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：
8584046
负责人：
DAVID J SHAPIRO
金额：
$ 19.96万
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 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8584046
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）：我们提出了一种新的方法，用于基于降低C-MYC和其他癌基因的表达，MDR1（多药耐药蛋白1）和NF-b？b来鉴定新的探针和潜在的抗癌剂，并通过靶向mRNA MRNA结合蛋白IMP-1/CRD-BP/IGD-BP/IGF2BP1。 IMP-1是一种癌端mRNA结合蛋白，可与编码C-MYC，K-RAS，ERK和其他Oncogenes，MDR1的mRNA结合并稳定并间接增加肿瘤启用因子Nf-？B的活性。？ - 蛋白质和C-MYC诱导IMP-1，它是Let-7 microRNA的关键调节靶标。通过RNAi敲低降低IMP-1水平，或通过表达Let-7 miRNA降低C- MYC水平，强烈抑制细胞增殖并逆转对抗癌药物的抗性。 Kaplan-Meier情节表明，IMP-1的表达与肺，卵巢癌和结肠癌的预后不良和降低的生存率有关。 IMP-1的小分子抑制剂尚未报道。 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站点并进行了成功的飞行员屏幕。为了过滤主要的命中，我们基于纯化的蛋白质，荧光素酶报告器和细胞增殖建立了验证测定。具体目的是：目标1。鉴定具有较高效力和特异性的小分子，以抑制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，并确定抑制纯化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计数器测试每个化合物是否单独降低了FL-MYC探针的信号，然后测试该化合物是否抑制IMP-1与FL-MYC探针的结合。（i）将使用FL-MYC同时验证主要命中率，并测试是否能够抑制IMP-1与FL-MDR1 mRNA结合位点（II）测试特异性的能力，我们将评估抑制孕酮受体（PR）与其FL-DNA响应元件（FL-PRE）结合的命中。（iii）将在剂量反应研究中评估命中的效力和功效。目标2。鉴定铅1 IMP-1抑制剂，以减少癌细胞的生长。最初的基于细胞的测定是基于荧光素酶的小分子的测定，这些分子抑制NF-？B-荧光素酶的表达和我们的荧光素酶MDR1 mRNA嵌合体以及抑制IMP-1阳性IGROV-1和ES-ES-2卵巢癌细胞的增殖，对IMP-1负PC-3细胞产生的影响很小或没有影响。为了接近抑制剂的作用部位，将测试铅化合物，以下调IMP-1蛋白，C-MYC和MDR1 mRNA和蛋白质。使用IMP-1负细胞的微阵列研究将评估铅抑制剂的脱靶作用。将确认铅结构，将铅重新合成，并启动与IMP-1的抑制剂的结构研究。