Development of Selective Inhibitors of DNA Polymerase Delta: A Thesis

This thesis is divided into three parts , united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol S). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs , selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol a) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol S , and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol S inhibitor on cellular DNA synthesis. Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2(pbutylanilino) -2' -deoxyadenosine (BUAdA), and related compounds , of Chinese hamster ovary (CHO) cell (3H) thymidine (( 3H) TdR) incorporation , was investigated. The potency of the compound could largely be explained by its potency (IC = 23 M) as an inhibitor of CHO cell ( H)TdR uptake. BuAdA inhibi ted incorporation by CHO cells of (32p) phosphate into DNA relatively weakly, displaying an IC so value of 80 Differential inhibition of DNA polymerases alpha and del ta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol derived from calf thymus selectively with respect to pol a derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol S weakly, but with greater potency than pol Based on this lead , an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol S with a potency (Ki = 1. M) twenty-two times greater than that displayed for pol a. Kinetic studies indicated that COMDP inhibited pol S competitively with the dNTP specified by the template but not competitively with the template: primer. Analogous experiments with pol a showed that the compound inhibited that enzyme uncompetitively with the dNTP , and not competitively with the template:primer. COMDP was a weak inhibitor of the 3' S' exonuclease activity of pol S displaying an ICSO value greater than 1 ro. Inhibition of permeabilized cell DNA synthesis bv a selective pol S inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (ICSO = 200 ~M) did not clearly indicate the participation of pol in cellular DNA replication. Prospectus. The thesis concludes with a prospectus for the development of pol S inhibitors with improved properties compared to COMDP.