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

描述(申请人提供)：我们提出了一种新的方法来识别新的探针和潜在的抗癌药物，其基础是通过靶向mRNA结合蛋白IMP-1/CRD-BP/IGF2BP1来减少c-Myc和其他癌基因、MDR1(多药耐药蛋白1)和NF-βB的表达。IMP-1是一种癌胚样mRNA结合蛋白，与编码c-Myc、K-RAS、ERK等癌基因mdr1的mRNAs结合并稳定，间接增加肿瘤激活因子NF-B-catenin和c-Myc诱导的IMP-1活性，是let-7 microRNA的关键调控靶点。通过RNAi敲除或表达let-7miRNA来降低IMP-1的水平，可以降低c-Myc的水平，强烈地抑制细胞增殖，逆转抗癌药物的耐药性。Kaplan-Meier曲线显示，在肺癌、卵巢癌和结肠癌中，IMP-1的表达与预后不良和生存率降低有关。IMP-1的小分子抑制剂尚未见报道。在初步研究中，我们确定了mdr1中的IMP-1结合和mRNA稳定部位，建立了分析IMP-1与其c-Myc和mdr1 mRNA靶标结合的(FAMA)荧光各向异性/偏振微孔板分析方法，建立了基于FAMA的高通量筛选方法(Z‘因子=0.64)，筛选IMP-1与c-Myc mRNA结合位点的抑制剂，并进行了成功的中试筛选。为了筛选初步的HIT，我们建立了基于纯化蛋白、荧光素酶报告和细胞增殖的验证分析。其具体目的是：1.寻找高效、特异的抑制IMP-1与其c-Myc mRNA结合位点结合的小分子。我们将使用大约180,000个小分子进行Fama HTS筛选，并鉴定抑制纯化的IMP-1与荧光素标记的c-Myc mRNA结合位点结合的化合物。为了减少假阳性的数量，我们的两步检测首先使用内部对筛来测试每个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探针的结合。(I)将同时使用FL-Myc验证初级HITS，并测试其抑制IMP-1与FL-MDR1mRNA结合位点的结合的能力。(Ii)为测试特异性，我们将评估HITS抑制孕激素受体(PR)与其FL-DNA反应元件(FL-PRE)的结合。(Iii)HITS的效力和疗效将在剂量反应研究中进行评估。目的2.寻找抑制癌细胞生长的先导型IMP-1抑制剂。最初的基于细胞的检测是基于荧光素酶的检测，检测抑制NF-B-荧光素酶和我们的荧光素酶-MDR1mRNA嵌合体表达的小分子，以及抑制IMP-1阳性的IGROV-1和ES-2卵巢癌细胞的增殖，而对IMP-1阴性的PC-3细胞几乎没有影响。为了接近抑制剂的作用部位，将测试先导化合物是否下调IMP-1蛋白、c-Myc和mdr1基因和蛋白的表达。使用IMP-1阴性细胞的微阵列研究将评估铅抑制剂可能的脱靶效应。铅的结构将得到确认，铅的重新合成和与IMP-1结合的抑制剂的结构研究将启动。

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.