Stat3 Probes that Target Breast Cancer Stem Cells

描述（申请人提供）：常规化疗在控制和逆转肿瘤生长方面初步有效。然而，残留的癌症总是会重新生长。我们最近来自配对人类乳腺癌样本的数据表明，标准治疗消除了分裂的子细胞，同时丰富了CD44？在乳腺细胞（MS）培养物中具有自我更新能力并在异种移植物移植后产生肿瘤的低推定乳腺癌干细胞（CSCs）。我们已经在这些CD44？/低，ms形成细胞，具有Stat3。利用虚拟配体筛选（VLS）技术，我们开发了靶向Stat3 Src同源性（SH） 2结构域的铅化学探针，并竞争性和选择性地抑制Stat3的激活。三维药效团分析采用最活性先导化合物（Cpd188）和Life Chemicals化合物文库；通过表面等离子体共振（SPR）测试了得分最高的化合物对Stat3与其磷酸肽配体结合的抑制作用。在39种被检测的化合物中，除6种外，其余化合物的IC50值均可测量，其中17种的IC50值等于或低于Cpd188。其中一种化合物（Cpd188-15）在MS形成抑制试验中的IC50值比Cpd188低一个对数。我们已经开发了2个高度集中的SPECIFIC AIMS来直接测试Stat3的小分子抑制剂可以特异性靶向乳腺癌干细胞的假设。目的1。开发具有更高结合亲和力和抑制活性的第三代Stat3化学探针。我们建议使用最活跃的第二代探针Cpd188-15作为制造第三代探针的支架。修改计划是基于对第二代探针进行的构效关系（SAR）分析的结果，并围绕着从磺酰氯和酰胺的面板直接合成磺胺。每个新的磺胺化合物将在SPR试验中进行检测，在高通量荧光显微镜（HTFM）试验中，将测试il -6刺激的细胞质到核易位的抑制作用以及它们对Stat3和Stat1的选择性。目标2。在临床前MS试验和人类异种移植物模型中，确定Stat3通路的抑制是否可以改善现有的癌症治疗。我们将通过MS形成抑制试验和人类乳腺癌异种移植模型，确定最活跃的第3代探针对Stat3的抑制是否会提高体外和体内常规治疗的疗效。除了确定Stat3对乳腺癌干细胞存活和自我更新的贡献外，这些临床前研究可能为未来临床试验的设计提供信息，并确定抑制干细胞自我更新和治疗耐药途径是否可以改善患者现有的乳腺癌治疗。

DESCRIPTION (provided by applicant): Conventional chemotherapy is effective initially in controlling and reversing tumor growth. However, residual cancers will invariably re-grow. Our recent data from paired human breast cancer samples indicates that standard therapy eliminates dividing daughter cells while enriching the residual tumor for CD44?/low putative breast "cancer stem cells" (CSCs) that have the ability to self-renew in mammosphere (MS) cultures, and to give rise to tumors upon xenograft transplantation. We have identified a cancer stem cell signature within these CD44?/low, MS-forming cells that features Stat3. Using virtual ligand screening (VLS), we developed lead chemical probes that target the Src homology (SH) 2 domain of Stat3 and that competitively and selectively inhibited Stat3 activation. 3-D pharmacophore analysis was performed using the most active lead compound (Cpd188) and a Life Chemicals compound library; the top scoring compounds identified were tested for inhibition of Stat3 binding to its phosphopeptide ligand by surface plasmon resonance (SPR). All but six of 39 compounds tested have measurable IC50s, with 17 having IC50 values equal to or less than Cpd188. One compound (Cpd188-15) has an IC50 value in MS formation inhibition assays that is one log lower than Cpd188. We have developed 2 highly focused SPECIFIC AIMS to directly test the hypothesis that small- molecule inhibitors of Stat3 can specifically target breast cancer stem cells. AIM 1. To develop third (3rd) generation Stat3 chemical probes with increased binding affinity and inhibitory activity. We propose using the most active 2nd generation probe, Cpd188-15, as a scaffold for making 3rd generation probes. Modifications planned are based on the results of a structure-activity relationship (SAR) analysis performed on 2nd generation probes and center around the straightforward synthesis of sulfamides from panels of sulfonyl chlorides and amides. Each novel sulfamide compound will be examined in the SPR assay, in a high throughput fluorescence microscopy (HTFM) assay that will test inhibition of IL-6-stimulated cytoplasmic-to-nuclear translocation and for their selectivity for Stat3 vs. Stat1. AIM 2. To determine whether suppression of the Stat3 pathway can improve existing cancer therapies in preclinical MS assays and human xenograft models. We will determine whether Stat3 inhibition by the most active 3rd generation probes will improve efficacy of conventional therapy in vitro and in vivo, using MS formation inhibition assays and human breast cancer xenograft models. In addition to establishing the contribution of Stat3 to breast cancer stem cell survival and self-renewal, these preclinical studies may inform the design of future clinical trials and determine whether suppressing stem cell self-renewal and treatment resistance pathways can improve existing breast cancer therapies in patients. PUBLIC HEALTH RELEVANCE: A small proportion of breast cancer cells fail to respond to the usual treatments and cause relapsing disease. This proposal outlines chemical and biological studies designed to develop small molecule probes that target these therapy-resistant cells. If these studies are successful, these small molecule probes may be combined with current treatments for breast cancer thereby preventing cancer relapse and resulting in a cure.