Mutant transgenic plant cells as a novel source of drugs

摘要 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。在野生型植物中，生物活性代谢物通常具有 通过突变和自然选择进化了数千年[1]，而在这里，“目标导向进化”可以 在几个月内改变突变植物细胞中的活性代谢物谱。概念验证已在 半边莲，含有复杂的生物碱洛宾碱。这是一种新型的多巴胺抑制剂 转运蛋白（DAT）[2]，帕金森病和药物依赖的分子靶点[3，4]。一、毛根 转化该物种的培养物以表达人DAT。这使得这些转基因（hDAT） 植物细胞对神经毒素MPP+诱导的毒性高度敏感，MPP+在细胞内积累 这些转基因细胞的功能获得突变体然后在一个培养皿中产生。 MPP+的浓度对非突变体是致命的。这种选择过程有利于突变体的生存， 过量产生抑制DAT的代谢物。结果，超过一半的>100 MPP+抗性突变体 与对照组相比，DAT抑制活性显著过量。在大多数突变体中， 增强的DAT抑制作用可归因于洛比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”HPLC峰，这些峰在突变体中没有观察到。 野生型，并且这些都含有DAT抑制活性。第一个具体目标是分离足够的量 用于化学鉴定和药理学分析（体外和体内DAT抑制）。 体内）。这将确定是否存在具有治疗潜力的新型DAT抑制代谢物， 变种人许多剩余的MPP+耐药群体（不会过度产生DAT抑制剂的突变体） 似乎过度产生抑制MPP+细胞内机制的代谢物。这些的摘录 突变体保护多巴胺能细胞系SH-SY 5 Y对抗MPP+，因此可能含有新的神经保护剂 代谢物。第二个具体目标是使用相同的方法， 对新的高效液相色谱峰进行分离和分析，以便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提供证明。 药物发现同样的方法也可以用来产生过度生产的突变体， 生物合成生产系统，并作为一种手段，优化生物合成的治疗代谢物， 药用植物此外，虽然该提案使用了人类DAT和L。以红雀为例， 技术可应用于许多其他目标和植物物种。如果全球制药公司采用 这可能是一项变革性的技术。申请者打算在第三阶段继续这样做， 与大型制药和/或生物技术公司建立伙伴关系。因此， 建议是技术开发，通过证明这种方法能够实现 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.