 Da （CNID-2）和17963 Da （CNID-3）的精确分子质量。建立纯化的CNID蛋白的氨基酸序列。CNID-1和CNID-3的肽片段充分重叠，从而对这两种蛋白进行测序。当CNID-1和CNID-3相互比对时，它们具有71%的序列相似性，当使用NCBI数据库与已知蛋白质进行比较时，它们没有显示出与任何已知蛋白质的显著序列同源性。所有三种CNID蛋白都能以浓度依赖性抑制病毒诱导的细胞杀伤，其EC50值为皮摩尔。这些CNIDs具有明显的抗HIV活性，从皮摩尔到低纳摩尔范围，平均而言，与从天然来源分离的其他抗病毒蛋白的活性相比，格里菲斯蛋白的活性较低或相当。在三种CNID蛋白中，CNID-1在CEM-SS细胞（EC50 = 85 pM）中最有效地保护免受hiv - 1rf诱导的细胞病变效应。cid -3对HIV-1ROJO原代分离物的抑制作用最强（EC50为1.5 nM），而cid -2的抑制作用最低。CNID-1与gp41和gp120的结合同样良好，这与我们之前发现的CV-N、SVN和GRFT不同。我也是两项临床试验指导委员会的成员，这两项试验是为评估griffithsin作为抗艾滋病毒杀菌剂而资助的。已开始对GRFT对尼帕病毒、埃博拉病毒和中东呼吸综合征的活性进行进一步的校内研究。我们还与外部合作开展了针对HSV-1和hsv - 2以及阴道毛瘤的活动。

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.