SCREENING AND IDENTIFICATION OF NATURAL PRODUCT LEADS FOR RESTORATION OF P53

实验室方案1的目标是检测、表征、 从自然资源或通过小规模的 恢复肿瘤功能的活性化合物的规模合成 抑癌基因，P53。活性化合物将从Xenova衍生 天然产品资源，目前由26000个微生物和 7500种植物。发酵条件和次生代谢物 提取程序的设计是为了揭示化学多样性 在各自的自然资源中与生俱来。这一系列将是 筛选具有以下三种活性之一的活性化合物：i) 恢复特定p53突变体的正常DNA结合活性；ii) 破坏P53和癌基因MDM2之间的相互作用；III) 能够模拟WAF1的细胞周期抑制活性(即 由P53诱导)。这些筛查将使用酶连接进行 实验室专门为这些目的开发的免疫分析方法 方案2.实验室方案2提供的化验格式如下 经过调整和验证，可作为高吞吐量自动屏幕运行 微生物发酵和植物提取物。活动样本，通常 由超过50,000个微生物发酵提取物和7,000个 植物提取物，将是最先进的生物信息学的优先事项 软件，并提交给生物测定引导的化学分级。 铅化合物功能的确认将使用CELL- 基础化验。将从结构上阐明活性化学物种。 使用分析技术的组合，主要是核磁共振和质量 光谱分析。继活性化合物、类似物的表征之后 将进行合成和放大生产，以提供 相关的药理和毒理学评价材料 建立动物模型，探讨药物对大鼠心脏功能的影响机制。 各自的目标(实验室方案2)。这些材料也将被 用于分析缔合的目标分子的晶体结构 对于推定的药物，一种有助于阐明其机制的方法 行动计划(实验室方案3)。

The objective of laboratory programme 1 is the detection, characterization, isolation, analysis and production from natural resources or via small scale synthesis of active compounds which restore the function of the tumor suppressor gene, p53. The active compounds will be derived from the Xenova natural product resource which currently consists of 26000 microbes and 7500 plant species. Fermentation conditions and secondary metabolite extraction procedures are designed to reveal the chemical diversity inherent in the respective natural resources. This collection will be screened for active compounds exhibiting one of three activities: i) restoration of normal DNA-binding activity to particular p53 mutants; ii) disruption of the interaction between p53 and the oncogene mdm2; iii) ability to mimic the cell cycle-inhibitory activity of WAF1 (which is induced by p53). These screens will be performed using enzyme-linked immunoassays developed specifically for these purposes by laboratory programme 2. The assay formats supplied by laboratory programme 2 will be adapted and validated to run as high throughput automated screens with microbial fermentation and plant extracts. Active samples, typically generated from in excess of 50,000 microbial fermentation extracts and 7000 plant extracts, will be prioritized by state-of-the-art bioinformatics software, and submitted to bioassay-guided chemical fractionation. Confirmation of lead compound functionality will be evaluated using cell- based assays. Active chemical species will be structurally elucidated using a combination of analytical techniques, principally NMR and mass spectrometry. Following characterization of active compounds, analogue synthesis and scale-up of production will be carried out to provide material for pharmacological and toxicological evaluation in relevant animal models and to explore the mechanisms of drug action on the respective targets (laboratory programme 2). This material will also be used to analyse the crystal structure of the target molecule in association with putative drug, an approach which will serve to elucidate the mechanism of action (Laboratory programme 3).