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证明，标准化疗后的残留肿瘤富含 CD44 / CD24-/低群体的乳腺癌干细胞或肿瘤起始细胞 (TIC)，这些细胞表现出增强的乳腺球形成效率 (MSFE) 并显示小鼠异种移植物生长的增加 (1,2,11)。此外，我们通过基因表达分析和 CD44 /CD24-/低乳腺球形成群体的 shRNA 文库筛选确定，参与乳腺 TIC 自我更新的主要经典途径包括 Stat3 信号传导 (3)。通过虚拟配体筛选，我们鉴定了三种小分子探针，它们靶向 Stat3 的 Src 同源 (SH) 2 结构域，并竞争性、选择性地抑制 Stat3 激活 (5)。其中最活跃的 C188 与标准化疗联合使用时，可显着减少人鼠乳腺癌肿瘤移植体积，并将移植小鼠的无瘤生存率提高 4 倍 (3)。我们使用 C188 的支架作为查询结构和商业化合物库（Life Chemicals）进行 2-D 相似性筛选，然后进行 3-D 药效团分析，并鉴定了 33 种活性衍生物。一种衍生物 (C188-9) 现归 StemMed 所有，在 SPR 测定中抑制 Stat3 结合和阻断配体介导的 Stat3 磷酸化方面，其活性比 C188 高 1 个对数级 (6)。此外，C188-9 在小鼠中表现出最小的毒性，剂量高达 100 mg/kg/天，持续 14 天，在体外不会抑制正常小鼠骨髓集落形成，并且在小鼠恶病质模型中预防体重减轻和肌肉减少症。在第一阶段 STTR 中将检验的具体假设是 C188-9 或其衍生物之一将对 Stat3 具有高度活性，在小鼠中具有良好的耐受性，并将在乳腺肿瘤移植模型中表现出功效。我们提出了 2 个紧密关注的具体目标来实现这一假设： 目标 1：基于 C188-9 先导结构的修饰合成候选第三 (3rd) 代 C188 探针，并识别那些具有增强效力和有利 ADMET 特征的探针。新探针的合成很简单；将使用成熟的测定法来鉴定具有更大效力和有利 ADMET 特征的探针。目的 2. 检查最有效且毒性最小的第三代 Stat3 探针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。我们将使用我们开发的 29 个乳腺肿瘤移植模型中信息最丰富的子集，检查两种最有效、毒性最小且具有最佳 ADMET 特征的 C188-9 衍生物与标准化疗联合使用时靶向 TIC 和抑制肿瘤生长的能力。这些研究将为 II 期 STTR 应用奠定基础，在该应用中，我们将提议对最有希望的先导化合物进行 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。 公共健康相关性：成功治愈对标准化疗有抵抗力的癌症将需要新药，包括针对被称为癌症干细胞的癌细胞子集的药物。癌症干细胞对标准化疗具有抵抗力，并且是肿瘤复发的原因。我们已经确定了乳腺癌干细胞中对其生存至关重要的途径，该途径涉及一种名为 Stat3 的蛋白质，并在本申请中提出开发强效且选择性的 Stat3 小分子抑制剂，以开发为治疗转移性乳腺癌的新型有效药物。

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.