Novel estrogen receptor ligands from plant genomics

描述（由申请人提供）：人核受体（HNR）配体作为药物治疗或化学预防剂具有潜在价值，可用于许多对肿瘤有反应的癌症。该STTR的主要目的是将“学术”植物基因组学技术转移到针对HNR配体的药物发现中。植物中的雌激素受体（ER）配体将用于证明应用，因为这些配体本身具有明确的商业和治疗价值。在第1阶段，表达人ER α/GFP构建体的转基因拟南芥幼苗用于筛选来自天然植物库的ER配体提取物。鉴定了9种先前未研究的水生物种，包括在哺乳动物细胞筛选中对ER具有激动剂或拮抗剂活性的物种。在第2阶段，将进一步研究这些物种的代表，使用分析指导的HPLC分馏分离活性化合物，将在体外和体内对乳腺癌细胞进行测试。然而，第二阶段的主要目标是应用新的植物基因组学技术来优化两种植物中ER配体的生产。在该技术中，产生植物物种的转基因毛状根培养物，其表达ER α/GFP构建体或新的ER β/GFP构建体。然后对这些培养物进行激活标签诱变（ATM），其诱导基因组中的随机功能获得性突变。现在，任何ER配体产生增加的个体突变体都应该通过明亮的绿色荧光来识别。这快速筛选了物种产生ER α或ER β配体的“基因组能力”。在第1阶段，在表达ER α/GFP的转基因拟南芥幼苗的突变体群体中实现了这种方法的概念证明，并且在第2阶段，我们最初提出将这种方法应用于大豆（大豆-已知含有高水平的ER配体）和Cacalia plantagelae（具有高水平的ER α/β激动剂活性的天然大蕉）的毛状根培养物。阳性培养物将通过其GFP表达和在ER活性的常规筛选中培养物提取物的活性来表征。还将尝试再生具有相同过量生产表型的完整突变体植物，以及来自突变体培养物或植物的活性级分，所述突变体培养物或植物如上对乳腺癌细胞进行评价。该方法可以用于任何HNR靶标，主要目的是表明该技术可以转移到抗癌天然产物发现中。 商业化计划旨在许可该技术，或与主要公司合作，将该技术应用于合作伙伴指定的目标。 公共卫生相关性：该STTR中的技术转让有望快速评估植物物种对人类核受体的适当治疗活性。所选择的实例是对两种类型的人雌激素受体（ER α和ER β）具有活性的植物化合物，这两种类型的人雌激素受体具有不同的治疗意义，特别是对于乳腺癌。这是女性最常见的癌症形式，也是美国60岁以下女性死亡的主要原因。许多乳腺癌（至少在最初）是“激素反应性的”，因为抑制ER的药物显著减缓癌细胞的生长，这是制药工业中非常活跃的研究领域。植物是已知的ER配体的丰富来源，但已经证明难以利用这种来源，因为常规的植物药物发现通常是耗时且昂贵的。这项技术有望通过将基因组学方法应用于这一领域的植物药物发现来改变这一点。

DESCRIPTION (provided by applicant): Human nuclear receptor (HNR) ligands have potential value as pharmacotherapies or chemopreventive agents for many hormone-responsive cancers. The primary aim of this STTR is to transfer an "academic" plant genomics technology into drug discovery, targeting HNR ligands. Estrogen receptor (ER) ligands in plants will be used for proof of application, because these have clear commercial and therapeutic value in their own right. In phase1 transgenic Arabidopsis thaliana seedlings expressing a human ERalpha/GFP construct were used to screen extracts from a native plant library for ER ligands. 9 previously uninvestigated aquatic species were identified, including species with agonist or antagonist activity at ERs in mammalian cell-based screens. In phase 2, representatives of these species will be investigated further, using assay-guided HPLC fractionation to isolate active compounds, which will be tested on breast cancer cells in vitro and in vivo. However, the major aim in phase 2 is to apply the novel plant genomics technology to optimize the production of ER ligands in two plant species. In this technology, transgenic hairy root cultures of a plant species are generated, expressing either the ERalpha/GFP construct, or a novel ERbeta/GFP construct. These cultures are then subjected to activation tagging mutagenesis (ATM), which induces random gain-of-function mutations in the genome. Now, any individual mutant in which the production of ER ligands is increased, should be identifiable by bright green fluorescence. This rapidly screens the "genomic capability" of the species to generate ligands for either ERalpha or ERbeta. In phase 1, proof of concept for this approach was achieved in a mutant population of transgenic A thaliana seedlings expressing ERalpha/GFP, and in phase 2 we propose initially to apply this approach to hairy root cultures of Glycine max (soybean - known to contain high levels of ER ligands) and Cacalia plantaginea (a native plantain with high levels of ERalpha/beta agonist activity). Positive cultures will be characterized by their GFP expression, and on the activity of culture extracts in conventional screens for ER activity. Attempts will also be made to regenerate intact mutant plants with the same over-producing phenotype, and active fractions from mutant cultures or plants evaluated on breast cancer cells as above. The approach can be used with any HNR target, and the major aim is to show that the technology can be transferred to anti-cancer natural product discovery. The commercialization plan is aimed at licensing the technology, or partnering with major companies to apply the technology to targets specified by the partner. PUBLIC HEALTH RELEVANCE: The technology transfer in this STTR promises to rapidly evaluate plant species for appropriate therapeutic activity at human nuclear receptors. The example chosen is for plant compounds with activity at the two types of human estrogen receptors (ERalpha and beta) which have different therapeutic implications, particularly for breast cancer. This is the most common form of cancer in women and is the major cause of death for women under 60 in the USA. Many breast cancers are (initially at least) "hormone responsive" in that drugs which inhibit ERs markedly slow the growth of the cancer cells, and this is a very active research area in the pharmaceutical industry. Plants are a known rich source of ligands for the ER, but it has proven difficult to exploit this source because conventional plant drug discovery is often time-consuming and expensive. This technology promises to change this by applying genomics approaches to plant drug discovery in this area.