Selection-driven plant metabolites for treatment of CNS diseases

选择驱动的植物代谢物用于治疗中枢神经系统疾病

• 批准号: 8905682
• 负责人: JOHN M. LITTLETON
• 金额: $ 9.85万
• 依托单位: NAPROGENIX, INC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-20 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8905682
• 关键词: 1-Methyl-4-phenylpyridinium; Agonist; Alkaloids; Amphetamines; Antioxidants; Award; Biological Assay; Biological Factors; Biotechnology; Brain; Cell Culture Techniques; Cell Line; Cells; Central Nervous System Diseases; Chemicals; Complex; Computer Simulation; Corpus striatum structure; Development; Drug Addiction; Drug Industry; Electrochemistry; Evaluation; Evolution; Free Radical Scavenging; Guanine + Cytosine Composition; High Pressure Liquid Chromatography; Hormonal; Human; In Vitro; Individual; Lead; Ligands; Lobelia; Medicine; Methods; Mitochondria; Modification; National Institute on Alcohol Abuse and Alcoholism; Natural regeneration; Neurodegenerative Disorders; Neurotoxins; Nicotine; Nicotinic Receptors; Norepinephrine; Nucleus Accumbens; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Plant Extracts; Plants; Population; Population Heterogeneity; Predisposition; Preparation; Process; Production; Proteins; Rattus; Resistance; Rest; Serotonin; Small Business Innovation Research Grant; Small Business Technology Transfer Research; Source; Structure; System; Techniques; Technology; Testing; Therapeutic Uses; Toxin; Transgenic Organisms; United States National Institutes of Health; base; chemical synthesis; design; directed evolution; dopamine transporter; gain of function; gain of function mutation; high throughput screening; improved; in vivo; inhibitor/antagonist; interest; killings; mutant; novel; pre-clinical; pressure; programs; public health relevance; radioligand; uptake

DESCRIPTION (provided by applicant): This project aims to demonstrate that the evolution of plant biosynthetic pathways can be accelerated and driven to favor the synthesis of ligands which interact with a specific human target protein. This is achieved by subjecting mutant plant cells to selection pressures favoring the survival of mutants with the phenotype of interest. As an example, this approach will be used to optimize pharmacological activity in Lobelia cardinalis, which inhibits the human dopamine transporter (hDAT), putatively by its ability to synthesize the complex alkaloid, lobinaline. A stable transgenic line of L. cardinalis plant cells expressing the hDAT has been established. These cells show increased sensitivity to toxins transported into the cell by the hDAT, including the neurotoxin MPP+. A large, genetically diverse, population of gain-of-function mutants expressing the hDAT has now been generated, and selected on medium containing 100uM MPP+, which kills the vast majority of the transgenic mutants. However, individual mutants that are over-producing inhibitors of the hDAT have a survival advantage, so that the MPP+-resistant population is greatly "enriched" in clones with this bioactivity. Preliminary GC/MS analysis of individual MPP+-resistant mutants with increased hDAT inhibitory activity indicates that many of these are overproducing lobinaline, but the rest are generating other metabolites, some of which are not detectable in the wild-type plant. Phase II is designed to demonstrate that this biotechnology can be used to (a) generate novel natural products with a specific valuable pharmacology (b) provide a biosynthetic production system for these compounds in mutant plants. The process should be of particular value when a plant-derived lead compound, such as lobinaline, is too complex for chemical synthesis. The first specific aim is to analyze the remaining MPP+-resistant population to determine those individuals in which lobinaline content cannot explain increased hDAT inhibitory activity. Separation (assay-guided preparative HPLC) and tentative identification (GC/MS) of active compounds will be followed by pharmacological evaluation in vitro, in comparison with lobinaline and a synthetic inhibitor of the hDAT. The most active compounds will then be tested for functional effects on the DAT in rat brain in vivo using electrochemistry. In parallel studies, the mutant clonal cultures which are overproducing active metabolites to the greatest extent will be regenerated to intact mutant plants, and extracts analyzed to establish whether the pharmacological /chemical phenotype is retained. The ultimate aim is to commercialize the technology as a platform for discovering and producing novel plant-derived natural products targeted on specific human CNS proteins.

描述（由申请人提供）：该项目旨在证明植物生物合成途径的演变可以加速并驱动以有利于与特定人类靶蛋白相互作用的配体的合成。这是通过使突变植物细胞的选择压力有利于具有感兴趣表型的突变体存活的选择压力来实现的。例如，该方法将用于优化Cardinalis洛贝利亚（Lobelia Cardinalis）的药理活性，该药理活性抑制了人类多巴胺转运蛋白（HDAT），这是其合成复杂生物碱洛比宁氨酸的能力。已经建立了表达HDAT的Cardinalis植物细胞的稳定转基因系。这些细胞显示出对HDAT转运到细胞的毒素的敏感性，包括神经毒素MPP+。现已生成一个大型，遗传多样的功能收益突变体，并在包含100UM MPP+的培养基上选择，该培养基杀死了绝大多数转基因突变体。但是，过量产生HDAT抑制剂的个体突变体具有生存优势，因此MPP+耐药的种群在具有这种生物活性的克隆中被极大地“富集”。 HDAT抑制活性增加的单个MPP+耐药突变体的初步GC/MS分析表明，其中许多是产生的洛比宁氨酸，但其余的正在产生其他代谢物，其中一些代谢物在野生型植物中无法检测到。 II期旨在证明该生物技术可用于（a）具有特定有价值的药理学（b）的新型天然产物（a）为突变植物中这些化合物提供生物合成生产系统。当植物衍生的铅化合物（例如洛比奈碱）对于化学合成太复杂时，该过程应具有特殊的价值。第一个具体目的是分析剩余的MPP+耐药人群，以确定那些无法解释HDAT抑制活性增加的个体。与洛比宁氨酸和HDAT的合成抑制剂相比，活性化合物的分离（测定引导的制剂HPLC）和活性化合物的初步鉴定（GC/MS）将在体外进行药理评估。然后，最活跃的化合物将使用电化学对大鼠脑中DAT的功能作用进行测试。在平行研究中， 突变的克隆培养物在最大程度上使活跃代谢产物过量产生活性代谢物，将被再生到完整的突变植物中，并分析了提取物以确定是否保留了药理 /化学表型。最终目的是将该技术商业化为发现和生产针对特定人类中枢神经系统蛋白的新型植物衍生天然产品的平台。