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.)
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. )
|Ketkar, Amit; Zafar, Maroof K; Maddukuri, Leena et al. (2013) Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity. Chem Res Toxicol 26:221-32|
|Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen et al. (2013) Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase ?. J Biol Chem 288:14247-55|
|Yamanaka, Kinrin; Dorjsuren, Dorjbal; Eoff, Robert L et al. (2012) A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase ?. PLoS One 7:e45032|