Selection-driven plant metabolites for treatment of CNS diseases

描述(申请人提供)：这个项目旨在证明植物生物合成途径的进化可以被加速和驱动，以有利于与特定人类目标蛋白相互作用的配体的合成。这是通过使突变的植物细胞受到有利于具有感兴趣表型的突变的生存的选择压力来实现的。作为一个例子，这种方法将被用来优化红半边莲的药理活性，它抑制人的多巴胺转运体(HDAT)，推测是通过它合成复杂生物碱-叶黄碱的能力来实现的。建立了稳定表达HDAT的红衣草转基因株系。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。现在已经产生了大量表达HDAT的遗传多样性的功能获得突变体，并在含有100uM MPP的培养基上进行选择，这会杀死绝大多数转基因突变体。然而，过量生产HDAT抑制剂的个别突变体具有生存优势，因此抗MPP的群体在具有这种生物活性的克隆中得到了极大的“丰富”。对个别具有HDAT抑制活性的MPP抗性突变体进行的初步GC/MS分析表明，其中许多突变体过量生产苯丙氨酸，但其余的突变体产生其他代谢产物，其中一些在野生型植物中检测不到。第二阶段的目的是证明这种生物技术可用于(A)产生具有特定价值的药理作用的新的天然产物(B)在突变植物中为这些化合物提供生物合成生产系统。当一种植物来源的铅化合物，如叶黄碱，对于化学合成来说过于复杂时，这一过程应该特别有价值。第一个具体目标是分析剩余的MPP耐药群体，以确定那些叶黄碱含量不能解释HDAT抑制活性增加的个体。活性化合物的分离(测定导向制备高效液相色谱)和初步鉴定(GC/MS)之后将进行体外药理学评估，与洛布林和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具活性的化合物对大鼠大脑DAT的功能影响。在平行研究中， 最大程度地产生活性代谢产物的突变克隆培养物将被再生为完整的突变植株，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质的新型植物来源天然产品的平台。

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.