Chemical probes that target Stat3 to treat cancer

描述（由申请人提供）：我们首次在人类乳腺癌患者中发现，标准化疗后残留的肿瘤富集了CD44+/CD24-/低水平的乳腺癌干细胞或肿瘤启动细胞（TIC），这些细胞在小鼠中表现出增强的乳腺球体形成效率（MSFE），并显示出异种移植物移植生长的增加（1,2,11）。此外，我们通过对CD44+/CD24-/低乳腺球体形成人群的基因表达分析和shRNA文库筛选确定，参与乳腺TIC自我更新的顶级典型途径包括Stat3信号传导(3)。通过虚拟配体筛选，我们确定了三种靶向Stat3 Src同源性（SH） 2结构域的小分子探针，并竞争性和选择性地抑制Stat3的激活(5)。其中最活跃的药物C188被证明可以显著减少人类植入小鼠的乳腺癌肿瘤移植物体积，并在与标准化疗联合使用时将移植物小鼠的无瘤生存率提高4倍(3)。我们利用C188骨架作为查询结构和商业化合物库（Life Chemicals）进行二维相似性筛选，然后进行三维药效团分析，鉴定出33个活性衍生物。其中一种衍生物（C188-9）在SPR实验中抑制Stat3结合和阻断配体介导的Stat3磷酸化的活性比C188高1倍(6)。此外，C188-9在100 mg/kg/天的剂量下，连续14天对小鼠表现出最小的毒性，在体外不抑制正常小鼠骨髓集落的形成，并且在小鼠恶病质模型中防止体重减轻和肌肉减少。在这个I期STTR中将检验的具体假设是，C188-9或其衍生物之一将对Stat3具有高活性，在小鼠中具有良好的耐受性，并将在乳腺肿瘤移植模型中证明有效性。我们提出了两个紧密聚焦的具体目标来实现这一假设：目标1：基于C188-9引线结构的修饰合成候选第三代C188探针，并鉴定出具有更高效力和有利ADMET特征的候选C188探针。新探针的合成很简单；具有更大效力和有利ADMET特征的探针将使用成熟的测定方法进行鉴定。目标2。研究最有效和毒性最小的第三代Stat3探针在与标准化疗联合使用时靶向乳腺tic和抑制肿瘤移植物生长的能力。两种C188-9衍生物是最有效的，毒性最小的，并且具有最好的ADMET谱，当使用我们开发的29种乳腺肿瘤移植模型中最具信息的亚群与标准化疗联合使用时，将检测其靶向TIC和抑制肿瘤生长的能力。这些研究将为II期STTR申请奠定基础，届时我们将提出最有希望的先导化合物的GMP合成和体内ADMET研究，为转移性乳腺癌的I期临床试验做准备。

DESCRIPTION (provided by applicant): We have shown for the first time in human breast cancer patients that residual tumors after standard chemotherapy are enriched for a CD44+/CD24-/low population of breast cancer stem or tumor-initiating cells (TIC) that exhibit enhanced mammosphere-forming efficiency (MSFE) and display an increase in xenograft transplant outgrowths in mice (1, 2, 11). In addition, we have determined from gene expression analysis and shRNA library screening of the CD44+/CD24-/low-mammosphere-forming population that the top canonical pathways involved in breast TIC self-renewal include Stat3 signaling (3). Using virtual ligand screening, we identified three small-molecule probes that target the Src homology (SH) 2 domain of Stat3 and that competitively and selectively inhibited Stat3 activation (5). The most active of these hits, C188, was shown to dramatically reduce human-into-mouse breast cancer tumorgraft volumes and to increase tumor-free survival of engrafted mice 4-fold when used in combination with standard chemotherapy (3). We performed 2-D similarity screening using the scaffold of C188 as the query structure and a commercial compound library (Life Chemicals) followed by 3-D pharmacophore analysis and identified 33 active derivatives. One derivative (C188-9), now owned by StemMed, is 1-log more active than C188 at inhibiting Stat3 binding in SPR assays and in blocking ligand-mediated Stat3 phosphorylation (6). In addition, C188-9 exhibited minimal toxicity in mice up to a dose of 100 mg/kg/day for 14 days, did not inhibit normal murine bone marrow colony formation in vitro, and, prevented weight loss and sarcopenia in a murine model of cachexia. The specific hypothesis that will be examined in this Phase I STTR is that C188-9 or one of its derivatives will be highly active against Stat3, will be well tolerated in mice, and will demonstrate efficacy in breast tumorgraft models. We have proposed 2 tightly focused specific aims to pursue this hypothesis: AIM 1: To synthesize candidate third (3rd) generation C188 probes based on modifications of the C188-9 lead structure and to identify those with increased potency and favorable ADMET features. Synthesis of new probes is straightforward; probes with greater potency and favorable ADMET features will be identified using well-established assays. AIM 2. To examine the most potent and least toxic 3rd generation Stat3 probes for the ability to target breast TICs and inhibi tumorgraft growth when used in combination with standard chemotherapy. Two C188-9 derivatives that are the most potent, least toxic, and have the best ADMET profile will be examined for the ability to target TIC and inhibit tumor growth when combined with standard chemotherapy using the most informative subset of the 29 breast tumorgraft models we have developed. These studies will set the stage for a Phase II STTR application in which we will propose GMP synthesis and in vivo ADMET studies of the most promising lead compound identified in preparation for a Phase I clinical trial in metastatic breast cancer. PUBLIC HEALTH RELEVANCE: Successfully curing cancers that are resistant to standard chemotherapy will require new drugs including those directed at a subset of cancer cells referred to as cancer stem cells. Cancer stem cells are resistant to standard chemotherapy and are responsible for tumor recurrence. We have identified a pathway in breast cancer stem cells essential for their survival that involves a protein called Stat3 and propose in this application t develop potent and selective small-molecule inhibitors of Stat3 for development into a novel and effective drug treatment for metastatic breast cancer.