Mutant transgenic plant cells as a novel source of drugs

突变转基因植物细胞作为新的药物来源

基本信息

批准号：
9253077
负责人：
JOHN M. LITTLETON
金额：
$ 45.58万
依托单位：
NAPROGENIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-22 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9253077
关键词：
1-Methyl-4-phenylpyridinium Adopted Alkaloids Anabolism Biotechnology Carbon Cell Line Cells Chemicals Complex Development Directed Molecular Evolution Dopaminergic Cell Drug Addiction Drug Industry Evaluation Freeze Drying High Pressure Liquid Chromatography Human In Vitro Industry Lobelia Mass Fragmentography Mass Spectrum Analysis Medicinal Plants Methanol Methodology Molecular Target Mutation N-Methylaspartate Natural Selections Neurons Neurotoxins Nuclear Magnetic Resonance Parkinson Disease Pharmaceutical Preparations Pharmacologic Substance Phase Plant Roots Plants Population Procedures Production Proteins Protons Resistance Resolution Source Spectrometry Spectrum Analysis Structure Synapses System Technology Testing Therapeutic Toxic effect Toxin Traditional Medicine Transgenic Organisms Transgenic Plants Work abstracting aqueous base chemical synthesis commercialization cytotoxicity dopamine transporter drug discovery drug production gain of function gain of function mutation in vitro activity in vivo inhibitor/antagonist mutant novel novel therapeutics technology development therapeutic target uptake

项目摘要

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.

抽象的目标是进一步开发生物技术，“进化”植物物种的生物合成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特定的分子靶点。经过数千年的突变和自然选择而进化[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已获得概念证明。半边莲，含有复杂的生物碱半边莲碱，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性的分子靶点[3,4]。该物种的培养物被转化以表达人类 DAT，这使得这些转基因 (hDAT) 成为可能。植物细胞对细胞内积累的神经毒素 MPP+ 诱导的毒性高度敏感然后通过 DAT 的活性获得这些转基因细胞的功能突变体。对非突变体致命的 MPP+ 浓度该选择程序有利于突变体的生存。过量产生抑制 DAT 的代谢物因此，超过一半的 >100 MPP+ 耐药突变体。在大多数突变体中，DAT 抑制相对活性显着过量。 DAT 抑制作用增强可归因于 lobinaline 或其他已知的活性代谢物，但 25 个突变体这些突变体中含有未知的 DAT 抑制剂，其中有 9 个“新的”HPLC 峰，这些峰在野生型，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数量。这些代谢物的化学鉴定和药理学分析（体外和体内 DAT 抑制）这将确定这些药物中是否存在具有治疗潜力的新型 DAT 抑制代谢物。许多剩余的 MPP+ 抗性群体（不会过度产生 DAT 抑制剂的突变体）这些提取物似乎过量产生抑制 MPP+ 细胞内机制的代谢物。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第二个具体目标是使用相同的方法来分离和分析这些代谢物。分离和分析新的 HPLC 峰，从而确定其潜在的治疗价值。成功完成这些目标将为该技术作为植物平台的应用提供证据药物发现也可用于产生过量生产的突变体。生物合成生产系统，并作为优化治疗代谢物生物合成的手段此外，虽然该提案以人类 DAT 和 L.cardinalis 为例，但如果被全球制药公司采用，该技术可以应用于许多其他目标和植物物种。这可能是一项变革性技术，申请人打算在第三阶段寻求这一技术。因此，与主要制药和/或生物技术公司建立伙伴关系。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型活性代谢物。