Screening-based design, synthesis and testing of potential Gα protein inhibitors as chemical probes for GPCR signaling

基于筛选的设计、合成和测试潜在的 Gα 蛋白抑制剂作为 GPCR 信号化学探针

• 批准号: 431459858
• 负责人: Professorin Dr. Diana Imhof, Ph.D.
• 金额: --
• 依托单位: Abteilung Pharmazeutische Biochemie und Bioanalytik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/431459858?language=en
• 关键词: Screening; based; design; synthesis; testing

Since decades heterotrimeric G proteins are known as key mediators for signal transduction in cells. It is, however, surprising that there are only few compounds available to selectively modulate their activity. From a pharmacological point of view this is still a major drawback concerning the study of the impact of individual G proteins and their subunits on signal transduction in varying physiological situations. Thus, there is a clear demand for inhibitors for the difficult or less druggable G alpha proteins to provide scientists with tools to study their interactions, mechanism of action, and crosstalks in signaling.As a first approach towards G protein inhibitor development we have established and performed a study of screening linear and (bi)cyclic combinatorial peptide libraries against Gi and Gs to first fulfill criteria such as feasibility and robustness of the strategy, while meeting the requirements for binding specificity and selectivity of potential G alpha protein ligands. In the subsequent step, to be pursued herein, one of the biggest challenge in this research will be addressed, i.e. the screening of three different Gα proteins (Gi, Gs, G12/13) in different conformational states (inactive vs. active) for at least two different cyclic peptide libraries. This will dramatically increase the outcome regarding potential specific ligands for the individual G alpha proteins. In a combination of different techniques and bioassays (binding studies, 2nd messenger production on membrane preparations, cell-based assays) the hits will be evaluated and classified according to their binding affinity and inhibitory activity. The top-ranked hits will be subjected to in-depth structural studies (NMR spectroscopy, molecular modeling, molecular docking) to obtain information about their three-dimensional structure and supposed binding site at the respective G alpha protein. Finally, compounds matching the aforementioned criteria will pass an optimization procedure in order to also improve features such as cell permeability and proteolytic stability. Apart from the mentioned experiments, recombinant expression of G12/13 is to be established in the course of the envisaged project to allow for investigation of this G alpha subunit, while protocols for Gi and Gs production were already established in our preliminary work. Altogether, this proposal aims at developing new tools for the inhibition of Gα proteins (Gi, Gs, G12/13, Gi) by combinatorial approaches which significantly increases the chance to succeed in finding promising candidates to study G protein mediated signal transduction pathways and, in turn, provides lead structures for drug development in the field of GPCR/G-protein related-diseases.

几十年来，异三聚体 G 蛋白被认为是细胞信号转导的关键介质。然而，令人惊讶的是，只有很少的化合物可用于选择性调节其活性。从药理学的角度来看，这仍然是研究单个 G 蛋白及其亚基在不同生理情况下对信号转导的影响的主要缺点。因此，对难以或难以成药的 G α 蛋白的抑制剂有明确的需求，为科学家提供研究其相互作用、作用机制和信号传导串扰的工具。作为 G 蛋白抑制剂开发的第一个方法，我们已经建立并进行了一项针对 Gi 和 Gs 筛选线性和（双）环组合肽库的研究，以首先满足诸如可行性和 该策略的稳健性，同时满足潜在 G α 蛋白配体的结合特异性和选择性的要求。在本文要追求的后续步骤中，将解决本研究中最大的挑战之一，即筛选不同构象状态（非活性与活性）的三种不同的 Gα 蛋白（Gi、Gs、G12/13）以获得至少两个不同的环肽库。这将极大地提高有关单个 G α 蛋白的潜在特异性配体的结果。结合不同的技术和生物测定（结合研究、膜制剂上的第二信使产生、基于细胞的测定），将根据其结合亲和力和抑制活性对命中进行评估和分类。排名靠前的热门产品将接受深入的结构研究（核磁共振波谱、分子建模、分子对接），以获得有关其三维结构和各自 G α 蛋白的假定结合位点的信息。最后，符合上述标准的化合物将通过优化程序，以改善细胞通透性和蛋白水解稳定性等特性。除了上述实验之外，G12/13 的重组表达将在设想的项目过程中建立，以便研究该 G α 亚基，而 Gi 和 Gs 生产的方案已经在我们的前期工作中建立。总而言之，该提案旨在通过组合方法开发抑制 Gα 蛋白（Gi、Gs、G12/13、Gi）的新工具，从而显着增加成功找到有希望的候选者以研究 G 蛋白介导的信号转导途径的机会，进而为 GPCR/G 蛋白相关疾病领域的药物开发提供先导结构。