Selection-driven plant metabolites for treatment of CNS diseases

描述(由申请人提供)：主要目标是将用于植物药物发现的生物技术平台从学术实验室转移到行业使用(即该应用程序直接响应“实验室到市场”路线图计划)。植物中的许多“防御性”代谢物是针对昆虫中枢神经系统中的关键蛋白质的，而这些蛋白质与人类蛋白质之间的同源性使这些代谢物具有潜在的中枢神经系统药物的价值。这些植物代谢物是在自然选择的反应下进化的，因此施加的选择压力应该会引导植物的次生代谢转向更有效的药物。实现这一目标的一种方法是在植物细胞中表达一种与毒性机制有关的人类目标蛋白。现在，产生这种蛋白质的抑制剂的细胞更有可能存活下来，反复的突变和选择应该引导“进化”朝着日益有效的抑制剂方向发展。例如，人类多巴胺转运体(HDAT)是药物依赖和神经退行性疾病的分子靶点。功能性HDAT在植物细胞中的表达(初步研究)，使这些细胞对由HDAT运输的神经毒素(MPTP，6-羟基多巴胺)敏感。相反，选择性DAT抑制剂GBR12909保护这些细胞。因此，在一个突变群体中，在这些毒素中幸存下来的转基因HDAT植物细胞应该在过量产生抑制HDAT的代谢物的克隆中“丰富”，或者以某种其他方式保护自己免受神经毒素的伤害。两者都具有潜在的治疗价值。为了确定适合这种方法的物种，对大约1000种本地植物的提取物进行了相关活性筛选。这确定了红衣藻是最好的候选者，而且这个物种也很容易培养和转化，使其成为应用的理想证据。经活化标签诱变的转基因红衣主教毛状根群体，现已进行MPTP选择。第一阶段的建议是筛选耐药突变亚群，以寻找存在抑制大鼠脑突触体内DAT的代谢物，或保护人类多巴胺能细胞系免受神经毒素的影响。阳性克隆(第一阶段交付成果)将在第二阶段用于化合物鉴定，评估复杂模型中的潜在治疗价值，并用于再生突变的药用植物品系。所有这些产品都有潜在的商业价值，但它说明了技术的一般价值，以及它从实验室转移到市场的过程，这对公司来说是最有商业价值的。 与公共健康相关：植物仍然是复杂生物活性化合物的非常有价值的来源，但目前植物药物发现的方法不能与组合化学和高通量药理筛选的制药工业技术竞争。这项建议使用了一种新的生物技术方法来定向进化和发现在人类中枢神经系统疾病中具有潜在价值的植物代谢物。这些疾病是美国和世界范围内最具破坏性和经济重要性的疾病之一。该提案的重点是治疗药物依赖和酒精中毒的潜在药物，以及治疗神经退行性疾病的药物，包括精神刺激剂引起的神经毒性，以及与衰老相关的疾病，包括帕金森症和帕金森病(美国第二常见的神经退行性疾病)。将使用的技术结合了植物分子生物学和分子药理学，它的支持将加速将这一学术研究引入商业部门。它的简单和快速表明，它可能会与现有的合成化学方法竞争，甚至可能重振植物作为治疗中枢神经系统疾病的药物来源的使用。

DESCRIPTION (provided by applicant): The primary aim is to transfer a biotechnology platform for plant drug discovery from the academic laboratory to industry use (i.e. this application is directly responsive to the "lab to marketplace" roadmap program). Many "defensive" metabolites in plants are targeted on key proteins in the insect CNS, and homology between these and human proteins, makes such metabolites potentially valuable as CNS drugs. These plant metabolites evolved in response to natural selection, and so an imposed selection pressure should direct plant secondary metabolism toward more effective drugs. One way this could be achieved is by the expression of a human target protein, linked to a toxic mechanism, in plant cells. Now, cells which are producing inhibitors of this protein are more likely to survive, and repeated mutagenesis and selection should direct "evolution" toward increasingly effective inhibitors. For example, the human dopamine transporter (hDAT) is a molecular target in drug dependence and neurodegenerative disease. Expression of the functional hDAT in plant cells (Preliminary studies), makes these susceptible to neurotoxins, (MPTP, 6hydroxydopamine) which are transported by the hDAT. Conversely, the selective DAT inhibitor, GBR12909, protects these cells. Therefore in a mutant population, transgenic hDAT plant cells that survive these toxins should be "enriched" in clones which over-produce metabolites that inhibit the hDAT, or protect against the neurotoxins in some other way. Both would be of potential therapeutic value. To identify a species for this approach, extracts from ~1000 native plants were screened for relevant activity. This identified Lobelia cardinalis as the best candidate, and this species is also easy to culture and transform, making it ideal for proof of application. A population of transgenic hDAT L. cardinalis hairy roots, mutagenized by activation tagging, has now been subjected to MPTP selection. The proposal for phase 1 is to screen the resistant mutant sub-population for the presence of metabolites which inhibit the DAT in rat brain synaptosomes, or protect a human dopaminergic cell line against neurotoxins. Positive clones (phase 1 deliverables) will be used in phase 2 for compound identification, assessment of potential therapeutic value in complex models, and for regeneration into mutant medicinal plant lines. All of these products have potential commercial value, but it is the illustration of the general value of the technology, and its transfer from lab to marketplace, which is of most commercial value to the company. PUBLIC HEALTH RELEVANCE: Plants remain a very valuable source of complex bioactive compounds, but current methods of plant drug discovery do not compete with the pharmaceutical industry techniques of combinatorial chemistry and high throughput pharmacological screening. This proposal uses a novel biotechnology approach to the directed evolution and discovery of plant metabolites with potential value in human CNS diseases. These are among the most devastating and economically important diseases in the USA and world wide. The proposal focuses on potential medications for drug dependence and alcoholism, as well as for neurodegenerative conditions including psychostimulant-induced neurotoxicity, and diseases associated with aging, including parkinsonism and Parkinson's Disease (the second most common neurodegenerative disease in the US). The technology to be used combines plant molecular biology and molecular pharmacology, and its support will accelerate the introduction of this academic research into the commercial sector. Its simplicity and rapidity suggests that it may compete with the existing synthetic chemistry approaches, and might even revitalize the use of plants as a source of medicines for CNS disease.