Mutant transgenic plant cells as a novel source of drugs

摘要 其目标是进一步发展生物技术，使植物物种的生物合成向 具有特定分子靶点的生物活性代谢物。在野生植物中，生物活性代谢物通常有 千百年来由突变和自然选择进化而来[1]，而在这里，“靶向进化”可以 在几个月内改变突变植物细胞中的活性代谢物。已在#年获得概念验证 红花半边莲(Lobelia Cardinis)中含有复杂的生物碱Lobinaline。这是一种新型的多巴胺抑制剂。 转运蛋白(DAT)[2]，帕金森氏病与药物依赖的分子靶点[3，4]。第一，毛状根 将该物种的培养物转化为表达人DAT。这使得这些转基因(HDAT) 植物细胞对细胞内积累的神经毒素MPP的毒性高度敏感 通过DAT的活动。这些转基因细胞的功能突变体的获得然后在 对非突变体致死的MPP浓度。这种选择程序有利于突变体的存活 过度产生抑制DAT的代谢物。结果，超过一半的&gt；100耐MPP突变体 与对照组相比，显著提高了DAT抑制活性。在这些突变体中的大多数中， 增强的DAT抑制可以归因于洛布林或其他已知的活性代谢物，但25个突变体 含有未知的DAT抑制剂。在这些突变体中，有9个新的高效液相色谱峰是在 野生型，这些都具有DAT抑制活性。第一个具体目标是分离出足够数量的 这些代谢物用于化学鉴定和药理分析(DAT体外抑制和 Vivo)。这将确定是否存在具有治疗潜力的新的DAT抑制代谢物 变种人。许多剩余的MPP耐药群体(不过量生产DAT抑制剂的突变株) 似乎过度产生代谢产物，从而抑制MPP的细胞内机制。摘自这些 突变体保护多巴胺能细胞系SH-SY5Y免受MPP的影响，因此可能含有新的神经保护作用 代谢物。第二个具体目标是使用相同的方法来分离和分析它们，基于 对新的高效液相色谱峰进行分离和分析，以确定其潜在的治疗价值。 这些目标的成功完成将为该技术作为工厂平台的应用奠定基础 药物发现。同样的方法也可以用来产生生产过剩的突变体 生物合成生产系统，作为优化治疗代谢物生物合成的一种手段 药用植物。此外，虽然这项提案以人的DAT和红衣藻为例，但 技术可以应用于许多其他目标和植物物种。如果被全球制药公司采用 工业：这可能是一项变革性的技术。申请者打算在第三阶段通过寻求 与主要制药和/或生物技术公司建立伙伴关系。因此，该项目的主要目的是 建议是技术开发，要通过证明这种方法能够 在突变转基因植物细胞中发现具有治疗潜力的新活性代谢物。

Abstract The objective is further development of a biotechnology that “evolves” biosynthesis in a plant species toward bioactive metabolites with a specific molecular target. In wild-type plants, bioactive metabolites have generally evolved by mutation and natural selection over millennia [1], whereas here, “target-directed evolution” can change the active metabolite profile in mutant plant cells within months. Proof of concept has been obtained in Lobelia cardinalis, which contains the complex alkaloid lobinaline. This is a novel inhibitor of the dopamine transporter (DAT) [2], a molecular target in Parkinson's Disease and drug dependence [3,4 ]. First, hairy root cultures of this species were transformed to express the human DAT. This made these transgenic (hDAT) plant cells highly susceptible to toxicity induced by the neurotoxin MPP+, which is accumulated intracellularly by activity of the DAT. Gain of function mutants of these transgenic cells were then generated in a concentration of MPP+ that is lethal to non-mutants. This selection procedure favors survival of mutants that overproduce metabolites that inhibit the DAT. As a result, more than half the >100 MPP+-resistant mutants were significantly overproducing DAT inhibitory activity relative to controls. In the majority of these mutants, enhanced DAT inhibition could be ascribed to lobinaline, or other known active metabolites, but 25 mutants contain unknown DAT inhibitors. In these mutants there are 9 “novel” HPLC peaks that are not observed in wild-type, and these all contain DAT inhibitory activity. The first specific aim is to separate sufficient quantities of these metabolites for chemical identification and pharmacological analysis (DAT inhibition in vitro and in vivo). This will establish whether novel DAT inhibitory metabolites with therapeutic potential exist in these mutants. Many of the remaining MPP+-resistant population (mutants that do not overproduce DAT inhibitors) appear to overproduce metabolites that inhibit the intracellular mechanism of MPP+. Extracts from these mutants protect the dopaminergic cell line SH-SY5Y against MPP+, and so may contain novel neuroprotective metabolites. The second specific aim is to separate and analyze these also, using the same approach based on separation and analysis of novel HPLC peaks, so that their potential therapeutic value can be ascertained. Successful completion of these aims will establish proof of application for the technology as a platform for plant drug discovery. The same approach can also be used to generate overproducing mutants to become biosynthetic production systems, and as a means of optimizing biosynthesis of therapeutic metabolites in medicinal plants. In addition, although this proposal uses the human DAT and L. cardinalis as examples, the technology can be applied to many other targets and plant species. If adopted by the global pharmaceutical industry this could be a transformative technology. The applicants intend to pursue this in phase III by seeking partnerships with major pharmaceutical and/or biotechnology companies. Thus, the main purpose of the proposal is technology development, to be achieved by demonstrating that this approach is capable of discovering novel active metabolites with therapeutic potential in mutant transgenic plant cells.