Isolation of bioactive proteins from natural product extracts

从天然产物提取物中分离生物活性蛋白

• 批准号: 10014695
• 负责人: Barry Okeefe
• 金额: $ 77.92万
• 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/10014695
• 关键词: Address; Agriculture; Animal Model; Anions; Antiviral Agents; Binding; Biochemical; Biological; Biological Assay; Biological Sciences; Cells; Centers for Disease Control and Prevention (U.S.); Chromatography; Clinical Trials; Cloning; Collaborations; Country; Drug Kinetics; Enzymes; Escherichia coli; HIV; HIV Envelope Protein gp120; HIV-1; Hydrophobic Interactions; In Vitro; Infection; Lectin; Licensing; Lytic; Malignant Neoplasms; Methods; Molecular Target; Monoclonal Antibodies; Names; National Institute of Allergy and Infectious Disease; Natural Products; Nipah Virus; Oryctolagus cuniculus; Patients; Periodicity; Phase I Clinical Trials; Plants; Porifera; Precipitation; Production; Proteins; Rabies; Reporting; Research Personnel; Resources; Rice; Structure; System; Systems Development; Techniques; Testing; Tobacco; Toxic effect; Virus; Work; anti-HIV microbicide; anticancer activity; aqueous; base; cancer cell; clinical development; coral; cost; efficacy study; expression vector; first-in-human; improved; in vivo evaluation; large scale production; mutant; nanomolar; novel; polyclonal antibody; screening; soy; three dimensional structure; transmission process

Structure-activity, biochemical and antiviral studies on griffithsin (GRFT), cyanovirin(CV-N) and scytovirin(SVN) continue. Currently, we are continuing the clinical development of GRFT as an anti-HIV microbicide. The first Phase I clinical trial has now been successfully completed with a second ( from another collaboration) currently accruing patients. We have also recently begun collaborating with the Centers for Disease Control on the use of these antiviral proteins against both Nipah virus and Rabies. To potentially use PCMBS lectins as anti-HIV microbicides, whose largest need is in resource-poor countries, it was necessary to find large-scale, low-cost methods of production. To address this requirement, we collaborated broadly with researchers to develop options for the agricultural production of these proteins. We have now completed large scale production of the proteins GRFT and CV-N in plants including tobacco, rice and soy. The PCMBS has steadily collaborated with the MCL to enable structure determination of the bioactive proteins we discover. We have worked with Dr. Alex Wlodawer's group to define the structure of CV-N, SVN and GRFT. During the current review period we have continued this collaboration to evaluate modified GRFT mutants and characterized several GRFT-tandemers with improved potency. GRFT is now entering first-in-human clinical trials as an anti-HIV microbicide with one Phase I clinical trial underway and a second slated to start in 2019. The PCMBS continues to isolate and characterize new anti-HIV proteins from aqueous natural product extracts. We have been working on a new protein isolated from the sponge Vagocia sp, from which no bioactive compounds have so far been reported. Initial studies on the protein fraction of this extract identified potent anti-HIV activity that appeared to bind to HIV-1 gp120. A protein, named vagocin, has been purified to homogeneity by a combination of precipitation techniques, hydrophobic interaction and anion exchange chromatography. Vagocin was shown to have an anti-HIV-1 EC50 of 4.4 nM in whole-cell anti-HIV assays. Additional anti-HIV screening of vagocin using the TzmBl assay system, performed with our collaborator ImQuest Biosciences, showed that the protein had activity against several clades of HIV-1 in the mid- to low-nanomolar range, with clade C being the most sensitive. Vagocin also inhibited cell-to-cell transmission of HIV and was found to be synergistic with the anti-HIV monoclonal antibody b12 and to a lesser extent with GRFT. We have produced rabbit polyclonal antibodies to vagocin which we will use to further define its binding interactions. We have also recently isolated and characterized a novel cyclic protein that allosterically inhibits the TDP1 enzyme. In addition, we have recently isolated a new anti-HIV protein from the soft coral Alertigorgia sp. This protein has low nano molar activity against HIV and is 10 kDa in size.

对 griffithsin (GRFT)、cyanovirin (CV-N) 和 scytovirin (SVN) 的结构活性、生化和抗病毒研究仍在继续。目前，我们正在继续将 GRFT 作为抗 HIV 杀菌剂进行临床开发。第一个 I 期临床试验现已成功完成，第二个临床试验（来自另一项合作）目前正在招募患者。我们最近还开始与疾病控制中心合作，使用这些抗病毒蛋白来对抗尼帕病毒和狂犬病。 PCMBS 凝集素潜在地用作抗 HIV 杀微生物剂（资源匮乏国家的最大需求），有必要找到大规模、低成本的生产方法。为了满足这一要求，我们与研究人员广泛合作，开发这些蛋白质的农业生产方案。我们现已在烟草、水稻和大豆等植物中完成了蛋白质 GRFT 和 CV-N 的大规模生产。 PCMBS 与 MCL 稳步合作，以实现我们发现的生物活性蛋白质的结构测定。我们与 Alex Wlodawer 博士的团队合作定义了 CV-N、SVN 和 GRFT 的结构。在当前的审查期间，我们继续这种合作来评估修饰的 GRFT 突变体，并表征了几种具有改进效力的 GRFT 串联体。 GRFT 作为一种抗 HIV 杀菌剂目前正在进入首次人体临床试验，其中一项 I 期临床试验正在进行中，第二期临床试验预计于 2019 年启动。PCMBS 继续从水性天然产物提取物中分离和表征新的抗 HIV 蛋白。我们一直在研究从海绵 Vagocia sp 中分离出的一种新蛋白质，迄今为止尚未报道其中的生物活性化合物。对该提取物的蛋白质部分的初步研究发现了有效的抗 HIV 活性，该活性似乎与 HIV-1 gp120 结合。通过结合沉淀技术、疏水相互作用和阴离子交换层析，一种名为 Vagocin 的蛋白质已被纯化至均质。在全细胞抗 HIV 检测中，Vagocin 的抗 HIV-1 EC50 值为 4.4 nM。与我们的合作者 ImQuest Biosciences 一起使用 TzmBl 测定系统对迷走菌素进行额外的抗 HIV 筛选，结果表明该蛋白对中低纳摩尔范围内的几个 HIV-1 分支具有活性，其中分支 C 最为敏感。 Vagocin 还可以抑制 HIV 的细胞间传播，并被发现与抗 HIV 单克隆抗体 b12 具有协同作用，与 GRFT 具有较小程度的协同作用。我们已经生产了针对 vagocin 的兔多克隆抗体，我们将用它来进一步确定其结合相互作用。我们最近还分离并鉴定了一种新型环状蛋白，它可以变构抑制 TDP1 酶。此外，我们最近从软珊瑚 Alertigorgia sp. 中分离出一种新的抗 HIV 蛋白。该蛋白具有低纳摩尔抗 HIV 活性，大小为 10 kDa。