Targeting Ral GTPases in Bladder Cancer

转移性膀胱癌（BC）治疗的最后一次重大进展发生在1997年吉西他滨的出现。尽管有这种进展，但内脏转移通常是致命的。拟议研究的总体目标是开发小分子抑制剂，阻断转移过程中的关键节点。我们发现Rai GTPases作为治疗上易于处理的信号通路的分子开关，使UC细胞在肺（最常见的内脏转移部位）中生长。该途径的临床意义和Rai作为治疗靶点的有效性得到了支持，发现肿瘤中Rai的高表达使患者具有更高的转移风险，并且在UC动物模型中发生肺转移需要Rai的表达。我们的指导假设是小分子靶向Rai为转移性UC提供有效的治疗。在MD Anderson膀胱孢子发育研究项目（DRP）的支持下，我们通过计算和组合筛选评估了bb50万种化合物结合RalA或RalB的能力，并选择了99种“命中”。这些在一系列的二级分析中进行了评估，使我们能够选择Rai结合化合物（RUC）8和10在本应用中进行研究。选择RUC8和10的原因是：1)在人UC细胞中抑制RalA与RalBPI的结合，并诱导RalA在小鼠胚胎成纤维细胞中扩散；2)抑制人UC细胞体外单层生长（IC50 0.5 ~ 1.9 pM）；3)通过核磁共振（NMR）直接结合RalB；4)在小鼠体内具有良好的药代动力学（PK）特性（Cmax 1.3-23 pM, T1/2 3.7-4.6 hrs）。为了开发这类新型药物，我们提出了以下具体目标：目标1：利用药物化学、基于计算片段的设计和化学数据库的相似性搜索，基于RUC8和10表征效力更高的第二代化合物。在不太可能的情况下，在Aim 1中没有发现更有效的化合物，我们将使用RUC8和10进行Aim 2和3，因为它们具有足够的IC50和体内PK。目标2：评估第二代化合物在新型人类UC内脏转移模型中的体内治疗效果。目标3：在人体组织中开发对安曲拉尔治疗反应的预测性生物标志物，这将使我们在项目结束时进行i期试验。Astra Zeneca在我们的工作中记录的兴趣提高了将我们的新型Rai抑制剂转化为临床抗癌治疗药物的整体成功机会。

The last major advance in the treatment of metastatic bladder cancer (BC) took place in 1997 with the advent of gemcitabine. Despite this advance, visceral metastases are usually fatal. The overall goal of the proposed studies is to develop small molecule inhibitors that block a critical node in the metastatic process. We found that Rai GTPases serve as the molecular switches of a therapeutically tractable signaling pathway that allows UC cells to grow in the lung, the most common visceral metastatic site. The clinical significance of this pathway and validity of Rai as a therapeutic target is supported by finding that high Rai expression in tumors places patients at higher risk for metastasis and the requirement of Rai expression for lung metastasis to occur in animal models of UC. Our Guiding Hypothesis for this application is that small molecules targeting Rai provide effective therapy for metastatic UC. With support from the MD Anderson Bladder SPORE Developmental Research Program (DRP), we evaluated >500K compounds for their ability to bind RalA or RalB in computational and combinatorial screens and selected 99 "hits". These were evaluated in a series of secondary assays allowing us to select Rai Binding Compound (RUC)8 and 10 to be pursued in this application. RUC8 and 10 were selected because they: 1) inhibit RalA to RalBPI binding in human UC cells and RalA induced spreading in murine embryo fibroblasts; 2) inhibit in vitro monolayer growth (IC50 0.5-1.9 pM) of human UC cells; 3) bind RalB directly by nuclear magnetic resonance (NMR) spectroscopy; and 4) have good pharmacokinetic (PK) properties in mice (Cmax 1.3-23 pM, T1/2 3.7-4.6 hrs). To develop this novel class of agents we propose the following Specific Aims: Aim 1: Characterize higher potency 2nd generation compounds based on RUC8 and 10 using medicinal chemistry, computational fragment-based design, and similarity search of chemical databases. In the unlikely situation that higher potency compounds are not found in Aim 1, we will pursue Aim 2 and 3 using RUC8 and 10, given their adequate IC50 and in vivo PK. Aim 2: Evaluate 2nd generation compounds for their in vivo therapeutic efficacy in novel human UC models of visceral metastasis. Aim 3: Develop predictive biomarkers of response to antlRal therapeutics in human tissues that will position us for Phase 1 trials by end of this project. Documented interest by Astra Zeneca in our work improves overall chances for success in translating our novel Rai inhibitors into the clinical setting as anticancer therapeutics.