Identification of chemical probes that specifically disrupt the GIV-Gi interface

描述（由申请人提供）：通过异源三聚体（以下简称三聚体）G蛋白的信号传导调节大多数（如果不是全部）生理功能，其失调是大量疾病的原因。超过30%的市售药物靶向G蛋白偶联受体（GPCR），这是三聚体G蛋白的经典激活剂。我们最近发现了一种新的信号转导机制，其中三聚体G蛋白被称为GIV（又名Girdin）的细胞质非受体蛋白而不是GPCR激活。这种机制具有重要的生物医学意义，因为它的上调与转移的发展密切相关。尽管转移导致约90%的癌症相关死亡，但这一过程仍然知之甚少，并且仍然无法治愈。因此，GIV激活G蛋白的一种新的、不依赖于受体的机制的存在不仅为G蛋白信号调节提供了新的视角，而且为药物干预开辟了新的机会。然而，没有现有的化学探针靶向GIV-G蛋白偶联。我们的目标是鉴定特异性抑制GIV-G蛋白相互作用的化学探针作为研究工具，以进一步了解G蛋白信号转导的新机制及其在疾病中的影响。背景技术背景：先前的工作已经确定GIV中约25 aa的短线性基序负责其作为G蛋白激活剂（鸟嘌呤）的生物活性 核苷酸交换因子，GEF）和作为肿瘤细胞中促转移行为的增强剂。我们已发表和未发表的数据提供了丰富的结构信息，表明GIV-G蛋白相互作用是一个易于处理的分子界面，可以特异性靶向。两者合计，这些结果提供了原则的证明，考虑GIV-G？I interface接口a bona真正molecular分子target目标in signaling信号and cancer癌症metastasis转移.我们的假设是，化学探针，专门抑制GIV-G？i接口将为研究转移过程中G蛋白激活的新机制提供有价值的研究工具，并为未来的靶向治疗奠定基础。研究报告：我们已确定了该活动所需的所有测定，包括中试筛选（Z 'e0.7）中筛选/反筛选测定的验证。在SA#1中，我们将使用荧光偏振（FP）对> 150，000种化合物进行高通量筛选（HTS）。命中将在基于AlphaScreen（R）的计数器筛选测定中进行测试，以消除假阳性，并将通过化合物的质量控制和药物化学评估进行分类。在SA#2中，我们将在酵母和哺乳动物细胞中进行后续试验，以评估疗效、特异性和毒性。过滤的化合物和类似物优化的效力将最终测试其阻断肿瘤细胞迁移的能力，作为替代措施的生物活性的目标（GIV-G？i）转移。HTS和试验自动化将在ICCB-Longwood筛选机构（哈佛医学院）进行，分类命中和合成类似物所需的药物化学工作将由共同研究者Aaron Beeler（波士顿大学）进行。

DESCRIPTION (provided by applicant): Signaling via heterotrimeric (henceforth trimeric) G proteins regulates most, if not all, physiological functions and its dysregulation is the cause of vast array of diseases. More than 30% of marketed drugs target G protein-coupled receptors (GPCRs), which are the classical activators of trimeric G proteins. We have recently identified a novel mechanism of signal transduction in which trimeric G proteins are activated by a cytoplasmic, non-receptor protein called GIV (aka Girdin) instead of by GPCRs. This mechanism has important biomedical implications because its upregulation is tightly associated with the development of metastasis. Despite that metastasis causes ~90% of cancer-related deaths, this process is still poorly understood and remains incurable. Thus, the existence of a novel, receptor-independent mechanism of G protein activation by GIV not only provides a new perspective on G protein signaling regulation but also opens new opportunities for pharmacological intervention. However, no existing chemical probe targets the GIV-G protein coupling. Our goal is to identify chemical probes that specifically inhibit the GIV-G protein interaction as research tools to further understand a novel mechanism of G protein signaling and its impact in disease. BACKGROUND: Previous work has established that a short linear motif of ~25 aa in GIV is responsible for its biological activity as a G protein activator (Guanine nucleotide Exchange Factor, GEF) and as an enhancer of prometastatic behavior in tumor cells. Our published and unpublished data provide a wealth of structural information indicating that the GIV-G protein interaction is a tractable molecular interface that can be specifically targeted. Taken together, these results provide the proof of principle to consider the GIV-G?i interface a bona fide molecular target in signaling and cancer metastasis. Our hypothesis is that chemical probes that specifically inhibit the GIV-G?i interface will be valuable research tools to investigate a novel mechanism of G protein activation during metastasis and will lay the foundation for future targeted therapies. RESEARCH PLAN: All the assays required for this campaign have been established in our hands, including validation of the screen/ counter-screen assays in a pilot screen (Z'e0.7). In SA#1 we will carry out a high throughput screen (HTS) of >150,000 compounds using a fluorescence polarization (FP). Hits will be tested in a counter-screen assay based on AlphaScreen(R) to remove false positives and will be triaged by quality control of compounds and medicinal chemistry assessments. In SA#2 we will perform follow-up assays in yeast and mammalian cells to assess efficacy, specificity and toxicity. Filtered compounds and analogs optimized for potency will be eventually tested for their ability to block tumor cell migration as a surrogate measure of the bioactivity of the target (GIV-G?i) in metastasis. The HTS and assay automations will be performed at the ICCB-Longwood screening facility (Harvard Medical School) and the medicinal chemistry efforts required to triage hits and synthesize analogs will be carried by the co-investigator Aaron Beeler (Boston University).