Bátora Daniel, Fischer Jérôme P, Kaderli Reto M, Varga Máté, Lochner Martin, Gertsch Jürg
Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland.
ACS Pharmacol Transl Sci. 2024 Apr 25;7(5):1557-1570. doi: 10.1021/acsptsci.4c00096. eCollection 2024 May 10.
The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency toward CaSR (EC < 45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in an FLIPR-based cellular functional assay. The specificity of the EvoSiR probes toward CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labeled the high CaSR expressing neuromasts of the lateral line . EvoSiR4 was also assessed in human parathyroid tissues , showing a specific absolute CaSR-associated fluorescence compared to that of parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small-molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.
钙敏感受体（CaSR）是一种广泛表达的G蛋白偶联受体（GPCR），可调节甲状旁腺中的细胞外钙信号。CaSR最近还与哮喘、肠道炎症和癌症等非降钙素相关的病理生理过程有关。迄今为止，缺乏能够对组织中的CaSR进行生物成像的分子工具。基于对CaSR配体现有构效关系数据的分析，我们设计并制备了临床批准药物依维卡塞的硅罗丹明（SiR）缀合物。在基于荧光成像板读数器（FLIPR）的细胞功能测定中，新探针EvoSiR4和EvoSiR6在依维卡塞羧基末端具有不同的连接子长度，与依维卡塞和依维卡塞-连接子缀合物相比，它们对CaSR的效力分别增加了6倍和3倍（EC<45 nM）。在活细胞实验中评估了EvoSiR探针与CaSR结合的特异性以及白蛋白的影响。两种探针均显示出强烈的白蛋白结合，这有助于清除非特异性结合相互作用。因此，在斑马鱼胚胎中，EvoSiR4特异性标记了侧线中高表达CaSR的神经丘。还在人甲状旁腺组织中评估了EvoSiR4，与甲状旁腺自发荧光相比，显示出与CaSR相关的特异性绝对荧光。总之，用SiR对依维卡塞进行功能化导致制备了高效且特异性的荧光CaSR探针。EvoSiR4是一种通用的小分子探针，可用于抗体不适用的与CaSR相关的生物医学分析。
