Isolation of bioactive proteins from natural product extracts

对griffithsin（GRFT）、cyanovirin（CV-N）和scytovirin（SVN）的结构-活性、生物化学和抗病毒研究仍在继续。目前，我们正在继续将GRFT作为抗HIV杀微生物剂进行临床开发。第一个I期临床试验现已成功完成，第二个（来自另一个合作）目前正在进行中。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质对抗尼帕病毒和狂犬病。为了潜在地将PCMBS凝集素用作抗HIV杀微生物剂，其最大需求在资源贫乏的国家，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，为这些蛋白质的农业生产开发选择。我们现在已经完成了在烟草、水稻和大豆等植物中大规模生产GRFT、SVN和CV-N蛋白。最近的其他研究表明，griffithsin、scytovirin和cyanovirin对包括SARSCoV-2在内的冠状病毒具有活性。PCMBS继续从水溶性天然产物提取物中分离和鉴定新的抗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，迄今为止还没有报道过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鉴定出似乎与HIV-1 gp 120结合的强效抗HIV活性。一种蛋白质，命名为迷走神经肽，已被纯化到均匀的结合沉淀技术，疏水相互作用和阴离子交换色谱。在全细胞抗HIV测定中，Vagocin显示具有4.4 nM的抗HIV-1 EC 50。与我们的合作者ImQuest Biosciences一起使用TzmBl测定系统进行的vagocin的额外抗HIV筛选显示，该蛋白质在中低纳摩尔范围内对HIV-1的几个进化枝具有活性，其中进化枝C是最敏感的。Vagocin也抑制HIV的细胞间传播，并被发现与抗HIV单克隆抗体b12协同作用，在较小程度上与GRFT协同作用。我们已经产生了兔多克隆抗体迷走蛋白，我们将使用进一步确定其结合相互作用。我们最近还分离和表征了一种新型的环状蛋白，其变构抑制TDP 1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。这种蛋白对HIV具有低纳米摩尔活性，大小为10 kDa。最后，我们已经分离并表征了一类新的小环状肽，其具有针对癌症靶标TDP-1的活性。

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.