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Specific Recognition of G-quadruplexes

G-四链体的特异性识别

基本信息

批准号：
10015231
负责人：
Hanbin Mao
金额：
$ 31.52万
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/10015231
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，我们将测试抑制人类端粒酶逆转转录的新化合物