Inhibitors of DNA polymerase kappa

描述（由申请人提供）：诱导链间DNA交联的化学品通常用于化疗方案。当细胞在含有链间交联的DNA上启动复制时，互补链不能分离，复制被阻断，细胞分裂被抑制，并且可以诱导细胞凋亡。虽然正常细胞可以通过几种机制启动链间交联的修复，但一些癌细胞对这些交联的细胞毒性作用更难抵抗。增加细胞耐受性的一种机制是在一条链在交联位点周围被双重切割后，使用专门的DNA聚合酶催化DNA合成通过链间交联的能力。第一个被鉴定为具有这种活性的聚合酶是人DNA聚合酶κ（pol：）。此外，pol：的表达水平随着神经胶质瘤等级的严重程度而增加，并且可能与这些肿瘤对治疗疗法的难治性相关。据推测，pol：特异性抑制剂与交联剂联合给药将增加交联诱导剂的治疗有效性。要标识波尔，请执行以下操作：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测定对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级测定法鉴定并验证初步命中，该测定法证实了HTS鉴定抑制剂的稳健性。三级生物测定已被试行，以扩大这些调查到基于细胞的研究。本研究的目的是：1）使用上述基于荧光的测定对约40万成员的分子库小分子库（MLSMR）集合进行HTS; 2）使用染料置换测定对混杂DNA结合剂进行反筛选; 3）对其它DNA聚合酶的混杂抑制进行反筛选; 4）进行放射性标记引物延伸正交验证测定; 5）通过类似物购买和药物化学来扩大命中率; 6）在生物测定中分析抑制剂的有效性。 公共卫生相关性：化学疗法方案通常包括DNA损伤诱导化合物，其是共价连接DNA的两条链的双功能化学品，形成链间DNA交联。细胞通过使用专门的DNA聚合酶复制经过这些位点来最小化对这些药物的细胞毒性。本研究的目的是鉴定一种DNA聚合酶的抑制剂，其增加细胞对这种化疗治疗的耐受性，从而增强肿瘤细胞的杀伤。)

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. )