腺苷酸环化酶抑制剂体外筛选模型的建立及应用

Establishment and application of a screening model of adenylate cyclase inhibitor in vitro

腺苷酸环化酶(adenylate cyclase,AC)通过调节环磷酸腺苷(cyclic adenosine cyclophosphate,cAMP)的合成从而调控细胞相关功能.在卵巢中,cAMP调控卵母细胞的发育及卵子的成熟,cAMP含量变化影响下游芳香化酶的表达,进而影响雌激素的生物合成.为开发靶向AC的候选药物,根据AC1、AC2两个活性区域,利用大肠杆菌系统克隆表达可溶ⅠC1-ⅡC2重组融合蛋白,使用ATP检测试剂盒,检测ⅠC1-ⅡC2的催化活性,从而建立有效的ⅠC1-ⅡC2蛋白体外筛选抑制剂模型.利用该模型筛选了由2 861个化合物组成的文库.化合物来那替尼(neratinib)可明显抑制ⅠC1-ⅡC2蛋白活性,且呈浓度依赖性,其IC50为10.2±0.152 2μmol/L.进一步研究发现,来那替尼可显著降低人卵巢颗粒细胞KGN细胞中芳香化酶的表达,并且抑制雌激素的生物合成.本研究通过建立蛋白体外筛选模型发现来那替尼具有抑制AC的活性,结果可为开发新的靶向芳香化酶治疗多囊卵巢综合征等疾病的候选药物提供参考.(图5表1参28)

Adenylate cyclase(AC) affects cellular functions by regulating the synthesis of cyclic adenosine monophosphate(cAMP), which regulates oocyte development and follicle maturation in the ovary. Changes in cAMP content modulates the expression of aromatase and the biosynthesis of estrogen. Therefore, inhibition of AC by small molecules may provide new therapeutic means for the treatment of estrogen-related diseases. In order to develop candidate drugs targeting AC, an Escherichia coli system was used to clone and express soluble ⅠC1-ⅡC2 recombinant fusion proteins based on the two active regions of AC1 and AC2. The catalytic activity of ⅠC1-ⅡC2 was evaluated using an ATP detection kit. A chemical library consisting of 2 861 compounds was screened for inhibitors of ⅠC1-ⅡC2. The results showed that the compound neratinib significantly inhibited ⅠC1-ⅡC2 activity in a concentration-dependent manner, with an IC50 value of 10.2 ± 0.1522 μmol/L. Furthermore, neratinib significantly inhibited the expression of aromatase and the biosynthesis of estrogen in human ovarian granulose-like KGN cells. This study established a high-throughput screening model of ⅠC1-ⅡC2 protein to find a highly active AC inhibitor in vitro, providing a good experimental foundation for the study of infertility treatment.