REGULATION OF GENE EXPRESSION BY DNA BINDING LIGANDS

序列特异性DNA结合小分子，可以渗透人类细胞，可能会调节特定基因的转录。当人们考虑到在未来几年内整个人类基因组将被绘制和测序的事实，再加上生物学和人类医学中将人类疾病与特定基因联系起来的显着发现，对这些DNA结合配体的基础研究可能会导致功能基因组学研究的试剂，重要的是，一类新的人类治疗方法。我们的目标是阐明设计小分子的化学原理，这些小分子以蛋白质的亲和力和特异性结合预定的双螺旋DNA序列，以靶向人类基因组内的预定位点。Py-Im-Hp聚酰胺是一项为期20年的化学计划的结果，旨在了解DNA识别的物理有机原理，是细胞可渗透的配体，其大小仅为蛋白质的百分之几，具有转录因子的亲和力和特异性。这些合成的DNA结合配体已被证明可以穿透人类细胞，运输到细胞核，找到转录活性基因的启动子DNA序列并抑制基因表达。在下一个资助期内，将研究这种方法在聚酰胺结构、大小、细胞类型和不同转录因子家族方面的范围和局限性。癌症和病毒基因对人类健康的重要性将成为目标。关于具体目标，聚酰胺将被设计和合成为：（1）抑制人乳腺癌基因的转录（Her-2/neu），（2）抑制单纯疱疹病毒（HSV）的转录，（3）调节小鼠中的c-fos启动子活性，（4）通过产生作为人工转录激活剂的聚酰胺-肽缀合物来上调转录，（5）抑制大肠杆菌和酵母中的DNA复制，（6）通过设计聚酰胺-丝裂霉素和氮芥缀合物化学修饰基因的编码区，（7）抑制鼠白血病病毒整合到宿主染色体中，和（8）通过具有膜易位肽序列的聚酰胺进一步增强细胞摄取。

Sequence specific DNA binding small molecules that can permeate human cells could potentially regulate transcription of specific genes. When one considers the fact that within the next few years the entire human genome will be mapped and sequenced and, coupled to the remarkable discoveries in biology and human medicine which link human disease to specific genes, fundamental research on these DNA binding ligands could lead to reagents for research in functional genomics and, importantly, a new class of human therapeutics. Our objective has been to elucidate chemical principles for the design of small molecules which bind predetermined double-helical DNA sequences with the affinity and specificity of proteins in order to target predetermined sites within the human genome. The Py-Im-Hp polyamides, the result of a 20-year chemistry program to understand the physical organic principles for DNA recognition, are cell permeable ligands only a few percent the size of a protein which have the affinity and specificity of transcription factors. These synthetic DNA binding ligands have been shown to penetrate human cells, traffic to the nucleus, find the promoter DNA sequences of transcriptionally active genes and inhibit gene expression. In this next funding period, the scope and limitations of this approach with regard to polyamide configuration, size, cell types, and different families of transcription factors will be examined. Cancer and viral genes important in human health will be targeted. With regard to specific aims, polyamides will be designed and synthesized to: (1) inhibit transcription of human breast cancer oncogenes (Her-2/neu), (2) inhibit transcription of Herpes Simplex Virus (HSV), (3) regulate c-fos promoter activity in a mouse, (4) up-regulate transcription by creation of polyamide-peptide conjugates as artificial transcription activators, (5) inhibit DNA replication in E coli and yeast, (6) chemically modify coding regions of genes by the design of polyamide-mitomycin and nitrogen mustard conjugates, (7) inhibit viral integration of murine leukemia virus into a host chromosome, and (8) enhance cell uptake further by polyamides with membrane translocation peptide sequences.