The activity of chromenones bearing heteroaryl groups at the

The activity of chromenones bearing heteroaryl groups at the 8-position is summarised in . Replacement of the 8-phenyl substituent of by a thiophen-2-yl group ( and ) did not improve DNA-PK inhibitory activity, although the 4-phenylthiophen-2-yl derivative (), together with the bithiophene analogue (), proved the most potent member of this series (IC=90nM). With the exception of the indolyl derivative (), larger heterocyclic groups on the thiophene ring were detrimental to potency, and where direct comparisons were possible ( with , and with ), replacement of thiophen-2-yl by thiazol-2-yl was not beneficial. Substitution of the 8-phenyl ring of by a 2- or 4-pyridyl group ( and ) resulted in a reduction in potency, and derivatives bearing other heterocycles on the pyridyl ring () were all also less active. In summary, we have identified a novel series of 8-biarylchromen-4-ones as inhibitors of DNA-PK that exhibit a range of potencies against the isolated enzyme. Notably, 8-(3-(thiophen-2-yl)phenyl)chromenone (), the most potent of these inhibitors, was also found to potentiate the cell killing of 2Gy of ionising radiation by a factor of 1.6 when used at concentration of 500nM in a Hela cervical carcinoma cell-based assay. This demonstrates that is cell permeable, and that cellular inhibition of DNA-PK is achievable with this GX-674 at pharmacologically relevant concentrations. Overall, the studies described in this letter have further elucidated an understanding of SARs for DNA-PK inhibition, and will provide a platform for ongoing efforts to optimise potency and in vitro/in vivo activity for this chemotype. Acknowledgment
Introduction Cells are constantly exposed to environmental and metabolic insults such as radiation, chemical agents and perturbation of DNA replication. Such exposure may generate DNA lesions that lead to mutations and DNA breaks and cause genomic instability. Potentially genotoxic lesions are recognized by damage-sensor kinases that are members of the phosphatidylinositol 3-kinase family: ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA–PK).1., 2. Replication-mediated DNA breaks are predominantly recognized by the ATM and ATR kinases, which induce a DNA damage S-phase checkpoint.3., 4., 5. The third kinase, DNA–PK, is primarily involved in the response to double-strand DNA breaks (DSBs) induced by replication independent lesions. In contrast to ATM and ATR, DNA–PK is not directly involved in the activation of the S-phase checkpoint. However, cells deficient in the catalytic subunit of DNA–PK are hypersensitive to replication inhibition by hydroxyurea (HU), suggesting that DNA–PK plays a role in the response to replication perturbation. The role of DNA-PK in the response to DSBs at replication forks has yet to be elucidated. DNA-PK consists of a catalytic subunit (DNA–PKcs) and of the Ku heterodimer (Ku70/Ku80) regulatory subunit. The DNA–PK complex plays a major role in activating non-homologous end-joining (NHEJ) repair in mammalian cells8., 9., 10. and is involved in induction of programmed cell death, telomere maintenance, and innate immunity.6., 9. The Ku subunit first binds to DNA ends and then recruits DNA–PKcs, which can tether broken DNA ends together. The assembled DNA–PK can phosphorylate the histone H2AX in the absence of ATM, forming foci of phosphorylated H2AX (γ-H2AX) in a manner akin to that described for ATM and ATR.12., 13., 14. The assembly of Ku and DNA–PKcs at the sites of DSBs is followed by recruitment of the DNA ligase IV–XRCC4 complex and ligation of the two DNA ends. Mammalian cells have two distinct DNA DSB repair pathways: homologous recombination (HR) and NHEJ. HR requires sequence homology at the sites of DNA breaks and functions at late S-phase and G2 phase when sister chromatids are present. In contrast, NHEJ plays a role at all phases of the cell cycle. HR is the predominant pathway that repairs replication-mediated DSBs7., 15. and plays an important role in the repair of stalled replication forks.16., 17. However, in both human fibroblasts and Chinese hamster ovary cells, the NHEJ pathway recognized DSBs earlier than the HR pathway.18., 19. Interestingly, HR or NHEJ (DNA–PKcs)-deficient Chinese hamster ovary cells are sensitive to HU but only HR-deficient cells are sensitive to thymidine. These observations suggest that the roles of HR and NHEJ in the recognition and repair of lesions caused by replication perturbations may differ depending on the replication stress.