Inhibitors of DNA polymerase kappa

DNA 聚合酶 kappa 抑制剂

基本信息

批准号：
8259424
负责人：
R. Stephen Lloyd
金额：
$ 3.85万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-21 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8259424
关键词：
Apoptosis Biochemical Biological Assay Bypass Cell Survival Cell division Cells Chemicals Chemistry Collaborations Collection Complex DNA DNA Binding DNA Binding Agent DNA Damage DNA Interstrand Crosslinking DNA Polymerase I DNA Polymerase Inhibitor DNA biosynthesis DNA-Directed DNA Polymerase Data Data Analyses Dyes Effectiveness Escherichia coli Exclusion Exhibits Exposure to Fluorescence Genetic Recombination Genomics Glioma Growth Individual Informatics Investigation Kinetics Label Lead Libraries Link Measures Mitomycins Modeling Molecular Bank Nature Normal Cell One-Step dentin bonding system Pathway interactions Pharmaceutical Chemistry Pharmaceutical Preparations Polymerase Primer Extension Process Protocols documentation Radio Radiolabeled Reaction Refractory Reporting Screening procedure Series Severities Side Site Small Interfering RNA Specificity Structure Structure-Activity Relationship Surgical incisions Testing Triage United States National Institutes of Health analog base cancer cell cell killing chemotherapeutic agent counterscreen crosslink cytotoxic cytotoxicity endonuclease follow-up high throughput screening human DNA inhibitor/antagonist killings member neoplastic cell public health relevance radiotracer repaired repository single molecule small molecule therapeutic effectiveness thiazole orange trend tumor

项目摘要

DESCRIPTION (provided by applicant): Chemicals that induce interstrand DNA crosslinks are routinely used in chemotherapeutic protocols. When a cell initiates replication on DNAs containing interstrand crosslinks, the complementary strands cannot separate, replication is blocked, cell division is inhibited, and apoptosis may be induced. Although normal cells can initiate repair of interstrand cross links by several mechanisms, some cancer cells are more refractory to the cytotoxic effects of these cross links. One mechanism for increased cellular tolerance is the ability to use specialized DNA polymerases to catalyze DNA synthesis past interstrand cross links after one of the strands has been dually incised around the cross linked site. The first polymerase identified to possess such an activity is human DNA polymerase kappa (pol :). Additionally, the level of expression of pol : increases with the severity of the grade of gliomas and may be correlated with the refractory nature of these tumors to treatment therapies. It is hypothesized that pol : specific inhibitors, given in conjunction with cross linking agents, will increase the therapeutic effectiveness of crosslink-inducing agents. To identify pol : inhibitors, preliminary high throughput screens (HTS) have been conducted on 16,000 compounds in collaboration with the NIH Chemical Genomics Center (NCGC) using a fluorescence-based primer extension and strand-displacement assay. Preliminary hits were identified and verified using a radio labeled primer extension secondary assay that confirmed the robust nature of HTS to identify inhibitors. Tertiary biological assays have been piloted to extend these investigations into cell-based studies. The aims of this investigation are to: 1) conduct HTS of the ~400,000-member Molecular Libraries Small Molecule Repository (MLSMR) collection using the fluorescence- based assay above; 2) counter screen for promiscuous DNA binding agents using a dye displacement assay; 3) counter screen for promiscuous inhibition of other DNA polymerases; 4) conduct radio labeled primer extension orthogonal confirmatory assay; 5) hit expansion by analog purchasing and medicinal chemistry; 6) analyze inhibitor effectiveness in biological assays.) PUBLIC HEALTH RELEVANCE: Chemotherapeutic protocols often include DNA damage-inducing compounds which are bi-functional chemicals that covalently link both strands of DNA, forming interstrand DNA cross links. Cells minimize cytotoxicity to these drugs by replicating past these sites using specialized DNA polymerases. The objective of this investigation is to identify inhibitors of one of the DNA polymerases that increase a cell's tolerance to such chemotherapeutic treatments and thereby enhance tumor cell killing. )

描述（由申请人提供）：诱导链间DNA交联的化学物质通常用于化学治疗方案中。当细胞对含有链间交联的DNA启动复制时，互补链不能分开，复制被阻断，细胞分裂被抑制并诱发凋亡。尽管正常细胞可以通过几种机制来启动链间交叉联系，但某些癌细胞对这些交叉链路的细胞毒性作用更难受。细胞耐受性提高的一种机制是能够使用专门的DNA聚合酶催化DNA合成，而在交叉链接的位点周围两次切开了一条链后的链间交叉连接。鉴定出具有这种活性的第一个聚合酶是人DNA聚合酶kappa（pol :)。另外，POL的表达水平：与神经胶质瘤等级的严重程度增加，并且可能与这些肿瘤的难治性与治疗疗法的难治性有关。假设POL：与交叉链接剂一起给出的特定抑制剂将提高交联诱导剂的治疗有效性。为了识别POL：抑制剂，使用基于荧光的底漆扩展和链 - 置换测定法对NIH化学基因组学中心（NCGC）进行了16,000种化合物的初步高吞吐量筛选（HTS）。使用标记为引物延伸的次级测定的无线电标记的次级测定，确定了HTS的稳健性以鉴定抑制剂的稳健性。三级生物学测定已被试用，以将这些研究扩展到基于细胞的研究中。这项研究的目的是：1）使用上面的基于荧光的测定法进行〜400,000成员分子库的HTS小分子存储库（MLSMR）收集； 2）使用染料位移测定法进行混杂DNA结合剂的反屏幕； 3）反筛查其他DNA聚合酶的滥用； 4）进行无线电标记为底漆延伸正交确认测定； 5）通过模拟购买和药物化学的刺激； 6）分析生物测定中的抑制剂有效性。）公共卫生相关性：化学治疗方案通常包括诱导DNA损伤的化合物，这些化合物是双功能化学物质，可连接DNA的两个链，形成链间DNA交叉链路。细胞通过使用专门的DNA聚合酶复制这些位点来最大程度地减少这些药物的细胞毒性。这项研究的目的是确定一种DNA聚合酶的抑制剂，这些DNA聚合酶增加了细胞对这种化学治疗的耐受性，从而增强了肿瘤细胞的杀伤。）