Targeting of non-canonical G protein signaling with small molecules

通过异源三聚体G蛋白的信号传导在 生理学和疾病的反应是由G蛋白偶联受体（GPCR），激活G蛋白偶联受体（GPCR）， 蛋白质是超过25%的FDA批准药物的靶点。有趣的是，我们和其他人最近的工作 已经导致鉴定了三聚体G蛋白激活的新机制， 非受体蛋白而不是膜结合的GPCR。虽然这一机制具有重要的 在人类疾病中的意义和靶向它是一个新的机会，开发治疗方法，它 从药理学的角度来看仍然完全未被开发。我们的目标是进行验证- 一流的GPCR小分子抑制剂早期药物开发的概念研究- G蛋白激活促进癌症转移的独立机制。更具体地说，我们的 将集中精力开发和表征破坏蛋白质-蛋白质的小分子， 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（PPI）。许多 独立的研究已经证明GIV在转移性癌中上调。关于GIV 过表达，GIV-Gαi PPI增强信号传导反应，导致肿瘤细胞迁移增加， 入侵因此，GIV-Gαi PPI的抑制是转移性肿瘤细胞的脆弱性，这可能提供了一种治疗方法。 治疗侵袭性转移性癌症的治疗窗口。这一点至关重要，因为 转移是>90%的癌症相关死亡的原因，并且其治疗选择非常有限。 初步数据和实验数据：在最近发表的工作中，我们描述了 GIV-Gαi界面的结构，并证明它可以被小分子破坏。我们有 随后进行了200，000种化合物的筛选。我们确认了在主屏幕上识别的匹配 通过正交试验筛选出化学成分不稳定的化合物，并对其进行验证 重新购买/重新合成后进行分析。在生物测定中评估后，我们已经鉴定了小的 分子基于四个不相关的，合成易处理的支架，破坏GIV-Gαi PPI，IC 50为 在~0.5-30 μM范围内，显示出预期的阻断肿瘤细胞迁移的生物活性， 不希望的非特异性毒性。我们假设这些化合物选择性地破坏GIV-Gαi PPI， 阻断PPI驱动肿瘤侵袭的信号传导和细胞行为过程， 通过优化，这些化合物将在转移的临床前模型中具有治疗效果。在目标1中， 将使用生物化学， 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前途的化合物 以增加其效力和类药性，从而在转移的小鼠模型中实现治疗效果。

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.