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上启动复制时，互补的链不能分离，复制被阻止，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几种机制启动链间交链的修复，但一些癌细胞对这些交链的细胞毒性作用更难耐受。提高细胞耐受性的一个机制是，在其中一条链在交联点周围被双重切割后，使用专门的DNA聚合酶催化DNA合成通过链间交联链。第一个被鉴定具有这种活性的聚合酶是人类DNA聚合酶kappa(Pola：)。此外，Pol1的表达水平随着胶质瘤级别的严重程度而增加，并可能与这些肿瘤对治疗方法的难治性有关。据推测，POL：特定的抑制剂与交联剂联合给予将增加交联剂的治疗效果。为了识别PolI：抑制剂，与NIH化学基因组中心(NCGC)合作，使用基于荧光的引物延伸和链置换分析，对16,000种化合物进行了初步高通量筛选(HTS)。使用放射性标记的引物延伸二次化验鉴定和验证初步命中，该二次化验证实了HTS用于鉴定抑制剂的强健性质。已经试行了第三级生物检测，以将这些研究扩展到基于细胞的研究。本研究的目的是：1)使用上述基于荧光的方法对~40万成员的分子文库小分子文库(MLSMR)进行HTS；2)使用染料置换法进行混杂DNA结合剂的计数器筛选；3)其他DNA聚合酶的混杂抑制的计数器筛选；4)进行放射性标记的引物延伸正交验证性试验；5)通过模拟购买和药物化学的HIT扩增；6)在生物检测中分析抑制剂的有效性。) 公共卫生相关性：化疗方案通常包括DNA损伤诱导化合物，这些化合物是双功能化学物质，共价连接DNA的两条链，形成链间DNA交联链。细胞通过使用特殊的DNA聚合酶复制通过这些位点，将对这些药物的细胞毒性降至最低。这项研究的目的是确定一种DNA聚合酶的抑制剂，它可以增加细胞对这种化疗的耐受性，从而增强肿瘤细胞的杀伤力。)

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. )