AST1306, A Novel Irreversible Inhibitor of the Epidermal Growth Factor Receptor 1 and 2, Exhibits Antitumor Activity Both In Vitro and In Vivo

Despite the initial response to the reversible, ATP-competitive quinazoline inhibitors that target ErbB-family, such a subset of cancer patients almost invariably develop resistance. Recent studies have provided compelling evidence that irreversible ErbB inhibitors have the potential to override this resistance. Here, we found that AST1306, a novel anilino-quinazoline compound, inhibited the enzymatic activities of wild-type epidermal growth factor receptor (EGFR) and ErbB2 as well as EGFR resistant mutant in both cell-free and cell-based systems. Importantly, AST1306 functions as an irreversible inhibitor, most likely through covalent interaction with Cys797 and Cys805 in the catalytic domains of EGFR and ErbB2, respectively. Further studies showed that AST1306 inactivated pathways downstream of these receptors and thereby inhibited the proliferation of a panel of cancer cell lines. Although the activities of EGFR and ErbB2 were similarly sensitive to AST1306, ErbB2-overexpressing cell lines consistently exhibited more sensitivity to AST1306 antiproliferative effects. Consistent with this, knockdown of ErbB2, but not EGFR, decreased the sensitivity of SK-OV-3 cells to AST1306. In vivo, AST1306 potently suppressed tumor growth in ErbB2-overexpressing adenocarcinoma xenograft and FVB-2/Nneu transgenic breast cancer mouse models, but weakly inhibited the growth of EGFR-overexpressing tumor xenografts. Tumor growth inhibition induced by a single dose of AST1306 in the SK-OV-3 xenograft model was accompanied by a rapid (within 2 h) and sustained (≥24 h) inhibition of both EGFR and ErbB2, consistent with an irreversible inhibition mechanism. Taken together, these results establish AST1306 as a selective, irreversible ErbB2 and EGFR inhibitor whose growth-inhibitory effects are more potent in ErbB2-overexpressing cells.尽管靶向ErbB家族的可逆ATP竞争性喹唑啉类抑制剂初显疗效，但此类癌症患者几乎无一例外都会产生耐药性。近期研究已提供了令人信服的证据，证明不可逆ErbB抑制剂有望克服这一耐药性。本研究发现，新型苯胺基喹唑啉化合物AST1306可在无细胞体系与细胞体系中，抑制野生型表皮生长因子受体（epidermal growth factor receptor, EGFR）、ErbB2以及EGFR耐药突变体的酶活性。尤为重要的是，AST1306属于不可逆抑制剂，其作用机制极有可能是分别与EGFR和ErbB2催化结构域中的Cys797、Cys805发生共价相互作用。进一步研究表明，AST1306可使上述受体下游的信号通路失活，进而抑制一组癌细胞系的增殖。尽管EGFR与ErbB2的酶活性对AST1306的敏感性相近，但过表达ErbB2的细胞系对AST1306的抗增殖效应始终表现出更高的敏感性。与此一致的是，敲低ErbB2而非EGFR，可降低SK-OV-3细胞对AST1306的敏感性。在体内实验中，AST1306可有效抑制过表达ErbB2的腺癌异种移植瘤及FVB-2/Nneu转基因乳腺癌小鼠模型的肿瘤生长，但对过表达EGFR的肿瘤异种移植瘤的生长抑制作用较弱。在SK-OV-3异种移植瘤模型中，单次给药AST1306所诱导的肿瘤生长抑制，伴随了EGFR与ErbB2的快速（2小时内）且持续（≥24小时）的抑制，这与不可逆抑制机制相符。综上，本研究结果证实AST1306是一种选择性不可逆ErbB2与EGFR抑制剂，其在过表达ErbB2的细胞中表现出更强的生长抑制活性。