Abstract：
Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release-activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.
摘要：
许多生理功能，如细胞分化，扩散,肌肉收缩,神经传递和受精，受Ca2+水平变化的调节。细胞中储存的主要Ca2+是内质网(ER)。某些细胞过程诱导内质网储存耗尽，例如通过激活IP3受体，这又引起了称为存储操作钙进入(SOCE)的存储再填充过程。这种重新填充过程需要ER膜中的Ca2感应基质相互作用分子（STIM）和质膜中的Orai蛋白之间的蛋白质-蛋白质相互作用。完全组装的STIM/Orai复合物然后形成高度选择性的Ca2通道，称为Ca2释放激活的Ca2通道（CRAC），Ca2离子通过该通道流入细胞质，然后通过肌质网/内质网钙ATP酶（SERCA）泵入ER。SOCE异常与许多人类疾病和癌症有关，因此，关键参与者STIM和Orai吸引了巨大的治疗兴趣。已经开发了几种有效的实验和临床候选化合物，并有助于研究各种细胞类型中的SOCE。我们基于已知的SOCE抑制剂GSK-7975A合成了多种新型小分子探针。这里我们介绍GSK-7975A衍生物，具有照片锁定功能，光交联，生物素和可点击部分，还含有氘标记。使用荧光成像板读数器（FLIPR）-基于Tetra的Ca2成像测定对这些GSK-7975A探针的评估表明，大多数合成修饰对SOCE抑制活性没有不利影响。光笼GSK-7975A也用于膜片钳电生理学实验。总之,我们开发了一些活跃的，基于GSK-7975A的分子探针具有有趣的特性，因此是在各种细胞和环境中研究SOCE的有用实验工具。
