Targeting c-Myc and MDR1 in Cancer Through Small Molecule Inhibitors of IMP-1

通过 IMP-1 小分子抑制剂靶向癌症中的 c-Myc 和 MDR1

基本信息

批准号：
8688973
负责人：
DAVID J SHAPIRO
金额：
$ 16.02万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8688973
关键词：
Affinity Antineoplastic Agents Apoptosis Binding Binding Proteins Binding Sites Biological Assay Cancer Cell Growth Cell Line Cell Proliferation Cells Characteristics Chimera organism Colon Carcinoma DNA DNA Binding Dose Down-Regulation Enabling Factors Fluorescein Fluorescence Anisotropy Germ Cells Hypoxia Illinois Inflammation Inhibition of Cell Proliferation Label Lead Luciferases Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of ovary Messenger RNA MicroRNAs Multi-Drug Resistance Nucleic Acid Binding Oncogenes P-Glycoprotein Preparation Progesterone Receptors Protein Binding Proteins Proto-Oncogene Proteins c-myc RNA Interference RNA Probes RNA-Binding Proteins Recombinants Reporter Reporting Resistance Response Elements Role Signal Transduction Site Specificity Structure Taxane Compound Testing Translations Ubiquitination Universities base c-myc Genes cancer cell cancer type counterscreen high throughput screening inhibitor/antagonist mRNA Decay mRNA Stability neoplastic cell novel strategies outcome forecast overexpression prostate cancer cell public health relevance response small molecule taxane therapeutic development therapeutic evaluation tumor tumor growth ubiquitin ligase

项目摘要

DESCRIPTION (provided by applicant): We propose a novel approach to identifying new probes and potential anticancer agents based on reducing expression of c-Myc and other oncogenes, MDR1 (multidrug resistance protein 1) and NF-?B by targeting the mRNA binding protein IMP-1/CRD-BP/IGF2BP1. IMP-1 is an oncofetal mRNA binding protein that binds to and stabilizes the mRNAs encoding c-Myc, K-Ras, ERK and other oncogenes, MDR1, and indirectly increases activity of the tumor enabling factor NF-?B. ?-catenin and c-Myc induce IMP-1 and it is a key regulatory target of let-7 microRNA. Reducing IMP-1 levels by RNAi knockdown, or by expression of let-7 miRNA, reduces c- Myc levels, strongly inhibits cell proliferation and reverses resistance to anticancer drugs. Kaplan-Meier plots show that expression of IMP-1 is associated with a poor prognosis and reduced survival in lung, ovarian and colon cancer. Small molecule inhibitors of IMP-1 have not been reported. In preliminary studies, we identified an IMP-1 binding and mRNA stabilizing site in MDR1 mRNA, established a (FAMA) fluorescence anisotropy/polarization microplate assay for analyzing binding of IMP-1 to its c-Myc and MDR1 mRNA targets, developed a robust (Z' factor=0.64) FAMA-based high throughput screen for inhibitors of binding of IMP-1 to a c-Myc mRNA binding site and carried out a successful pilot screen. To filter the primary hits, we established verification assays based on purified protein, luciferase reporters and cell-proliferation. The Specific Aims are: Aim 1. To identify small molecules with high potency and specificity that inhibit binding of IMP-1 to its c-Myc mRNA binding site. We will implement a FAMA HTS screen using ~180,000 small molecules and identify compounds that inhibit binding of purified IMP-1 to a fluorescein-labeled (fl) c-Myc mRNA binding site. To reduce the number of false positives, our two-step assay first uses an internal counterscreen to test whether each compound reduces the signal from the fl-Myc probe alone and then tests whether the compound inhibits binding of IMP-1 to the fl-Myc probe. (i) Primary hits will be simultaneously verified using fl-Myc and tested for ability to inhibit bindingof IMP-1 to the fl-MDR1 mRNA binding site (ii) To test for specificity, we will evaluate hits for inhibition of binding of progesterone receptor (PR) to its fl-DNA response element (fl-PRE). (iii) Potency and efficacy of hits will be evaluated in dose-response studies. Aim 2. To identify lead IMP-1 inhibitors which reduce the growth of cancer cells. Initial cell-based assays are luciferase-based assays for small molecules that inhibit expression of NF-?B-luciferase and our luciferase- MDR1 mRNA chimera and for inhibition of proliferation of IMP-1 positive IGROV-1 and ES-2 ovarian cancer cells with little or no effect on IMP-1 negative PC-3 cells. To approach inhibitor sites of action, lead compounds will be tested for down-regulation of IMP-1 protein and c-Myc and MDR1 mRNA and protein. Microarray studies using IMP-1 negative cells will assess possible off-target effects of the lead inhibitor. Lead structures will be confirmed, leads resynthesized and structural studies of inhibitor bound to IMP-1 will be initiated.

描述（由申请人提供）：我们提出了一种新方法，通过靶向 mRNA 结合蛋白来减少 c-Myc 和其他癌基因、MDR1（多药耐药蛋白 1）和 NF-κB 的表达，从而识别新探针和潜在抗癌药物IMP-1/CRD-BP/IGF2BP1。 IMP-1 是一种癌胎儿 mRNA 结合蛋白，可结合并稳定编码 c-Myc、K-Ras、ERK 和其他癌基因 MDR1 的 mRNA，并间接增加肿瘤促进因子 NF-κB 的活性。 β-catenin 和 c-Myc 诱导 IMP-1，它是 let-7 microRNA 的关键调控靶点。通过RNAi敲低或let-7 miRNA的表达来降低IMP-1水平，可以降低c-Myc水平，强烈抑制细胞增殖并逆转抗癌药物的耐药性。 Kaplan-Meier 图显示 IMP-1 的表达与肺癌、卵巢癌和结肠癌的不良预后和生存率降低相关。 IMP-1小分子抑制剂尚未见报道。在初步研究中，我们确定了 MDR1 mRNA 中的 IMP-1 结合和 mRNA 稳定位点，建立了 (FAMA) 荧光各向异性/偏振微孔板测定法，用于分析 IMP-1 与其 c-Myc 和 MDR1 mRNA 靶标的结合，开发了一种稳健的方法(Z'因子=0.64)基于FAMA的高通量筛选IMP-1与c-Myc mRNA结合位点结合的抑制剂，并进行了成功的试点筛选。为了过滤主要命中，我们建立了基于纯化蛋白、荧光素酶报告基因和细胞增殖的验证测定。具体目标是：目标 1. 鉴定具有高效力和特异性的小分子，可抑制 IMP-1 与其 c-Myc mRNA 结合位点的结合。我们将使用约 180,000 个小分子实施 FAMA HTS 筛选，并鉴定抑制纯化 IMP-1 与荧光素标记 (fl) c-Myc mRNA 结合位点结合的化合物。为了减少假阳性的数量，我们的两步测定首先使用内部反筛选来测试每种化合物是否单独减少来自 fl-Myc 探针的信号，然后测试该化合物是否抑制 IMP-1 与 fl-Myc 的结合探测。 (i) 将使用 fl-Myc 同时验证初级命中，并测试抑制 IMP-1 与 fl-MDR1 mRNA 结合位点结合的能力 (ii) 为了测试特异性，我们将评估抑制孕酮受体结合的命中(PR) 与其 fl-DNA 响应元件 (fl-PRE) 的关系。 (iii) 命中的效力和功效将在剂量反应研究中进行评估。目标 2. 鉴定可减少癌细胞生长的先导 IMP-1 抑制剂。最初的基于细胞的测定是基于荧光素酶的测定，用于抑制 NF-κB-荧光素酶和我们的荧光素酶-MDR1 mRNA 嵌合体表达的小分子，以及抑制 IMP-1 阳性 IGROV-1 和 ES-2 卵巢癌细胞的增殖对 IMP-1 阴性 PC-3 细胞影响很小或没有影响。为了接近抑制剂的作用位点，将测试先导化合物对 IMP-1 蛋白以及 c-Myc 和 MDR1 mRNA 和蛋白的下调。使用 IMP-1 阴性细胞的微阵列研究将评估先导抑制剂可能的脱靶效应。将确认先导结构、重新合成先导化合物并启动与 IMP-1 结合的抑制剂的结构研究。