异常活化的Hedgehog (Hh) 信号通路介导了各种肿瘤的发生和发展[1]。目前，针对Hh信号通路组分Smoothened (Smo) 的三种药物已经上市，用于基底细胞肿瘤或急性髓系白血病的临床治疗。然而，耐药性是这些药物普遍面临的问题，因此研究能够克服耐药性的Smo抑制剂对于临床辅助药物具有重要指导意义。本研究使用MTT试验、克隆形成试验和EdU试验检测药物对肿瘤细胞的增殖抑制活性。采用流式细胞术检测B13对细胞周期和凋亡的影响。采用急性毒性试验检测B13在体内的毒性，并采用异种移植瘤模型检测B13在体内的疗效。采用BODIPY-环硅烷竞争结合试验和分子对接研究B13与Smo的结合情况。通过Western blot和免疫荧光法研究了B13对Smo下游靶基因Gli1/2的表达和定位的影响。构建了SmoD473H突变细胞株以研究B13对耐药性的影响。   (1) B13对结肠癌细胞的抑制活性最强。 (2) B13能有效抑制结肠癌细胞的克隆形成和EdU阳性率。 (3) B13可以阻止细胞进入G2/M期，并导致细胞凋亡。 (4) B13在体内的毒性低，其疗效在体内优于Vismodegib。 (5) 分子对接和BODIPY-环硅烷实验表明，B13能够与Smo蛋白结合。 (6) B13可以抑制Smo下游靶标Gli1的蛋白表达，并阻止其进入细胞核。 (7) B13可以抑制SmoD473H在HEK293细胞中的Gli1表达，分子对接结果表明B13可以与SmoD473H蛋白结合。通过MTT试验筛选出了最佳的抗肿瘤活性B13。在体外药效学实验中，B13能够有效抑制结肠癌细胞的增殖和转移，诱导细胞周期停滞，并诱导细胞凋亡。在体内药效学实验中，B13在抗肿瘤活性方面优于Vismodegib，并且在体内毒性低。机制研究表明，B13可以结合Smo蛋白，抑制下游的Gli1的表达和进入细胞核。值得注意的是，B13克服了由SmoD473H突变引起的耐药性。版权所有 © 2023 Elsevier Inc. 保留所有权利。

Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. SmoD473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with SmoD473H, and the molecular docking results showed that B13 could bind SmoD473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by SmoD473H mutations.Copyright © 2023 Elsevier Inc. All rights reserved.