High throughput screen for cyclin D1 inhibitors (RMI)

描述（由申请人提供）：敲除编码D型细胞周期蛋白的基因的实验证明，小鼠中由neu或ras癌基因诱导的乳腺癌绝对依赖于细胞周期蛋白D1。 大多数人乳腺癌过表达细胞周期蛋白Dl：大约50%的人乳腺癌具有扩增的或以其他方式过表达的c-neu，其依赖于细胞周期蛋白Dl用于细胞周期激活，并且大约20%的人乳腺癌具有扩增的细胞周期蛋白Dl。 治疗neu-、ras-和/或细胞周期蛋白ZX？的逻辑方法与乳腺癌相关的药物是抑制细胞周期蛋白D1的功能。 由于敲除细胞周期蛋白D1对成年小鼠生理学的影响有限，细胞周期蛋白D1的特异性抑制可能具有非常有利的治疗指数。 细胞周期蛋白D1通过与其他蛋白质直接相互作用发挥功能。 蛋白质-蛋白质相互作用已被成功地抑制使用小分子库筛选中确定。 为了使治疗指数最大化，应当以排除细胞周期蛋白D2和细胞周期蛋白D3相互作用的抑制剂的方式设计用于鉴定细胞周期蛋白D1相互作用的特异性抑制剂的筛选。 考虑到D型细胞周期蛋白之间的序列差异，在酵母中使用反向双杂交筛选来鉴定不抑制细胞周期蛋白D2和细胞周期蛋白D3相互作用的细胞周期蛋白D1相互作用的抑制剂应该是可能的。 该提议中的筛选被设计为通过将这些相互作用与对酵母有条件毒性的基因的表达相联系来选择细胞周期蛋白Dl相互作用的抑制剂。 为了排除细胞周期蛋白D2和细胞周期蛋白D3的抑制剂，这些细胞周期蛋白的相互作用将与酵母生长所需的基因的表达相关联。 酵母已被成功地用于药物筛选，因为它们是可渗透的化合物在化学图书馆，平均小于500 Da。反向双杂交筛选是简单的构建和验证，并可以适用于高通量筛选的小分子库在96孔板，或筛选肽适体库的固体介质。 细胞周期蛋白D1的特异性抑制剂可以在乳腺癌细胞培养物和小鼠模型中进一步测试，并且可以用作neu-、ras-和/或细胞周期蛋白D1-相关乳腺癌的研究工具和潜在治疗剂。

DESCRIPTION (provided by applicant): Experiments with knockouts of the genes encoding the D-type cyclins demonstrate that breast cancers induced by the neu or ras oncogenes in mice are absolutely dependent on cyclin Dl. Most human breast cancers overexpress cyclin Dl: approximately 50% of human breast cancers have amplified or otherwise overexpress c-neu, which depends on cyclin Dl for cell-cycle activation, and approximately 20% of human breast cancers have amplified cyclin Dl. A logical approach to the treatment of neu-, ras-, and/or cyclin ZX?-associated breast cancers would be to inhibit cyclin Dl function. Because knocking out cyclin Dl has a limited impact on adult mouse physiology, specific inhibition of cyclin Dl would likely have a very favorable therapeutic index. Cyclin Dl functions by direct interaction with other proteins. Protein-protein interactions have been inhibited successfully using small molecules identified in library screens. To maximize therapeutic index, screens to identify specific inhibitors of cyclin Dl interactions should be designed in such a way as to exclude inhibitors of cyclin D2 and cyclin D3 interactions. Given the sequence divergence among the D-type cyclins, it should be possible, using reverse two-hybrid screening in yeast, to identify inhibitors of cyclin Dl interactions that do not inhibit cyclin D2 and cyclin D3 interactions. The screens in this proposal are designed to select for inhibitors of cyclin Dl interactions by linking these interactions to the expression of genes conditionally toxic to yeast. To exclude inhibitors of cyclin D2 and cyclin D3, the interactions of these cyclins will be linked to the expression of genes conditionally required for yeast to grow. Yeast have been used successfully in drug screens, as they are permeable to compounds in chemical libraries, which average less than 500 Da. Reverse two-hybrid screens are straightforward to construct and validate, and can be adapted to high-throughput screening of small-molecule libraries in 96-well plates, or to screening peptide-aptamer libraries on solid media. Specific inhibitors of cyclin Dl could be tested further in mammalian-cell-culture and mouse models, and would be useful both as research tools and as potential therapeutic agents for neu-, ras-, and/or cyclin Dl -associated breast cancers.