Selection-driven plant metabolites for treatment of CNS diseases

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促进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经受有利于具有感兴趣的表型的突变体存活的选择压力来实现的。作为一个例子，这种方法将用于优化半边莲的药理活性，半边莲抑制人类多巴胺转运蛋白（hDAT），通过其合成复杂生物碱，洛宾碱的能力。获得了一个稳定的转基因株系。已经建立了表达hDAT的红雀属植物细胞。这些细胞显示出对通过hDAT转运到细胞中的毒素（包括神经毒素MPP+）的敏感性增加。现在已经产生了表达hDAT的功能获得性突变体的大量遗传多样性群体，并在含有100 μ M MPP+的培养基上进行选择，所述MPP+杀死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存活优势，使得MPP+抗性群体在具有这种生物活性的克隆中大大“富集”。初步GC/MS分析个别MPP+抗性突变体与增加hDAT抑制活性表明，许多这些是过量生产洛宾碱，但其余的产生其他代谢产物，其中一些是在野生型植物中检测不到的。第二阶段旨在证明这种生物技术可用于（a）产生具有特定有价值药理学的新型天然产物（B）在突变植物中为这些化合物提供生物合成生产系统。当植物来源的铅化合物，如山胡椒碱，太复杂而不能进行化学合成时，该过程应该特别有价值。第一个具体目标是分析剩余的MPP+抗性群体，以确定其中山扁豆碱含量不能解释hDAT抑制活性增加的那些个体。活性化合物的分离（分析指导的制备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。然后使用电化学测试最具活性的化合物对大鼠脑中DAT的体内功能效应。在平行研究中， 将最大程度过量产生活性代谢物的突变克隆培养物再生为完整的突变植物，并分析提取物以确定是否保留了药理学/化学表型。最终目标是将该技术商业化，作为发现和生产针对特定人类CNS蛋白质的新型植物源天然产物的平台。

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.