Selective Targeting Survivin for Cancer Therapy

 描述（由申请人提供）：生存素的表达与癌症耐药性和患者生存率低呈正相关。 Survivin在大多数类型的癌症中普遍表达，但在正常组织中表达非常低。 因此，开发新的选择性生存素抑制剂作为潜在的癌症治疗是非常重要的。 我们最近发现了一种由化合物UC-112提供的新支架。 UC-112强烈抑制癌细胞增殖，在其他IAP中选择性降解生存素，并有效抑制体内黑色素瘤肿瘤生长。 基于这些初步研究，我们的总体假设是UC-112的新型支架对生存素具有精细的选择性。 本项目的目标是使用整合的结构生物学、分子建模、药物化学和分子生物学方法来优化UC-112支架。 目标1. 基于UC-112支架进行计算机辅助药物设计，迭代优化抗癌活性。 目标1.1：使用生存素-Smac复合物的晶体结构和我们对UC-112支架的预测分子模型，我们将设计和合成集中的UC-112类似物组，以优化其活性并阐明结构-活性关系。 目标1.2：筛选 使用一组人黑色素瘤细胞系和三维集落形成测定来鉴定多达20种最佳UC-112类似物的新化合物（标准：IC 50 < 100 nM并且可以克服多药耐药性）以推进到Aim 2。 目标2. 定义新开发的UC-112类似物的生物学作用机制。 目的2.1：UC-112类似物与生存素和其他IAP相互作用的结构表征。 我们将使用ITC和SPR测量来确认在其他IAP中的靶向存活素抑制和选择性。 我们还将解决生存素与强效UC-112类似物复合物的X射线晶体结构。 这些信息将反馈到目标1，以优化分子模型，从而实现更有效的结构优化。 目标2.2：使用泛素化、脉冲追踪和蛋白质印迹分析评价UC-112对生存素稳定性和/或生存素和半胱天冬酶之间的阻断的影响。 目的2.3：研究UC-112类似物是否在具有不同存活素表达水平的癌细胞中诱导分化程度的细胞凋亡。 目标2.4：使用下拉和抗体阵列分析确定新类似物的任何脱靶效应以及在其他IAP或新UC-112类似物的额外潜在靶标中的选择性。 目标3。 确定选择性生存素抑制剂的体内抗癌活性。 目的 第3.1节：评价化合物的稳定性、药代动力学（PK）和药效学（PD）特性，以选择多达6种最佳UC-112类似物用于后续体内疗效研究。 目标3.2：评价所选生存素抑制剂对体内（小鼠）人黑色素瘤肿瘤生长的抗癌活性以及对正常细胞/组织的潜在毒性。 目标3.3：在我们建立的实验性肺转移模型中评价我们的选择性生存素抑制剂治疗体内黑色素瘤转移的能力。

 DESCRIPTION (provided by applicant): The expression of survivin positively correlates with cancer drug resistance and poor patient survival. Survivin is ubiquitously expressed in most types of cancer, but has very low expression in normal tissues. Thus, developing novel selective survivin inhibitors as potential cancer therapies is highly significant. We recently discovered a new scaffold provided by compound UC-112. UC-112 strongly inhibits cancer cell proliferation, selectively degrades survivin among other IAPs, and potently suppresses melanoma tumor growth in vivo. Based on these preliminary studies, our overall hypothesis is that the novel scaffold of UC-112 is exquisitely selective for survivin. The objective of this project is to optimize the UC-112 scaffold using integrated structural biology, molecular modeling, medicinal chemistry, and molecular biology approaches. Aim 1. Perform computer-aided drug design based on the UC-112 scaffold and iteratively optimize the anticancer activity. Aim 1.1: Using the crystal structure of survivin-Smac complex and our predictive molecular models for the UC-112 scaffold, we will design and synthesize focused sets of UC-112 analogs to optimize their activity and elucidate the structure-activity relationships. Aim 1.2: Screen the new compounds using a panel of human melanoma cell lines and three-dimensional colony formation assays to identify up to 20 best UC-112 analogs (criteria: IC50 < 100 nM and can overcome multidrug resistance) to be advanced to Aim 2. Aim 2. Define the biological mechanism(s) of action of newly developed UC-112 analogs. Aim 2.1: Structural characterization of UC-112 analogs interacting with survivin and other IAPs. We will confirm the on-target survivin inhibition and selectivity among other IAPs using ITC and SPR measurements. We will also solve the X-ray crystal structures of survivin in complex with potent UC-112 analogs. This information will feed back to Aim 1 to optimize the molecular models for more efficient structural optimization. Aim 2.2: Evaluation of the effect of UC-112 on survivin stability and/or blockage between survivin and caspases using ubiquitination, pulse-chase, and Western blot analyses. Aim 2.3: Investigation of whether UC-112 analogs induce differentiated degrees of apoptosis in cancer cells with distinct survivin expression levels. Aim 2.4: Determination of any off-target effects of the new analogs and selectivity among other IAPs or additionally potential targets of the new UC-112 analogs using pulldowns and antibody array analysis. Aim 3. Determine the anticancer activity of selective survivin inhibitors in vivo. Aim 3.1: Evaluate compound stability, pharmacokinetics (PK), and pharmacodynamics (PD) properties to select up to six best UC-112 analogs for subsequent in vivo efficacy studies. Aim 3.2: Evaluate anticancer activities of the selected survivin inhibitors against human melanoma tumor growth in vivo (in mice) and the potential toxicity to normal cells/tissues. Aim 3.3: Evaluate the ability of our selective survivin inhibitors to treat melanoma metastasis in vivo usin our established experimental lung metastasis model.