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)在肿瘤微环境中经常上调，激活受体 酪氨酸激酶MET在癌细胞上表达，促进肿瘤进展并提供治疗作用 抵抗。因为癌细胞对HGF/MET信号上瘾，所以HGF和MET都是有效的 治疗靶点。尽管已经开发了各种针对HGF/MET信号转导的药物，但仍有 目前，还没有批准的药物可以特异性地抑制HGF或阻断MET活性。 HGF/MET信号转导中的限速步骤是前HGF蛋白分解为活性HGF。 三种丝氨酸蛋白酶中的一种或多种，即基质酶、肝素或肝细胞生长因子激活剂(HGFA)。我们已经开发出 首批模拟内源性HGF活性的HGF激活小分子抑制剂 激活、HAI-1和HAI-2。这些肝素、遮盖酶和HGFA的三重抑制剂来自两种化学物质 一系列酮苯并噻唑(KBTS)和环脲苯甲胺(CUBS)。我们证实了这些抑制剂 阻断HGF的激活，因此将其称为合成HAI(Shais)。我们已经证明了Shais抑制MET 在多种类型的癌细胞中传递信号并防止HGF介导的散射、迁移和存活。 我们已经证明，铅Shai，PTX2173，在体外克服了对EGFR抑制剂的耐药性，并阻碍了 HGF依赖的肺癌体内生长。此应用程序的目标是继续引领优化和 以证实其在结肠癌中的体内活性。我们将展示PTX2173阻断HGF介导的生长和 结肠癌的转移和体内预防/克服对EGFR抑制剂的耐药性。我们的具体目标是： 目的1：优化Shais以改善代谢稳定性和药代动力学(PK)性质。至 合理设计、合成和评估类似物的HGFA、Mattritase和Hepsin抑制活性： A)环肽和b)含有KBT抑制剂的非天然氨基酸。 C)测定沙伊斯铅的体外代谢稳定性、物理性质和体内PK。 目的2：证明PTX2173阻断HGF依赖的肿瘤生长/转移并克服 结肠癌患者对EGFR抑制剂的耐药性。 A)显示PTX2173在两个同基因结肠癌模型中抑制肿瘤生长和转移 由HGF、CT26和MC38驱动。 B)确认PTX2173预防HGF依赖的原发耐药和克服肿瘤 微环境介导的体内EGFR靶向药物治疗耐药。 总的来说，这些研究将为将Shais纳入阻断HGF-1的治疗方案提供理论基础。 依赖肿瘤进展，并防止或克服HGF依赖的靶向治疗耐药性， 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.