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: 8589434
• 负责人: JOHN M. LITTLETON
• 金额: $ 51.67万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8589434
• 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进化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承受有利于具有感兴趣表型的突变体生存的选择压力来实现的。例如，该方法将用于优化半边莲的药理活性，半边莲可抑制人类多巴胺转运蛋白（hDAT），推测是通过其合成复杂生物碱半边莲碱的能力。已经建立了表达 hDAT 的 L.cardinalis 植物细胞的稳定转基因系。这些细胞对 hDAT 转运到细胞内的毒素（包括神经毒素 MPP+）表现出更高的敏感性。现在已经产生了表达 hDAT 的大量遗传多样性的功能获得性突变体，并在含有 100uM MPP+ 的培养基上进行了选择，该培养基杀死了绝大多数转基因突变体。然而，过量产生 hDAT 抑制剂的个体突变体具有生存优势，因此 MPP+ 抗性群体在具有这种生物活性的克隆中大大“富集”。对 hDAT 抑制活性增加的单个 MPP+ 抗性突变体的初步 GC/MS 分析表明，其中许多突变体过量产生洛宾碱，但其余的则产生其他代谢物，其中一些在野生型植物中无法检测到。第二阶段旨在证明该生物技术可用于（a）生成具有特定有价值药理学的新型天然产物（b）为突变植物中的这些化合物提供生物合成生产系统。当植物源性先导化合物（例如洛布那林）对于化学合成来说过于复杂时，该过程应该具有特别的价值。第一个具体目标是分析剩余的 MPP+ 耐药群体，以确定洛豆碱含量无法解释 hDAT 抑制活性增加的个体。活性化合物的分离（测定引导的制备型 HPLC）和初步鉴定（GC/MS）之后将进行体外药理学评价，与洛比那林和 hDAT 的合成抑制剂进行比较。然后将使用电化学方法在体内测试最活跃的化合物对大鼠大脑中 DAT 的功能影响。在平行研究中， 最大限度地过量产生活性代谢物的突变体克隆培养物将再生为完整的突变体植物，并对提取物进行分析以确定是否保留了药理/化学表型。最终目标是将这项技术商业化，作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源性天然产品的平台。