Deciphering the Inhibition of the Neuronal Calcium Sensor 1 and the Guanine Exchange Factor Ric8a with a Small Phenothiazine Molecule for the Rational Generation of Therapeutic Synapse Function Regulators

Protein–protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.蛋白质-蛋白质相互作用（Protein–protein interactions, PPIs）在神经元钙传感器1（neuronal calcium sensor 1, NCS-1）与鸟苷酸交换因子Ric8a（guanine exchange factor Ric8a）之间发挥不可或缺的调控作用，参与突触功能的调控，该相互作用已成为脆性X综合征（fragile X syndrome, FXS）等突触疾病的可药物靶向界面。近期研究发现吩噻嗪类化合物FD44可作为该PPIs的抑制剂，能够降低FXS动物模型中异常增多的突触数量，并增强其关联学习能力。本研究整合了先进的实验与计算研究手段，解析了果蝇NCS-1与FD44识别过程的关键结构信息，以阐明其亲和力与特异性的分子基础，并开发优化的PPIs调节剂。通过该研究，我们成功鉴定出一种新型类药小分子化合物IGS-1.76，该化合物可有效抑制人源NCS-1/Ric8a复合物，且结合效力得到提升。果蝇NCS-1/IGS-1.76复合物的晶体结构显示，尽管该新型抑制剂与FD44的化学结构存在差异，但二者具有相同的作用机制，可作为治疗FXS的新型候选命中化合物。