Isolation of bioactive proteins from natural product extracts

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，我们正在继续进行GRFT作为抗hiv杀菌剂的临床开发。第一阶段临床试验现已成功完成，第二阶段（来自另一合作伙伴）正在进行中。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防治尼帕病毒和狂犬病。PCMBS凝集素的最大需求是在资源贫乏的国家，为了潜在地使用PCMBS凝集素作为抗hiv杀菌剂，有必要找到大规模、低成本的生产方法。为了满足这一需求，我们与研究人员广泛合作，开发这些蛋白质的农业生产选择。目前，我们已经在烟草、水稻和大豆等植物中完成了GRFT、SVN和CV-N蛋白的大规模生产。最近的其他研究表明，griffithsin、scytovirin和cyanovirin对包括SARSCoV-2在内的冠状病毒有活性。PCMBS继续从水相天然产物提取物中分离和表征新的抗hiv蛋白。我们一直致力于从海绵Vagocia sp中分离出一种新的蛋白质，迄今为止尚未报道从这种蛋白质中分离出生物活性化合物。对该提取物的蛋白质部分的初步研究发现，它似乎与HIV-1 gp120结合，具有强大的抗hiv活性。采用沉淀技术、疏水相互作用和阴离子交换色谱相结合的方法纯化了一种名为迷走球蛋白的蛋白质。在全细胞抗hiv检测中，迷走球蛋白具有4.4 nM的抗hiv -1 EC50。与我们的合作伙伴ImQuest Biosciences一起使用TzmBl检测系统对迷走草素进行了额外的抗hiv筛查，结果显示该蛋白在中低纳摩尔范围内对几种HIV-1进化支具有活性，其中C进化支最敏感。迷走球蛋白还能抑制HIV的细胞间传播，并被发现与抗HIV单克隆抗体b12有协同作用，与GRFT有较小程度的协同作用。我们已经生产了针对迷走球蛋白的兔多克隆抗体，我们将使用它来进一步确定其结合相互作用。我们最近也分离和表征了一种新的环蛋白，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，该蛋白具有低纳米摩尔抗HIV活性，大小为10 kDa。最后，我们分离并鉴定了一类新的具有抗肿瘤靶点TDP-1活性的小环肽。

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 underway. 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, SVN and CV-N in plants including tobacco, rice and soy. Additional recent studies have shown the activity of griffithsin, scytovirin and cyanovirin against coronaviruses including SARSCoV-2. 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. Finally, we have isolated and characterized a new class of small cyclic peptide which has activity against the cancer target TDP-1.