Anoctamin1 (ANO1), a calcium-activated chloride ion channel (CaCC), is associated with various physiological functions including cancer progression and metastasis/invasion. ANO1 has been considered as a promising target for cancer therapeutics as ANO1 is over-expressed in a variety of cancers including glioblastoma (GBM) and inhibition of ANO1 has been reported to suppress cell proliferation, migration and invasion in GBM. GBM is one of the most common and aggressive cancers with poor prognosis with median survival for 15 months. Lack of effective treatment options against GBM emphasizes urgent necessity of effective GBM therapeutics. In an effort to discover potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and synthesized a series of new 2-aminothiophene-3-carboxamide derivatives and performed SAR studies using both fluorescent cellular membrane potential assay and whole-cell patch-clamp recording. We observed that among these substances, 9c and 10q strongly suppress ANO1 channel activities and possess remarkable selectivity over ANO2. Unique structural feature of 10q, a cyclopentane-fused thiophene-3-carboxamide derivative, is the presence of benzoylthiourea functionality which dramatically contributes to activity. Both 9c and 10q suppress more strongly proliferation of GBM cells than four reference compounds including 3, Ani-9 and are also capable of inhibiting much more strongly colony formation than reference compounds in both 2D colony formation assay and 3D soft agar assay using U251 glioma cells. In addition, 9c and 10q suppress far more strongly migration/invasion of GBM cells than reference compounds. We, for the first time, found that the combination of ANO1 inhibitor (9c or 3) and temozolomide (TMZ) brings about remarkable synergistic effects in suppressing proliferation of GBM cells. Our study may provide an insight into designing selective and potent ANO1 inhibitors aiming at GBM treatment.

Anoctamin1（ANO1）是钙激活的氯离子通道（CaCC），与多种生理功能相关，包括癌症进展和转移/侵袭。ANO1被认为是癌症治疗的有希望的靶标，因为ANO1在多种疾病中过度表达，包括胶质母细胞瘤（GBM），据报道，抑制ANO1可抑制GBM中的细胞增殖，迁移和侵袭。GBM是最常见的侵袭性癌症之一，预后差，中位生存期为15个月。缺乏针对GBM的有效治疗选择强调了有效GBM治疗剂的迫切必要性。为了发现能够抑制GBM细胞的有效和选择性ANO1抑制剂，我们设计并合成了一系列新的2-氨基噻吩-3-羧酰胺衍生物，并使用荧光细胞膜电位测定法和全细胞膜片钳记录进行了SAR研究。我们观察到在这些物质中，9c和10q强烈抑制ANO1通道的活性，并具有明显优于ANO2的选择性。环戊烷稠合的噻吩-3-羧酰胺衍生物10q的独特结构特征是苯甲酰基硫脲功能的存在，该功能极大地促进了活性。既9C和10q的抑制更强烈增殖GBM细胞的比四个参考化合物，包括3，ANI-9，并且也能够使用U251神经胶质瘤细胞抑制强烈得多的集落形成比在2D集落形成试验和3D软琼脂测定参照化合物的。此外，9c和10q抑制GBM细胞的迁移/侵袭远比参考化合物强。我们首次发现ANO1抑制剂（9c或3）和替莫唑胺（TMZ）的组合在抑制GBM细胞增殖方面具有显着的协同作用。我们的研究可能为设计针对GBM治疗的选择性和有效ANO1抑制剂提供了见识。