Specific Recognition of G-quadruplexes

在这个项目中，我们将测试抑制人类端粒酶逆转录酶转录的新化合物， 通过特异性结合在hTERT启动子中形成的G-四链体（GQ），在转录酶（hTERT）中表达。 端粒酶在癌细胞中过度表达，延长端粒，避免程序性细胞死亡 （凋亡）。目前的端粒酶靶向方法已经证明了杀死癌细胞的功效， 导致几种药物正在临床试验中进行测试。然而，这些方法直接抑制了典型的 端粒酶的活性，导致端粒缩短，只有在很长一段时间后才能杀死癌细胞， 不适合于治疗应用。该领域的一个新兴战略是针对 端粒酶我们最近的发现表明，通过抑制hTERT转录，癌细胞被杀死， 通过干扰非典型的端粒酶活性，减少氧化应激， 线粒体，避免细胞凋亡。 虽然G-四链体（GQ）在人类细胞中的存在已被证明，但生物信息学已经证实了它的存在。 揭示了GQ宿主序列在许多癌基因的启动子区域中的富集。在hTERT核心 启动子，我们的实验室已经发现形成两个串联的GQ可以抑制端粒酶的转录。 在黑色素瘤患者中发现的突变位于该区域，导致GQ结构受损 和转录沉默的丧失。鉴于端粒酶在黑色素瘤患者中过度表达， hTERT启动子中GQ的形成抑制端粒酶表达的有力证据。我们有 还发现促进这些沉默物GQ折叠的小分子可以降低端粒酶活性， 这证实了hTERT启动子中GQ形成与端粒酶产生减少之间的联系。 特异性是通过G-四链体靶向hTERT启动子抑制端粒酶的主要障碍。 在人类基因组中，超过716，000个位点可以形成GQ。由于芳族堆积的一般性质， 由于GQ结合配体中采用的静电相互作用，目前的小分子不识别特异性 四倍体，这会带来副作用。由于hTERT启动子GQs两侧是双链DNA区域，我们 将共价偶联四链体结合配体，如吡啶抑制素，双链体DNA识别 元素，如聚酰胺。虽然双重靶向增加了结合亲和力，但选择性识别结合蛋白的能力降低了。 相邻的双链体DNA为hTERT启动子中的G-四链体提供特异性。我们将使用单一- 分子机械分析方法来评估这些新化合物的特异性和亲和力， hTERT GQ。在这些方法中，通过配体的机械稳定性来测量配体的效力。 结合的G-四链体，其充当马达蛋白如RNA聚合酶的机械块。这些 单分子结果将通过补充生物化学和细胞测定进一步验证，包括 荧光素酶、转录/翻译和端粒酶测定。

In this project, we will test new compounds that inhibit transcription of human telomerase reverse transcriptase (hTERT) through specific binding of G-quadruplexes (GQs) formed in the hTERT promoter. Over-expressed in cancer cells, telomerase elongates telomere and avoids programmed cell death (apoptosis). Current telomerase targeting approaches have demonstrated efficacy to kill cancer cells, which results in several drugs being tested in clinical trials. However, these approaches directly inhibit the canonical activity of telomerase, leading to the shortening of telomere that kills cancer cells only after a long lag time that is not suitable for therapeutic applications. An emerging strategy in the field is to target the biogenesis of telomerase. Our recent finding has indicated that by inhibition of hTERT transcription, cancer cells are killed within days through interfering with non-canonical telomerase activities that reduce oxidative stress in mitochondria and avoid apoptosis. While the presence of G-quadruplex (GQ) in human cells has been proven, bioinformatics have revealed the enrichment of GQ hosting sequences in promoter regions of many oncogenes. In the hTERT core promoter, our labs have found that formation of two tandem GQs can inhibit telomerase transcription. Mutations identified in melanoma patients are located in this region, resulting in compromised GQ structures and loss of transcriptional silencing. Given that telomerase is overexpressed in melanoma patients, it provides strong evidence that the formation of GQs in the hTERT promoter inhibits telomerase expression. We have also found that small molecules facilitating the folding of these silencer GQs can decrease telomerase activity, which corroborates the link between GQ formation in hTERT promoter and reduced telomerase production. Specificity presents a major hurdle to inhibit telomerase via G-quadruplex targeting in hTERT promoter. In human genome, over 716,000 sites can form GQs. Due to the generic nature of aromatic stacking and electrostatic interactions employed in GQ-binding ligands, current small molecules do not recognize specific quadruplexes, which brings side effects. Since hTERT promoter GQs are flanked by duplex DNA regions, we will covalently conjugate quadruplex-binding ligands, such as pyridostatin, to duplex DNA recognition elements, such as polyamides. While the dual targeting increases binding affinity, selective recognition of neighboring duplex DNA offers specificity for the G-quadruplexes in the hTERT promoter. We will use single- molecule mechanoanalytical approaches to evaluate the specificity and affinity of these new compounds to hTERT GQs. In these methods, the potency of a ligand is measured by the mechanical stability of ligand- bound G-quadruplexes, which serve as mechanical blocks to motor proteins such as RNA polymerase. These single-molecule results will be further validated by complementary biochemical and cellular assays including luciferase, transcription/translation, and telomerase assays on Taxotere-resistant prostate cancer cell lines.