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

描述（由申请人提供）：我们提出了一种新的方法，以确定新的探针和潜在的抗癌药物的基础上减少表达的c-Myc和其他癌基因，MDR 1（多药耐药蛋白1）和NF-？B通过靶向mRNA结合蛋白IMP-1/CRD-BP/IGF 2BP 1。IMP-1是一种癌胚mRNA结合蛋白，其结合并稳定编码c-Myc、K-Ras、ERK和其他癌基因、MDR 1的mRNA，并间接增加肿瘤使能因子NF-？B。?- catenin和c-Myc诱导IMP-1，并且IMP-1是let-7 microRNA的关键调节靶标。通过RNAi敲低或通过let-7 miRNA的表达降低IMP-1水平，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曲线显示IMP-1的表达与肺癌、卵巢癌和结肠癌的预后不良和生存率降低相关。IMP-1的小分子抑制剂尚未报道。在初步研究中，我们确定了IMP-1在MDR 1 mRNA中的结合和mRNA稳定位点，建立了（法马）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MDR 1 mRNA靶点的结合，开发了一个强大的（Z'因子=0.64）基于FAMA的IMP-1与c-受体结合的抑制剂的高通量筛选Myc mRNA结合位点并进行了成功的中试筛选。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。鉴定抑制IMP-1与其c-Myc mRNA结合位点结合的高效价和特异性小分子。我们将使用约180，000个小分子实施法马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降低来自fl-Myc探针的信号，然后测试化合物是否抑制IMP-1与fl-Myc探针的结合。(i)使用fl-Myc同时验证初步命中，并测试抑制IMP-1与fl-MDR 1 mRNA结合位点结合的能力。（ii）为了测试特异性，我们将评价抑制孕酮受体（PR）与其fl-DNA反应元件（fl-PRE）结合的命中。(iii)将在剂量反应研究中评价命中的效价和有效性。目标2.确定减少癌细胞生长的IMP-1抑制剂。最初的细胞为基础的检测是基于酶的检测小分子抑制NF-？B-荧光素酶和我们的荧光素酶-MDR 1 mRNA嵌合体，并抑制IMP-1阳性IGR-1和ES-2卵巢癌细胞的增殖，对IMP-1阴性PC-3细胞几乎没有或没有影响。为了接近抑制剂的作用位点，将检测先导化合物对IMP-1蛋白、c-Myc和MDR 1 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的可能脱靶效应。将确认电极导线结构，重新合成电极导线，并启动与IMP-1结合的抑制剂的结构研究。

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.