HTS-Based Identification of Novel Rce1p Inhibitors(RMI)

描述(由申请人提供)：我们的长期目标是确定RAS转换酶(Rce1p)的酶性质，Rce1p是CAAX蛋白-biosythens所需的一种相对未知的蛋白酶。CAAX蛋白是脂类修饰的分子，通常在重要的细胞通路中起信号分子的作用。RAS和RhoB是关键的例子。由于CAAX蛋白在细胞转化中的作用(例如，活化形式的RAS与30%的癌症有关)，调控这些蛋白的生物合成的策略正在被探索为新的抗癌疗法。Rce1P是这些策略中的一个新靶点。Rce1p是一种非典型的蛋白水解酶，具有多个膜跨度，缺乏一个典型的蛋白水解酶基序。到目前为止，Rce1p的作用机制仍然不清楚，部分原因是缺乏可以用作探针来研究其催化性质的抑制剂。我们已经开发了一套连贯的生化和遗传、化学和分子分析方法，用于确定Rce1p的活性部位和酶性质，并用于鉴定和表征新型Rcelp抑制剂。我们的一种检测方法用于监测猝灭的荧光肽底物的Rce1p依赖的切割。我们已经使用这种方法来评估新的抑制剂和突变对RCELP动力学参数的影响。这种检测方法很容易适用于HTS，本提案的目的之一是通过优化信噪比、确定重复性和进行Rce1p抑制剂的中试筛选，全面评估该检测方法在HTS中的实用性。作为第二个目标，我们建议将我们的其他Rce1p检测整合到二级筛查中，用于确认HTS HITS，并进一步评估候选抑制剂的靶标特异性。该项目的最终目标是鉴定新的Rce1p抑制剂，这些抑制剂可以作为深入了解Rce1p机制的工具；其中一些化合物也可能具有潜在的治疗价值。

DESCRIPTION (provided by applicant): Our long-term goal is to define the enzymatic properties of the Ras Converting Enzyme (Rce1p), a relatively uncharacterized protease that is required for CaaX protein-biosythensis. CaaX proteins are lipid-modified molecules that often function as signaling molecules in important cellular pathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e. g. activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesis of these proteins are being explored as novel anti-cancer therapies. Rce1P is a new target in these strategies. Rce1p is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rce1p remains undefined, in part due to a lack of inhibitors that can be used as probes to investigate its catalytic properties. We have developed a coherent set of biochemical and genetic, chemical, and molecular assays that we are using for defining the active site and enzymatic properties of Rce1p and for identifying and characterizing novel Rcelp inhibitors. 1 of our assays is used to monitor the Rce1p-dependent cleavage of a quenched fluorescent peptide substrate. We have used this assay to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Rcelp. This assay is readily adaptable for HTS, and 1 of the aims of this proposal is fully assess the utility of this assay for HTS by optimizing the signal-to-noise ratio, determining reproducibility, and conducting a pilot screen for Rce1p inhibitors. As a second aim, we are proposing to integrate our other Rce1p assays into secondary screens that can be used to confirm HTS hits and to further assess the target specificity of candidate inhibitors. The ultimate goal of this project is the identification of novel Rce1p inhibitors that can serve as tools for providing insight into the mechanism of Rce1p; some of these compounds may also be of potential therapeutic value.