Selection-driven plant metabolites for treatment of CNS diseases

描述（由申请人提供）：本项目旨在证明植物生物合成途径的进化可以加速，并有利于与特定人类靶蛋白相互作用的配体的合成。这是通过将突变植物细胞置于有利于具有感兴趣表型的突变体存活的选择压力下实现的。作为一个例子，这种方法将被用于优化红半边莲的药理活性，红半边莲抑制人类多巴胺转运蛋白（hDAT），据推测是通过其合成复杂生物碱的能力。已经建立了一个稳定的表达hDAT的红草植物细胞转基因系。这些细胞对hDAT输送到细胞内的毒素（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基因多样化的表达hDAT的功能获得突变体，并在含有100uM MPP+的培养基上选择，这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+抗性种群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产生洛比碱，但其余突变体产生其他代谢物，其中一些在野生型植物中未检测到。第二阶段旨在证明这种生物技术可用于(a)产生具有特定药理价值的新型天然产物(b)在突变植物中为这些化合物提供生物合成生产系统。当植物衍生的先导化合物，如洛比林因过于复杂而无法进行化学合成时，该工艺应具有特别的价值。第一个具体目标是分析剩余的MPP+耐药人群，以确定那些洛林碱含量不能解释hDAT抑制活性增加的个体。活性化合物的分离（测定导向制备HPLC）和初步鉴定（GC/MS）将随后进行体外药理学评价，并与洛比林和合成的hDAT抑制剂进行比较。然后使用电化学方法测试最有效的化合物对大鼠脑内DAT的功能影响。在平行研究中，

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.