Molecular Basis for p70S6 Kinase and JARID1B Demethylase Activity and Inhibition

该项目基于该项目的工作假设，即p70S6激酶和JARID1B组蛋白去甲基酶在黑色素瘤细胞的生长和存活中发挥关键作用。这里提出的研究将描绘这些酶活性的生化和结构基础，并利用这些信息开发小分子抑制剂，通过该计划的其他项目使用黑色素瘤模型在体内研究这些蛋白质的功能。这些研究还将为黑色素瘤治疗的开发提供先导化合物。目标1将专注于p70S6K，目标2将专注于 JARID1B。对于目标1，我们制备了具有活性的重组全长p70S6激酶和几个截断的蛋白结构，制备了选择性良好的先导p70S6K有机金属抑制剂支架(复合EMS)，IC{50}值在中纳摩尔范围内，并测定了与EMS抑制剂结合的p70S6激酶结构域的晶体结构。我们建议：(A)对p70S6激酶的催化活性进行生化表征；(B)围绕EMS制备一个聚焦文库 (D)利用基于结构的设计、药物化学、激酶图谱和细胞研究(与项目2一起)，开发高效和选择性的第二代有机金属和有机p70S6激酶抑制剂；(C)进行有机p70S6激酶抑制剂的高通量溶液筛选，建立抑制模式，并确定与有机抑制剂结合的p70S6激酶结构域的X射线晶体结构。对于目标2，我们制备了具有酶活性的重组JARID1B和酵母菌Jhd2p，用于生化和晶体分析，并初步制备了JARID1B和Jhd2p晶体。我们现在建议(A)对JARID1B和Jhd2p的催化活性进行生化表征；(B)确定JARID1B和Jhd2p的X射线晶体结构并进行基于结构的突变，以便于结构-功能分析；(C)使用重组JARID1蛋白(JARID1B和Jhd2p)和 为便于高通量解决方案和在电子计算机中筛选小分子抑制剂，JARID1抑制剂将分别与该计划的项目1一起在细胞中进行测试；以及(D)将最有效的JARID1抑制剂与JARID1蛋白质结晶，以进行基于结构的抑制剂优化。总之，这些研究将提供新的分子洞察力和小分子试剂来描述两个新的蛋白质靶点p70S6K和JARID1B在黑色素瘤中的作用，并将为开发治疗黑色素瘤的治疗药物提供先导小分子化合物。

This project is based on the working hypothesis of the program project that the p70S6 kinase and the JARID1B histone demethylase enzymes play key roles in both melanoma cell growth and survival. The studies proposed here will delineate the biochemical and structural basis for the activities of these enzymes and use this information to develop small molecule inhibitors to study the function of these proteins in vivo using melanoma models through the other projects of the program. These studies will also provide lead compounds for development for therapy of melanoma. Aim 1 will focus on p70S6K and Aim 2 will focus on JARID1B. For Aim 1, we have prepared active recombinant full-length p70S6 kinase as well as several truncated protein constructs, prepared a lead p70S6K organometallic inhibitor scaffold (compound EMS) with promising selectivity profile and an IC{50} value in the mid-nanomolar range, and determined the crystal structure of the p70S6 kinase domain bound to the EMS inhibitor. We propose to: (a) biochemically characterize the catalytic activity of the p70S6 kinase; (b) prepare a focused library around the EMS organometallic p70S6 kinase inhibitor for screening in vitro and in vivo (together with Project 2) and determine the X-ray crystal structure of p70S6K/inhibitor complexes; (c) carry out a high-throughput solution screen for organic p70S6 kinase inhibitors, establish the mode of inhibition, and determine the X-ray crystal structure of the p70S6 kinase domain bound to lead organic inhibitors; and (d) use structure-based design, medicinal chemistry, kinase profiling and cell-based studies (together with Project 2) to develop potent and selective second-generation organometallic and organic p70S6 kinase inhibitors. For Aim 2, we have prepared recombinant and enzymatically active JARID1B and the yeast Jhd2p ortholog for biochemical and crystallographic analysis and have prepared preliminary JARID1B and Jhd2p crystals. We now propose to (a) biochemically characterize the catalytic activity of JARID1B and Jhd2p; (b) determine the X-ray crystal structures of JARID1B and Jhd2p and carry out structure-based mutagenesis to facilitate a structure-function analysis; (c) use the recombinant JARID1 proteins (JARID1B and Jhd2p) and structures to facilitate high throughput solution and in silico small molecule inhibitor screening, respectively, and JARID1 inhibitors will be tested in cells together with Project 1 of the program; and (d) crystallize the most potent JARID1 inhibitors with the JARID1 proteins for structure-based inhibitor optimization. Together, the studies proposed here will provide novel molecular insights and small molecule reagents to delineate the roles of two novel protein targets, p70S6K and JARID1B, in melanoma and will provide lead small molecule compounds to develop therapeutic agents to treat melanoma.