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部分（如果不是全部）生理功能，其失调是大量疾病的原因。超过30%的上市药物靶向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蛋白被一种称为GIV（又名Girdin）的细胞质非受体蛋白激活，而不是由gpcr激活。这一机制具有重要的生物医学意义，因为它的上调与转移的发生密切相关。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，一种新的、不依赖受体的GIV激活G蛋白的机制的存在，不仅为G蛋白信号调控提供了新的视角，也为药物干预开辟了新的机会。然而，目前还没有针对GIV-G蛋白偶联的化学探针。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蛋白信号传导的新机制及其在疾病中的影响。背景：先前的研究已经确定，GIV中~ 25aa的短线性基序是其作为G蛋白激活剂（鸟嘌呤）的生物活性的原因

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 Our published and unpublished data provide a wealth of structural information indicating that the GIV-G protein interaction is a tractable molecular interface that can be specifically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).