Polyamide-Chl inhibition of cancer cell proliferation

本申请旨在开发一类新的小分子疗法 人类癌症。合成的吡咯-咪唑聚酰胺已被证明可以结合预定的 具有亚纳摩尔亲和力的DNA序列，可与天然 转录调节蛋白；此外，这些分子抑制转录因子-DNA 在体外和培养细胞中的相互作用和基因表达。一种特殊的聚酰胺-DNA 烷化剂(氯苯丁腈)共轭化合物已被确定能改变形态和生长 结肠癌细胞在培养过程中的特性及其在G2/M期的滞留 处于细胞周期的阶段，没有任何明显的细胞毒性。用这种化合物处理的细胞不能 在软琼脂中生长，在裸鼠体内不形成肿瘤，这表明聚酰胺处理的细胞不再具有致瘤性。重要的是，这种化合物在体内是活跃的，逆转了增殖 免疫低下小鼠转移性结肠癌细胞的潜能。基因芯片分析结果表明，只有一个基因被该聚酰胺显著下调，RT-PCR和Western blotting实验证实该聚酰胺确实下调了组蛋白H4的mRNA和蛋白的表达。为了跟进这些观察，将在细胞培养和各种人类癌症的动物模型中筛选聚酰胺-氯氨丁苯偶联物对癌细胞增殖的影响。抑制癌细胞生长的聚酰胺的基因组靶点将使用高密度DNA微阵列/杂交方法进行评估，并通过其他方法进行验证。聚酰胺抑制转录的机制将在体外、细胞培养和动物模型中进行评估。

This application is aimed at the development of a new class of small molecule therapeutics for human cancer. Synthetic pyrrole-imidizole polyamides have been shown to bind predetermined DNA sequences with subnanomolar affinities, comparable to the binding affinities of natural transcriptional regulatory proteins; moreover, these molecules inhibit transcription factor-DNA interactions and gene expression both in vitro and in cultured cells. A specific polyamide-DNA alkylator (chlorambucil) conjugate has been identified that alters the morphology and growth characteristics of colon carcinoma cells in culture, and causes these cells to arrest in the G2/M stage of the cell cycle, without any apparent cytotoxicity. Cells treated with this compound fail to grow in soft agar, and do not form tumors in nude mice, indicating that polyamide-treated cells are no longer tumorigenic. Importantly, this compound is active in vivo, reversing the proliferating potential of metastatic colon carcinoma cells in immunocompromised mice. Microarray analysis has given us the striking result that only one gene is significantly down regulated by this polyamide, and RT-PCR and western blotting experiments confirm that histone H4 mRNA and protein is indeed down regulated by this polyamide. To follow up on these observations, polyamide-chlorambucil conjugates will be screened for their effects on cancer cell proliferation both in cell culture and in animal models for various human cancers. The genomic targets of polyamides that inhibit cancer cell growth will be assessed using high density DNA microarray/hybridization methodology, and verified by other methods. The mechanism of inhibition of transcription by polyamides will be assessed both in vitro, in cell culture and in animal models.