ABSTRACT

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein G_i. An in vitro [³⁵S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gα_i subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gα_s family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca²⁺ levels and ERK/MAPK phosphorylation, both of which are regulated by Gβγ, following its dissociation from Gα_i. Our observations suggest that NPD9055 targets Gα_i and thus regulates Gβγ-dependent cellular processes, most likely by causing the dissociation of Gβγ from Gα_i.

摘要

发现具有独特表型变化的小分子诱导剂并结合随后的靶标鉴定常常为细胞功能提供新的见解。在这里，我们将基于细胞形态学和蛋白质组学变化的综合分析应用于化合物筛选。我们鉴定了茚满衍生物NPD9055，其机理与具有已知作用方式的参考化合物在机理上不同。然后，采用化学蛋白质组学方法，我们表明NPD9055结合异三聚体G蛋白G _i的亚基。的体外[ ³⁵ S]GTPγS结合测定显示，NPD9055抑制上的GαGDP / GTP交换_我亚基通过G蛋白偶联型受体激动剂诱导，但不从另一G蛋白的Gα_小号家庭。在完整的HeLa细胞，诱导NPD9055胞内Ca增加²⁺水平和ERK / MAPK磷酸化，这两者都是通过Gβγ调节，以下从Gα其解离_我。我们的观察表明NPD9055目标Gα_我并由此调节Gβγ依赖的细胞过程，最有可能从Gα造成Gβγ的离解_我。