Inhibitors of pro‐HGF activation overcome resistance to anti‐EGFR therapy

摘要 肿瘤微环境中的因素促进肿瘤的生长并影响其对治疗的反应。 肝细胞生长因子（HGF）在肿瘤微环境中频繁上调，激活受体 在癌细胞上表达的酪氨酸激酶MET，其有助于肿瘤进展并赋予治疗作用。 阻力由于癌细胞对HGF/MET信号成瘾，因此HGF和MET都是有效的 治疗目标尽管已经开发了各种试剂来靶向HGF/MET信号传导，但仍存在一些缺陷。 目前没有批准的药物可以特异性抑制HGF或阻断MET活性。 HGF/MET信号传导中的限速步骤是促HGF蛋白水解加工成活性HGF， 三种丝氨酸蛋白酶、间质蛋白酶、肝蛋白酶或HGF激活剂（HGFA）中的一种或多种。我们已经开发 模拟HGF内源性抑制剂活性的第一种HGF活化小分子抑制剂 激活，HAI-1和HAI-2。这些肝蛋白酶、间质蛋白酶和HGFA的三链体抑制剂来自两种化学物质， 系列酮苯并噻唑（KBTS）和环脲苯甲脒（Cubs）。我们证实这些抑制剂 阻断HGF活化，因此将它们称为合成HAI（sHAI）。我们已经证明，sHAI抑制MET 在多种类型的癌细胞中，HGF介导的扩散、迁移和存活。 我们已经表明，先导sHAI，PTX 2173，克服了体外对EGFR抑制剂的耐药性，并阻碍了EGFR抑制剂的释放。 体内肺癌的HGF依赖性生长。此应用程序的目标是继续铅优化和 以证实其在结肠癌中的体内活性。我们将证明PTX 2173阻断HGF介导的生长， 结肠癌转移和预防/克服体内对EGFR抑制剂的抗性。我们的具体目标是： 目的1：优化sHAI以改善代谢稳定性和药代动力学（PK）特性。到 合理设计、合成和评估类似物的HGFA、间质蛋白酶和肝蛋白酶抑制活性： a）环肽和B）含有非天然氨基酸的kbt抑制剂。 c）确定铅sHAI的体外代谢稳定性、物理性质和体内PK。 目的2：证明PTX 2173阻断HGF依赖性肿瘤生长/转移，并克服 结肠癌中EGFR抑制剂的耐药性。 a）为了显示PTX 2173在两种同源结肠癌模型中阻断肿瘤生长和转移， 由HGF、CT 26和MC 38驱动。 B）证实PTX 2173预防HGF依赖性原发性抗性并克服肿瘤 体内微环境介导的对EGFR靶向剂的治疗抗性。 总的来说，这些研究将提供将sHAI纳入治疗方案以阻断HGF的基本原理。 依赖性肿瘤进展以及预防或克服对靶向治疗的HGF依赖性抗性， 显著改善结肠癌患者的预后。

ABSTRACT Factors in the tumor microenvironment promote the growth of tumors and impact their response to therapy. Hepatocyte Growth Factor (HGF), frequently upregulated in the tumor microenvironment, activates the receptor tyrosine kinase MET expressed on cancer cells which contributes to tumor progression and confers therapeutic resistance. Because cancer cells become addicted to HGF/MET signaling, both HGF and MET are valid therapeutic targets. Although various agents have been developed to target HGF/MET signaling, there are currently no approved drugs that would inhibit HGF or block MET activity specifically. The rate-limiting step in the HGF/MET signaling is the proteolytic processing of pro-HGF to active HGF by one or more of the three serine proteases, matriptase, hepsin or HGF activator (HGFA). We have developed the first small molecule inhibitors of HGF activation which mimic the activity of the endogenous inhibitors of HGF activation, HAI-1 and HAI-2. These triplex inhibitors of hepsin, matriptase and HGFA are from two chemical series of ketobenzothiazoles (kbts) and cyclic urea benzamidines (cubs). We confirmed that these inhibitors block HGF activation and thus refer to them as synthetic HAIs (sHAIs). We have shown that sHAIs inhibit MET signaling and prevent HGF-mediated scattering, migration and survival in multiple types of cancer cells. We have shown that the lead sHAI, PTX2173, overcomes resistance to EGFR inhibitors in vitro and impedes HGF-dependent growth of lung cancer in vivo. The goal of this application is to continue lead optimization and to confirm its in vivo activity in colon cancer. We will show that PTX2173 blocks HGF-mediated growth and metastasis of colon cancer and prevents/overcomes resistance to EGFR inhibitors in vivo. Our specific aims are: Aim 1: To optimize sHAIs for improved metabolic stability and pharmacokinetic (PK) properties. To rationally design, synthesize and evaluate analogues for their HGFA, matriptase and hepsin inhibitory activity: a) cyclic peptide and b) unnatural amino acid containing kbt Inhibitors. c) To determine the in vitro metabolic stability, physical properties and in vivo PK of lead sHAIs. Aim 2: To demonstrate that PTX2173 blocks HGF-dependent tumor growth/metastasis and overcomes resistance to EGFR inhibitors in colon cancer. a) To show that PTX2173 blocks tumor growth and metastasis in two syngeneic colon cancer models that are driven by HGF, CT26 and MC38. b) To confirm that PTX2173 prevents HGF-dependent primary resistance and overcomes tumor microenvironment-mediated therapeutic resistance to EGFR targeting agents in vivo. Collectively, these studies will provide a rationale to include sHAIs into treatment regimens to block HGF- dependent tumor progression and to prevent or to overcome HGF-dependent resistance to targeted therapy, significantly improving the outcome of colon cancer patients.