Small-Molecule Antagonists of Ral GTPases in Cancer

 描述（由申请人提供）：Ral GTP酶调节的信号传导网络控制多种细胞过程，例如胞吐作用、胞吞作用、转录因子激活、 和肌动蛋白细胞骨架重组。 大量证据，包括我们自己的工作，支持Ral在Ras驱动的癌症如非小细胞肺癌（NSCLC）中的作用。 我们发现，一个小分子（BQU 57），通过促进其GDP结合状态的灭活Ral显着损害非小细胞肺癌的增殖在锚定依赖性和独立的测定。 相对于GTP酶Ras和RhoA，BQU 57对Ral具有选择性，并且抑制肿瘤异种移植物生长的程度与使用RNA干扰消耗Ral的程度相似。 除了BQU 57之外，我们还鉴定了Ral及其效应蛋白RalBP 1之间的蛋白质-蛋白质相互作用的小分子正构拮抗剂。 该化合物损害NSCLC细胞中的Ral活化，并在锚定依赖性和非依赖性测定中抑制增殖。 我们的长期目标是开发适用于临床治疗NSCLC的小分子Ral拮抗剂。 我们的短期目标是开发我们的两个亲本结构的衍生物，以探测Ral在体内的功能，并为有前途的化合物的临床研究奠定基础。 我们的中心假设是，Ral拮抗剂衍生物的结构指导的计算设计将导致小分子表现出更高的亲和力，更好的药代动力学（PK）特性和更大的体内功效。 我们的初步数据有力地证明了我们的假设。 BQU 57已经在体内表现出功效，并且我们的正构抑制剂显示出对NSCLC中的Ral活性和细胞活力的有希望的抑制。 在我们的第一个目标中，我们采用基于结构的计算方法和吸收，分布，代谢和排泄（ADME）预测来设计每一类小分子的衍生物。 在第二个目标中，我们每年合成20-25种衍生物，用于两种结构类别中的每一种，并使用生物化学和生物物理方法评估这些化合物的结合和抑制作用。 1H-15 N横向弛豫优化（TROSY）NMR光谱和X射线晶体学用于提供直接结合的结构证据。 在第三个目标中，我们评估化合物对Ral激活的影响，并使用锚定依赖性和独立测定法评估其在一组NSCLC细胞系中的活性。 我们为两类Ral拮抗剂中的每一类选择最有前途的化合物，并表征体外稳定性和体内PK参数。 将首先使用人类异种移植物进行功效研究，然后在转基因小鼠模型中评价最有希望的化合物。 我们还进行了Ral拮抗剂与Ras信号通路抑制剂的联合研究。 我们预期这项工作将导致具有纳摩尔范围亲和力的小分子Ral拮抗剂，其是口服生物可利用的，具有合适的PK性质，并且在阻断Ral驱动的肿瘤形成和生长中表现出显著的功效。

 DESCRIPTION (provided by applicant): Ral GTPase-regulated signaling networks control diverse set of cellular processes such as exocytosis, endocytosis, transcription factor activation, and actin cytoskeletal reorganization. Substantial evidence, including from our own work, supports a role for Ral in Ras-driven cancers such as non-small cell lung cancer (NSCLC). We found that a small molecule (BQU57) that inactivates Ral by promoting its GDP-bound state significantly impaired NSCLC proliferation in anchorage-dependent and independent assays. BQU57 was selective for Ral relative to the GTPases Ras and RhoA and inhibited tumor xenograft growth to a similar extent to the depletion of Ral using RNA interference. In addition to BQU57, we identified a small-molecule orthosteric antagonist of the protein-protein interaction between Ral and its effector protein RalBP1. The compound impairs Ral activation in NSCLC cells and inhibits proliferation in anchorage-dependent and independent assays. Our long-term objective is to develop small-molecule Ral antagonists that are suitable for treating NSCLC in the clinic. Our short-term objective is to develop derivatives of our two parental structures to probe Ral function in vivo and set the stage for clinical investigation of promising compounds. Our central hypothesis is that structure-guided computational design of derivatives of Ral antagonists will lead to small molecules that exhibit higher affinity, better pharmacokinetic (PK) properties and greater efficacy in vivo. Our preliminary data strongly positions us to test our hypothesis. BQU57 already exhibits efficacy in vivo and our orthosteric inhibitors show promising inhibition of Ral activity and cell viability in NSCLC. In our first aim, we employ structure-based computational methods and absorption, distribution, metabolism, and excretion (ADME) predictions to design derivatives for each class of small molecules. In the second aim, we synthesize 20-25 derivatives every year for each of the two structural classes and evaluate these compounds for binding and inhibition using biochemical and biophysical approaches. 1H-15N transverse relaxation-optimized (TROSY) NMR spectroscopy and X-ray crystallography are used to provide structural evidence of direct binding. In the third aim, we evaluate compounds for their effect on Ral activation, and assess their activity in a panel of NSCLC cell lines using anchorage-dependent and independent assays. We select the most promising compound for each of the two classes of Ral antagonists and characterize stability in vitro and PK parameters in vivo. Efficacy studies will be carried out first using human xenografts, followed by evaluation in transgenic mouse models for the most promising compounds. We also perform combination studies of our Ral antagonists with inhibitors of Ras signaling pathways. We expect that this work will lead to small-molecule Ral antagonists with nanomolar range affinity that are orally bioavailable, possess suitable PK properties, and exhibit substantial efficacy in blocking Ral driven tumor formation and growth.