DNA damage tolerance mechanisms promote genome stability and cell survival through bypass of unrepaired blocks to DNA replication. Chemotherapeutic strategies involving platinum-based drugs rely on DNA damage to limit tumor growth. Activation of DNA damage response pathways can modulate patient response to these types of therapies. For example, translesion DNA synthesis (TLS) by human DNA polymerase (pol) eta (η) can render platinum-based chemotherapy ineffective through the direct bypass of DNA damage. Therefore, targeting hpol η with small-molecule inhibitors is a promising strategy for combating chemoresistance in certain types of cancer, such as ovarian cancer, that rely on hpol η to survive genotoxic chemotherapy. The main objective of this study was to investigate mechanisms of TLS and identify small molecule inhibitors of TLS pols that modulate the efficacy of genotoxic anti-cancer drugs in an effort to improve patient outcomes. Towards that end, a quantitative structure activity relationship (QSAR) was performed to develop small molecule inhibitors of hpol η. A fluorescence-based assay was used to measure the catalytic activity of hpol η in the presence of eighty-five novel indole thio-barbituric acid (ITBA) and indole barbituric acid (IBA) derivatives. Several compounds that inhibit hpol η activity with low micromolar IC50 values were identified from this initial screen. One of these compounds, PNR-7-02, an ITBA derivative with both N-napthoyl moiety and 5-chloro substituent on the indole ring inhibited hpol η activity with an IC50 value of 8 μM. PNR-7-02 also inhibited hRev1 and hpol lambda (λ) with low micromolar IC50 values, but inhibition by PNR-7-02 was approximately ten-fold more potent against hpol η than B-family pols. To further improve the specificity of PNR-7-02, several modifications were made to the indole ring to yield PNR-9-59. Substituting the N-napthoyl moiety with N-bromobenzene and the 5-chloro substituent with 4-(2-ethoxyethyl) morpholine moiety improved the specificity for hpol η both within the Y-family and against other pol families. PNR-9-59 showed greater than five-fold specificity for hpol η compared to hpol η and hpol η. Michaelis-Menten kinetic analysis revealed a partial competitive mechanism of inhibition of hpol η activity with PNR-7-02 and a competitive mechanism of inhibition with PNR-9-59. Protein footprinting results identified a region of the little finger domain that contacts the template strand as the binding site for PNR-7-02, which further supported the kinetic mechanism of inhibition. A target-dependent effect on cell viability was observed when comparing hpol η-proficient and hpol η-deficient HAP-1 cells co-treated with cisplatin and PNR-7-02. The calculated combination index values indicated a strong synergy between cisplatin and PNR-7-02 in hpol η-proficient cells. This synergy was not evident in the hpol η knockout cells, supporting the idea that PNR-7-02 modulates cisplatin toxicity in a hpol η-dependent manner. Immunoblotting showed that combination of PNR-7-02 and CDDP also activate DNA damage and replication stress response in cancer cells. Results from cell cycle analysis were consistent with synergy observed between cisplatin and PNR-7-02. In summary, our lab has performed the first QSAR for TLS pols to identify potent inhibitor of hpol η that sensitizes cancer cells to cisplatin treatment.
|Advisor:||Eoff, Robert L.|
|Commitee:||Crooks, Peter A., Davidson, Mari K., Diekman, Alan B., Raney, Kevin D.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 78/12(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry, Oncology|
|Keywords:||Chemotherapy, Cisplatin, DNA, Polymerase eta inhibitors, PrimPol, Translesion DNA synthesis|
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