Rb-Raf1 Disrupters as Anti-Cancer Drugs

视网膜母细胞瘤肿瘤抑制蛋白Rb的失活对细胞周期的进展至关重要 从G1阶段到S阶段。Rb基因或其基因产物在多种癌症中失活，要么是通过 基因突变或蛋白质功能失活。在正常的细胞周期进程中，Rb是 通过与D和E型相关的激酶介导的一系列磷酸化事件而失活 旋风队。我们实验室的研究表明，生长因子对静止细胞的刺激会导致结合 细胞信号转导蛋白Raf-1与Rb蛋白结合。Raf-1与Rb的这种结合先于 Cyclins和CDKs，似乎对Rb随后的磷酸化是必不可少的。扰乱了 使用多肽抑制剂将Raf-1与Rb结合阻止了Rb的磷酸化，细胞周期进展， 裸鼠体内血管生成及肿瘤生长的研究。基于这些发现，我们建议确定和 开发能够破坏Rb-Raf-1相互作用的小分子。这类化合物可以预期会 具有抗增殖和抗肿瘤活性，可能用作抗癌剂。的确， 我们对药库的初步筛选使我们鉴定出了两个能够破坏 Raf-1与Rb的选择性结合，并具有合理的效力。他们在逮捕细胞方面很有效 其中一种化合物可抑制或延缓裸鼠体内肿瘤的生长。基于这些 结果，我们拟开展以下研究：(1)鉴定和合成小分子化合物 阻断Rb-Raf-1相互作用(2)体外评价Rb-Raf-1抑制剂的特异性和有效性(3) 评价Rb-Raf-1干扰物对细胞增殖和血管生成的影响。(四)评估效果 Rb-Raf-1干扰物对体内肿瘤生长和转移的影响。尼克·劳伦斯博士的实验室生病了 根据药物筛选的初始命中结果合成组合药物文库。额外的筛选 还将建立更大的文库，以确定比已确定的更有效的抑制剂。这些 化合物作为抗肿瘤药物的有效性将受到广泛的体外和体内分析。 此外，这些化合物将被用来研究早期细胞内发生的生物事件。 细胞周期的各个阶段。这些化合物还将被广泛用于研究其他信号通路。 在本PO-1申请的姊妹项目中进行了审查。

Inactivation of the retinoblastoma tumor suppressor protein, Rb, is essential for the progression of cell cycle from G1 to the S phase. Rb gene or its gene product is inactivated in a wide variety of cancers, either by mutation of the gene or functional inactivation of the protein. During normal cell cycle progression, Rb is inactivated by a cascade of phosphorylation events, mediated by kinases associated with the D and E type cyclins. Studies from our lab have shown that growth factor stimulation of quiescent cells leads to the binding of a cellular signaling kinase, Raf-1, to the Rb proteins. This binding of Raf-1 to Rb precedes the binding of cyclins and cdks and appears to be essential for the subsequent phosphorylation of Rb. Disruption of the binding of Raf-1 to Rb using a peptide inhibitor prevented Rb phosphorylation, cell cycle progression, angiogenesis as well as tumor growth in nude mice. Based on these findings, we propose to identify and develop small molecules that can disrupt the Rb-Raf-1 interaction. Such compounds can be expected to have anti-proliferative as well as anti-neoplastic activities and might be of use as anti-cancer agents. Indeed, our preliminary screen of a drug library led tot he identification of two small molecules capable of disrupting the binding of Raf-1 to Rb selectively and with reasonable potency. They were effective in arresting cell proliferation and one of the compounds could inhibit or retard tumor growth in nude mice. Based on these results, we propose to carry out the following studies: (1) To identify and synthesize small molecules to disrupt Rb-Raf-1 interaction (2) To assess the specificity and efficacy of the Rb-Raf-1 inhibitors in vitro (3) To evaluate the effects of Rb-Raf-1 disrupters on cell proliferation and angiogenesis. (4) To evaluate the effect of Rb-Raf-1 disrupters on tumor growth and metastasis in vivo. Dr. Nick Lawrence's laboratory w ill synthesize combinatorial drug libraries based on initial hits from the drug screen. Additional screening of larger libraries will also be done to identify more potent inhibitors than the ones already identified. These compounds will be subjected to extensive in vitro and in vivo analysis for their efficacy as anti-tumor drugs. Further, these compounds will be used to study the biological events taking place in the cell during early stages of cell cycle. These compounds will also be used extensively to study other signaling pathways examined in the sister projects of this PO-1 application.