Targeting of non-canonical G protein signaling with small molecules

异源三聚体G蛋白信号转导的重要性 生理和疾病的反映是G蛋白偶联受体(GPCRs)，它激活G 在FDA批准的药物中，蛋白质是超过25%的药物靶标。有趣的是，我们和其他人最近的工作 导致了一种新的三聚体G蛋白激活机制的发现，这种机制是由细胞质， 非受体蛋白，而不是膜结合型GPCRs。尽管这一机制具有重要的 对人类疾病的影响和针对它是开发治疗方法的新机会，它 从药理学的角度来看，仍然是完全未开发的。我们的目标是进行 GPCR一类小分子抑制剂早期药物开发的概念研究 G蛋白激活促进肿瘤转移的独立机制。更确切地说，我们的 努力将重点放在开发和表征破坏蛋白质-蛋白质的小分子上 G蛋白GαI与其非受体激活剂GIV(又名Girdin)之间形成的相互作用。许多 独立研究表明，GIV在转移性癌中表达上调。在GIV上 过表达，GIV-GαI PPI增强信号反应，导致肿瘤细胞迁移和 入侵。因此，抑制GIV-GαI PPI是转移性肿瘤细胞的一种脆弱性，可能提供 治疗侵袭性转移性癌症的窗口。这一点意义重大，因为 转移是癌症相关死亡的90%的原因，治疗它的选择非常有限。 初步数据和实验计划：在最近发表的工作中，我们描述了 他对GIV-GαI界面的结构进行了研究，并证明了它可以被小分子破坏。我们有 随后进行了20万种化合物的筛选。我们确认了主屏幕中确认的点击数 通过正交生化分析，筛选出具有化学易感性的化合物并进行验证 在重新购买/重新合成后进行分析。在生物检测中进行评估后，我们发现了小分子 基于四个无关的、合成的易处理的支架的分子，这些支架破坏了GIV-GαI PPI与IC_(50)‘S在 在~0.5-30微米范围内，且显示出预期生物活性 有害的非特异性毒性。我们推测，这些化合物选择性地干扰GIV-GαI PPI以 阻断这种PPI驱动肿瘤侵袭性的信号和细胞行为过程，并在 优化后，这些化合物将在临床前转移模型中具有治疗作用。在目标1中，我们 将利用生化手段全面表征新发现的抑制剂的作用模式， 生物物理和基于细胞的方法，而在目标2中，我们将优化我们最有希望的化合物 增强其药效和类药物特性，以达到在小鼠肿瘤转移模型中的治疗效果。

The high importance of signaling via heterotrimeric G proteins in physiology and disease is reflected by the fact that G protein-coupled receptors (GPCRs), which activate G proteins, are the target for more that >25% of FDA-approved drugs. Interestingly, recent work by us and others has led to the identification a novel mechanism of trimeric G protein activation that is mediated by cytoplasmic, non-receptor proteins instead of membrane-bound GPCRs. Although this mechanism has important implications in human disease and targeting it is a novel opportunity to develop therapeutic approaches, it remains completely unexploited form a pharmacological standpoint. Our goal is to carry out proof-of- concept studies for early stage drug development of first-in-class small molecule inhibitors of a GPCR- independent mechanism of G protein activation that promotes cancer metastasis. More specifically, our efforts will be focused on developing and characterizing small molecules that disrupt the protein-protein interaction (PPI) formed between the G protein Gαi and its non-receptor activator GIV (aka Girdin). Many independent studies have demonstrated that GIV is upregulated in metastatic carcinomas. Upon GIV overexpression, the GIV-Gαi PPI enhances signaling responses that lead to increased tumor cell migration and invasion. Thus, inhibition of the GIV-Gαi PPI is a vulnerability of metastatic tumor cells that might provide a therapeutic window to treat aggressive metastatic cancers. This is of paramount significance because metastasis is the cause of >90% of cancer related deaths and there are very limited therapeutic options for it. PRELIMINARY DATA AND EXPERIMENTAL PLAN: In recently published work, we have characterized the structure of the GIV-Gαi interface and demonstrated that it can be disrupted by small molecules. We have subsequently performed a screen of 200,000 compounds. We confirmed hits identified in the primary screen with an orthogonal biochemical assay, filtered out compounds with chemical liabilities and validated them analytically after re-purchase/re-synthesis. After evaluation in biological assays, we have identified small molecules based on four unrelated, synthetically tractable scaffolds that disrupt the GIV-Gαi PPI with IC50's in the ~0.5-30 µM range and that display the expected biological activity of blocking tumor cell migration without undesired non-specific toxicity. We hypothesize that these compounds selectively disrupt the GIV-Gαi PPI to block the signaling and cell behavioral processes by which this PPI drives tumor invasiveness, and that upon optimization, these compounds will have therapeutic effects in pre-clinical models of metastasis. In Aim 1 we will comprehensively characterize the mode of action of the newly identified inhibitors using biochemical, biophysical and cell-based approaches, while in Aim 2 we will optimize the most promising of our compounds to increase its potency and druglike properties to achieve therapeutic effects in mouse models of metastasis.