Selective Targeting Survivin for Cancer Therapy

 描述(申请人提供)：Survivin的表达与癌症耐药性和患者生存不良呈正相关。Survivin在大多数类型的癌症中普遍表达，但在正常组织中表达很低。因此，开发新的选择性Survivin抑制剂作为潜在的癌症治疗具有重要意义。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，选择性降解Survivin等IAP，并在体内有效地抑制黑色素瘤的生长。基于这些初步研究，我们的总体假设是UC-112的新型支架对Survivin具有良好的选择性。该项目的目标是使用集成的结构生物学、分子建模、药物化学和分子生物学方法来优化UC-112支架。目的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。目的1.1：利用Survivin-Smac复合体的晶体结构和我们对UC-112支架的预测分子模型，我们将设计和合成一组有针对性的UC-112类似物，以优化其活性并阐明其构效关系。目标1.2：筛选 利用一组人黑色素瘤细胞株和三维集落形成试验筛选出最好的20个UC-112类似物(标准：IC_(50)和lt；100 nm，可以克服多药耐药)，以推进到目标2.目的2.确定新开发的UC-112类似物的生物学作用机制(S)。目的2.1：UC-112类似物与Survivin和其他IAP相互作用的结构特征。我们将使用ITC和SPR测量来确认靶上Survivin对其他IAP的抑制和选择性。我们还将解决Survivin与强大的UC-112类似物的络合物中的X射线晶体结构。这些信息将反馈给目标1，以优化分子模型，以实现更有效的结构优化。目的2.2：通过泛素化、脉冲追逐和Western印迹分析评价UC-112对Survivin稳定性和/或Survivin和caspase之间的阻断作用。目的2.3：研究UC-112类似物是否诱导不同Survivin表达水平的癌细胞发生不同程度的凋亡。目标2.4：使用下拉列表和抗体阵列分析，确定新类似物的任何非靶点效应以及在其他IAP或新UC-112类似物的额外潜在靶点中的选择性。目的3.体内检测选择性Survivin抑制剂的抗癌活性。目标 3.1：评估化合物的稳定性、药代动力学(PK)和药效学(PD)特性，选择最多六种最好的UC-112类似物进行后续的体内疗效研究。目的3.2：评价所选Survivin抑制剂在体内(小鼠)对人黑色素瘤生长的抑制作用及其对正常细胞/组织的潜在毒性。目的3.3：在我们建立的实验性肺转移模型中，评估我们的选择性Survivin抑制剂在体内治疗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.