Inhibitors of DNA polymerase kappa

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疗方案。当细胞在含有链间交联的dna上开始复制时，互补链不能分离，复制被阻断，细胞分裂被抑制，并可能诱导细胞凋亡。虽然正常细胞可以通过多种机制启动链间交联的修复，但一些癌细胞对这些交联的细胞毒性作用更为顽固。增加细胞耐受性的一种机制是，在其中一条链在交联位点周围被双切口后，利用专门的DNA聚合酶催化DNA合成通过链间交联的能力。第一个确定具有这种活性的聚合酶是人类DNA聚合酶kappa （pol:）。此外，pol：的表达水平随着胶质瘤等级的严重程度而增加，并可能与这些肿瘤对治疗方法的难治性有关。假设pol：特异性抑制剂与交联剂联合使用，将增加交联诱导剂的治疗效果。为了鉴定pol：抑制剂，与NIH化学基因组学中心（NCGC）合作，使用基于荧光的引物延伸和链位移测定法对16,000种化合物进行了初步的高通量筛选（HTS）。使用无线电标记引物扩展二级试验确定并验证了初步命中点，证实了HTS识别抑制剂的强大特性。三级生物分析已试行将这些调查扩展到基于细胞的研究。本研究的目的是：1)利用上述基于荧光的分析方法对约40万分子库小分子库（MLSMR）进行HTS；2)用染料置换法筛选混杂的DNA结合剂；3)混杂抑制其他DNA聚合酶的反筛；4)进行无线电标记引物延伸正交验证试验；5)通过模拟物采购和药物化学进行冲击扩张；6)在生物试验中分析抑制剂的有效性。

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. )