Small-Molecule Antagonists of Ral GTPases in Cancer

 描述(申请人提供)：Ral GTP酶调节的信号网络控制多种细胞过程，如胞吐、内吞、转录因子激活、 和肌动蛋白的细胞骨架重组。大量证据，包括我们自己的工作，支持RAR在RAS驱动的癌症中的作用，如非小细胞肺癌(NSCLC)。我们发现，在锚定依赖和独立分析中，一个小分子(BQU57)通过促进其GDP结合状态来灭活Ral，显著削弱了NSCLC的增殖。BQU57是相对于Ras和RhoA的GTP酶选择性的Ral，抑制肿瘤移植瘤生长的程度与使用RNA干扰去除Ral的程度相似。除BQU57外，我们还鉴定了一种小分子正构体拮抗剂，该拮抗剂可以抑制Ral及其效应蛋白RalBP1之间的蛋白质-蛋白质相互作用。在锚定依赖和非锚定实验中，该化合物可损害NSCLC细胞的RAR活性并抑制其增殖。我们的长期目标是开发适合于临床治疗非小细胞肺癌的小分子受体拮抗剂。我们的短期目标是开发我们的两种亲本结构的衍生物，以探索在体内的al功能，并为有前景的化合物的临床研究奠定基础。我们的中心假设是，结构导向的计算设计的受体拮抗剂的衍生物将导致小分子表现出更高的亲和力，更好的药代动力学(PK)性质和更好的体内疗效。我们的初步数据有力地证明了我们的假设。BQU57已经在体内显示出疗效，我们的邻位构型抑制剂显示出良好的抑制非小细胞肺癌细胞活性和细胞活力的前景。在我们的第一个目标中，我们使用基于结构的计算方法和吸收、分布、代谢和排泄(ADME)预测来为每一类小分子设计衍生物。在第二个目标中，我们每年为两个结构类别中的每一个合成20-25个衍生物，并使用生化和生物物理方法评估这些化合物的结合和抑制作用。用1H-15N横向弛豫优化(TROSY)核磁共振谱和X射线结晶学提供了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，并使用锚定依赖和独立分析评估它们在一组非小细胞肺癌细胞系中的活性。我们为这两类受体拮抗剂中的每一类选择了最有前景的化合物，并对其体外稳定性和体内PK参数进行了表征。有效性研究将首先使用人类异种移植进行，然后在转基因小鼠模型中评估最有希望的化合物。我们还进行了我们的RAR拮抗剂与RAS信号通路抑制剂的联合研究。我们希望这项工作将导致具有纳摩尔范围亲和力的小分子RAL拮抗剂的口服生物利用，具有合适的PK特性，并在阻断RAL驱动的肿瘤形成和生长方面显示出实质性的效果。

 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.