Small-Molecule Antagonists of Ral GTPases in Cancer

癌症中 Ral GTP 酶的小分子拮抗剂

• 批准号: 9236177
• 负责人: Samy Meroueh
• 金额: $ 59.09万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9236177
• 关键词: Actins; Affinity; Binding; Bioavailable; Biochemical; Biological Assay; Biology; Biophysics; Blood - brain barrier anatomy; Caco-2 Cells; Calorimetry; Cancer cell line; Cell Proliferation; Cell Survival; Cell physiology; Cells; Clinic; Clinical Trials; Computing Methodologies; Cultured Cells; Cytoskeletal Modeling; Data; Development; Docking; Drug Kinetics; Endocytosis; Ensure; Enzyme-Linked Immunosorbent Assay; Evaluation; Excretory function; Exhibits; Exocytosis; Fluorescence Polarization; Free Energy; GTP Binding; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Impairment; In Vitro; Investigational Drugs; KRAS2 gene; Lead; Libraries; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of urinary bladder; Mediating; Metabolic; Metabolism; Mission; Mutation; NMR Spectroscopy; Non-Small-Cell Lung Carcinoma; Oral; Parents; Patients; Penetration; Permeability; Positioning Attribute; Process; Property; Proteins; Public Health; RALGDS gene; RNA Interference; Ras Inhibitor; Ras Signaling Pathway; Relaxation; Risk; Role; Signal Transduction; Solubility; Solvents; Structure; Surface Plasmon Resonance; Testing; Therapeutic; Therapeutic Agents; Titrations; Toxic effect; Transgenic Mice; United States National Institutes of Health; Work; X-Ray Crystallography; Xenograft Model; Xenograft procedure; absorption; base; biophysical techniques; cancer cell; clinical investigation; combinatorial; design; efficacy study; expectation; flexibility; in vivo; inhibitor/antagonist; lung tumorigenesis; mortality; mouse model; nanomolar; novel; protein protein interaction; public health relevance; receptor; screening; small molecule; small molecule inhibitor; tool; transcription factor; tumor; tumor growth; tumor xenograft; virtual

 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.

 描述（由申请人提供）：Ral GTPase 调节的信号网络控制多种细胞过程，例如胞吐作用、内吞作用、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)）中发挥作用。 我们发现，在锚定依赖性和独立测定中，一种小分子（BQU57）通过促进 Ral 的 GDP 结合状态来灭活 Ral，显着损害 NSCLC 增殖。 相对于 GTPases Ras 和 RhoA，BQU57 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 干扰消除 Ral 的程度相似。 除了 BQU57 之外，我们还鉴定了 Ral 及其效应蛋白 RalBP1 之间蛋白质-蛋白质相互作用的小分子正构拮抗剂。 该化合物会损害 NSCLC 细胞中的 Ral 活化，并在贴壁依赖性和独立测定中抑制增殖。 我们的长期目标是开发适合临床治疗NSCLC的小分子Ral拮抗剂。 我们的短期目标是开发两个亲本结构的衍生物，以探测 Ral 的体内功能，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，Ral 拮抗剂衍生物的结构引导计算设计将产生具有更高亲和力、更好的药代动力学 (PK) 特性和更大体内功效的小分子。 我们的初步数据使我们能够检验我们的假设。 BQU57 已经在体内表现出功效，我们的正构抑制剂在 NSCLC 中显示出对 Ral 活性和细胞活力的有希望的抑制作用。 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) 预测来设计每类小分子的衍生物。 在第二个目标中，我们每年为两个结构类别合成 20-25 个衍生物，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-15N 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。 第三个目标是，我们评估化合物对 Ral 激活的影响，并使用贴壁依赖性和独立测定法评估它们在一组 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。 首先将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估最有前途的化合物。 我们还对 Ral 拮抗剂与 Ras 信号通路抑制剂进行联合研究。 我们预计这项工作将产生具有纳摩尔范围亲和力的小分子 Ral 拮抗剂，这些拮抗剂可口服生物利用，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表现出显着功效。