Specific Recognition of G-quadruplexes

G-四链体的特异性识别

基本信息

批准号：
9770818
负责人：
Hanbin Mao
金额：
$ 30.57万
依托单位：
KENT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9770818
关键词：
Address Affinity Apoptosis BCL2 gene Binding Biochemical Biogenesis Bioinformatics Biological Biological Assay Cell division Cells Cellular Assay Circular Dichroism Clinical Clinical Trials Complex DNA DNA Damage DNA Structure DNA-Directed RNA Polymerase Development Disease Dissociation Drug Screening Drug Targeting Electrostatics Elements Ensure Evaluation Event G-Quartets Gene Expression Gene Silencing Genes Genetic Transcription Hour Human Human Genome Kinetics Length Ligand Binding Ligands Link Luciferases Malignant Neoplasms Measures Mechanics Methods Mitochondria Molecular Motor Mutation Nature Normal Cell Nylons Oncogenes Oxidative Stress Patients Pharmaceutical Preparations Pharmacologic Substance Play Process Production Promoter Regions Property Proteins RNA-Directed DNA Polymerase Reactive Oxygen Species Resistance Resolution Role S Phase Side Site Solid Specificity Structure Telomerase Telomerase inhibition Telomere Shortening Testing Therapeutic Thermodynamics Time Translations base c-myc Genes cancer cell cancer therapy cell killing docetaxel ds-DNA efficacy testing fighting gel electrophoresis innovation instrument laser tweezer melanoma nucleic acid structure overexpression promoter prostate cancer cell prostate cancer cell line receptor response screening senescence side effect single molecule small molecule telomere

项目摘要

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.

在这个项目中，我们将测试抑制人端粒酶反向转录的新化合物通过在HTERT启动子中形成的G四链体（GQ）的特异性结合的转录酶（HTERT）。端粒酶在癌细胞中过度表达，延长端粒并避免编程的细胞死亡（凋亡）。当前的端粒酶靶向方法已证明杀死癌细胞的功效导致在临床试验中测试了几种药物。但是，这些方法直接抑制了规范端粒酶的活性，导致端粒的缩短，该端粒仅在长期滞后时间杀死癌细胞不适合治疗应用。该领域的新兴策略是针对端粒酶。我们最近的发现表明，通过抑制HTERT转录，癌细胞被杀死在几天之内通过干扰降低氧化应激的非经典端粒酶活性线粒体并避免凋亡。尽管已证明了人类细胞中G Quadruplex（GQ）的存在，但生物信息学具有揭示了许多肿瘤基因的启动子区域中GQ托管序列的富集。在HTERT核心启动子我们的实验室发现，两个串联GQ的形成可以抑制端粒酶转录。黑色素瘤患者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表达，它提供了有力的证据表明，HTERT启动子中GQ的形成抑制端粒酶表达。我们有还发现，促进这些消音器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形成与端粒酶生产减少之间的联系。特异性提出了通过HTERT启动子中的G四链体靶向抑制端粒酶的主要障碍。在人类基因组中，超过716,000个地点可以形成GQ。由于芳香堆积的通用性和 GQ结合配体中采用的静电相互作用，当前的小分子不识别特定四链体，带来副作用。由于HTERT启动子GQ是双链DNA区域的两侧，我们将共轭四链体结合配体（例如吡啶斯汀）以双链DNA识别元素，例如聚酰胺。双重靶向增加了结合亲和力，但选择性识别相邻的双链DNA为HTERT启动子中的G四链体提供了特异性。我们将使用单一分子机械分析方法，以评估这些新化合物的特异性和亲和力 HTERT GQS。在这些方法中，配体的效力是通过配体的机械稳定性测量的结合的G四链体，作为运动蛋白（例如RNA聚合酶）的机械块。这些单分子结果将通过互补的生化和细胞测定法进一步验证荧光素酶，转录/翻译和端粒酶测定在耐纳税的前列腺癌细胞系上。