DNA-binding polyamides as Hedgehog inhibitors in human carcinoma

描述（由申请人提供）：癌症仍然是公共卫生的一个重要问题，2009年估计有近150万新病例和50万死亡病例。最近，在包括乳腺癌、前列腺癌和肺癌在内的大量人类癌症中，已经报道了Hedgehog （HH）信号通路的过度活性。此外，一些研究支持HH通路在恶性细胞的增殖、存活和转移过程中具有活性。因此，HH信号是治疗开发的一个有吸引力的靶点，值得在细胞培养模型中进一步进行机制研究，以评估其在癌症发生和进展中的作用。在正常的HH信号传导中，外源性HH配体与细胞表面受体结合，导致信号级联，最终激活Gli2转录因子。Gli2可促进超过130个基因的转录，其中一些具有抗凋亡和促增殖活性。这些基因的选择性调节将使研究人员能够仔细地探测过度活跃的HH信号的下游影响。dna结合聚酰胺是一种小分子，是研究基因特异性转录抑制的独特有效工具。通过杂环和脂肪残基的可编程组合，聚酰胺结构以高序列选择性和亲和力结合DNA的小凹槽。外源添加后，聚酰胺已被证明通过干扰细胞核中转录因子- dna相互作用来调节癌细胞培养中的基因表达。因此，我们建议开发一个针对Gli2调控基因的聚酰胺小文库，以便在基因、全基因组和细胞水平上研究HH信号在癌症中的作用。该提案的具体目标是：1)开发一个小的聚酰胺库，以序列特异性的方式中断Gli2- DNA相互作用；2)研究这些化合物在健康和肿瘤细胞培养中改变mRNA表达水平的能力；3)评估聚酰胺转录抑制剂对全基因组表达、细胞增殖和细胞活力的影响。首先，我们将合成六种针对癌症相关基因的聚酰胺，并在体外测量它们的dna结合亲和力以及它们从这些基因的启动子序列中取代Gli2的能力。然后，我们将在健康细胞以及基底细胞癌和前列腺癌细胞模型中研究这些化合物对其靶基因mRNA水平的影响，以及它们与染色质结合DNA结合的能力。聚酰胺转录抑制的全基因组效应将使用标准微阵列进行分析，总体细胞效应将通过显微镜、商业增殖试验和细胞分选进行测量。这些结果将与使用通用HH通路抑制剂的类似研究进行比较。Gli2抑制聚酰胺的开发将为研究HH信号在癌症中的作用提供新的工具，并有助于深入了解这一途径在基因靶向癌症治疗中的适用性。

DESCRIPTION (provided by applicant): Cancer remains an important concern for public health, with nearly 1.5 million new cases and 500,000 deaths estimated for 2009. Recently, overactivity of the Hedgehog (HH) signaling pathway has been reported in a large number of human carcinomas, including breast, prostate, and lung cancer. In addition, several studies support the requirement of an active HH pathway for the proliferation, survival and metastasis of malignant cells. HH signaling is therefore an attractive target for therapeutic development and merits further mechanistic investigation in cell culture models to evaluate its role in the development and progression of cancer. In normal HH signaling, exogenous HH ligands bind to cell surface receptors, resulting in a signaling cascade that culminates in the activation of the Gli2 transcription factor. Gli2 promotes transcription of over 130 genes, several with anti-apoptotic and proliferation-promoting activity. Selective regulation of these genes would allow researchers to carefully probe the downstream effects of overactive HH signaling. DNA-binding polyamides are small molecules that represent uniquely effective tools for the investigation of gene-specific transcriptional inhibition. Through a programmable combination of heterocyclic and aliphatic residues, polyamide structures bind the minor groove of DNA with high sequence selectivity and affinity. Upon exogenous addition, polyamides have been shown to regulate gene expression in cancer cell cultures by interfering with transcription factor-DNA interactions in the nucleus. It is thus proposed that the development of a small library of polyamides targeted to genes regulated by Gli2 will allow for the role of HH signaling in cancer to be studied at the gene, genome-wide, and cellular level. The specific aims of this proposal are: 1) to develop a small library of polyamides that interrupt Gli2- DNA interactions in a sequence-specific manner; 2) to investigate the ability of these compounds to alter mRNA expression levels in healthy and tumor cell cultures; and 3) to assess the effect of polyamide transcriptional inhibitors on global genome expression, cell proliferation and cell viability. To begin, we will synthesize six polyamides targeted to cancer-relevant genes and measure their DNA-binding affinity as well as their ability to displace Gli2 from the promoter sequences of these genes in vitro. We will then study the effects of these compounds on the mRNA levels of their target genes, as well as their ability to bind to chromatin- bound DNA, in healthy cells as well as basal cell carcinoma and prostate cancer cell models. The genome wide effects of polyamide transcriptional inhibition will be analyzed using standard microarrays, and the gross cell effects will be measured through microscopy, commercial proliferation assays, and cell sorting. These results will be compared to similar studies with a generic HH pathway inhibitor. The development of Gli2- inhibiting polyamides will provide novel tools for the investigation of HH signaling in cancer and lend insight into the suitability of this pathway for gene-targeted cancer therapy.