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

几十年来，异源三聚体G蛋白被认为是细胞内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以选择性地调节它们的活性。从药理学的角度来看，这仍然是研究单个G蛋白及其亚单位在不同生理情况下对信号转导的影响的一个主要缺陷。因此，对难于或不易下药的Gα蛋白的抑制剂有明显的需求，为科学家提供工具来研究它们之间的相互作用、作用机制和信号传递中的串扰。作为开发G蛋白抑制剂的第一途径，我们建立并进行了针对GI和Gs的线性和(双)环组合多肽库的筛选研究，以首先满足该策略的可行性和稳健性等标准，同时满足潜在Gα蛋白配体的结合特异性和选择性的要求。在接下来的步骤中，将解决这项研究中最大的挑战之一，即为至少两个不同的环肽文库筛选三种不同构象状态(非活性与活性)的不同Gα蛋白(Gi、Gs、G12/13)。这将极大地提高关于单个Gα蛋白的潜在特定配体的结果。在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、基于细胞的分析)的组合中，将根据它们的结合亲和力和抑制活性对HITS进行评估和分类。排名靠前的HITS将接受深入的结构研究(核磁共振光谱、分子建模、分子对接)，以获得有关它们的三维结构和推测的Gα蛋白结合位置的信息。最后，符合上述标准的化合物将通过优化程序，以便也改善细胞渗透性和蛋白质降解稳定性等特征。除了上述实验外，G12/13的重组表达将在设想的项目过程中建立，以便对Gα亚基进行研究，而GI和Gs的生产方案已经在我们的初步工作中建立。综上所述，该建议旨在通过组合方法开发抑制Gα蛋白(GI、Gs、G12/13、GI)的新工具，从而显著增加成功地找到有前途的候选基因来研究G蛋白介导的信号转导途径的机会，并反过来为G蛋白/G蛋白相关疾病领域的药物开发提供主导结构。

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.