Chemosensitizing effects and Molecular Dynamic study of Substituted Benzo1,3-oxazines as Novel DNA-PK, PI3K Inhibitors

Submission note: A thesis submitted in total fulfilment of the requirements for the Degree of Doctor of Philosophy to the School of Pharmacy and Applied Science, Faculty of Science, Technology and Engineering, La Trobe University, Bendigo.

A theoretically agreed concept to fight against tumour resistance is to use chemosensitizers that selectively sensitize tumour cells to chemotherapeutic drugs without affecting normal tissue. In this study, the chemosensitizing potential of novel benzoxazine derivatives in combination with Doxorubicin, a DNA damaging chemotherapeutic drug was evaluated. The primary aims were to elucidate the molecular pathways by which these compounds impart chemosensitization and to set down the particular protein and ligand determinants that explain the enzyme specificity of these compounds. The results of this study confirmed that LTUSi116 and LTUR6 were potent chemosensitizers of Doxorubicin in colon cancer cell lines, HCT116 and HT29; the possible mechanism being inhibition of DNA double strand breaks (DSB) repair. Results also showed that both LTUSi116 and LTUR6 delayed the resolution of Doxorubicin-induced γH2AX, a specific marker of unrepaired DNA DSB, and prolonged cell cycle arrest in both cell lines. This eventually led to DNA fragmentation, caspase activation and ultimately apoptosis in LTUSi116 and LTUR6 co-treated cell lines. Results of western blot analysis revealed that LTUSi116 and LTUR6 significantly inhibited the phosphorylation of DSB repair enzymes DNA-PK and AKT, in response to Doxorubicin-induced DSB. Further to this, protein array analyses uncovered changes to a subset of DNA-PK/AKT substrates affecting cell cycle arrest and apoptosis which help to explain the mechanism of action of these inhibitors. Molecular dynamics studies unravelled the ligand determinants of PI3K subtype specificity of these inhibitors enabling an explanation of the PI3Kδ specificity exhibited by LTUR6. Overall, the identification of molecular pathways through which these inhibitors cause chemosensitization combined with computational analysis of their interactions with target enzymes will aid in the development of new, potent and specific PI3K/AKT inhibitor as lead candidates for drug discovery.