Mammalian cells have two distinct DNA repair pathways
Mammalian erk definition have two distinct DNA repair pathways for DSBs, NHEJ and HR. Our data are consistent with the suggestion that the DNA–PK-mediated NHEJ pathway recognizes DSBs faster than the HR pathway and acts before the activation of the DNA damage S-phase checkpoint. The activation of NHEJ by DNA-PK and XRCC4 before recruitment of Rad51 to sites of replication inhibition might underlie the roles of DNA–PK and XRCC4 in suppressing spontaneous HR when spontaneous HR is caused by stalled or collapsed replication forks.15., 60. Our DNA fiber analysis suggests that when DNA–PK is active, replication can proceed slowly even in the presence of low levels of DNA polymerase inhibition. Presumably, under these conditions, low levels of DNA breaks are repaired via the NHEJ pathway. Although we cannot formally rule out that DNA–PK is involved in another repair pathway unrelated to NHEJ, our data are consistent with the suggestion that the ATR-Chk1-mediated S-phase checkpoint is activated in the absence or insufficiency of NHEJ and that the active S-phase checkpoint serves to stabilize replication forks to facilitate the alternative HR repair pathway.
Materials and Methods
DNA-dependent protein kinase (DNA-PK), a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family, is a multi-component serine/threonine protein kinase that plays a key role in the repair of mammalian DNA double-strand breaks (DSBs) via the non-homologous end joining pathway of DNA repair., Human cell lines defective in DNA-PK function are hypersensitive to agents that elicit DNA DSBs., By impeding DNA DSB repair, selective DNA-PK inhibitors have potential application as radio- and chemo-potentiators in the treatment of cancer., , , , In the absence of structural biology information for the enzyme, we have conducted extensive structure–activity relationships studies (SARs) using homology modelling based on the known crystal structure of PI3Kγ and employing 2-morpholino-8-phenyl-4-chromen-4-one (, LY294002) as a template for inhibitor design. Interestingly, a number of potent DNA-PK inhibitors have been developed from this structural class., , , , Previously we have described the incorporation of a dibenzothiophen-4-yl substituent at the 8-position of 2-morpholino-4-chromen-4-one, which conferred excellent inhibitory activity against DNA-PK (, IC=28nM). Crucially, chromenone has been demonstrated to sensitise a human tumour cell line to both ionising radiation and the topoisomerase II inhibitor etoposide in vitro and in vivo. Further refinement of this template has provided the highly potent DNA-PK inhibitor KU-0060648 (; IC=5nM). With a view to optimising the biological and pharmaceutical properties of NU7441 (), and to expand SARs, both the core chromenone scaffold and the dibenzothiophen-4-yl moiety have been systematically modified. To this end, we have recently reported the identification of 8-biarylchromen-4-ones (e.g., ; IC=18nM), which offered the opportunity to further probe regions of the ATP-binding domain of the enzyme and exhibited good potency against DNA-PK. Interestingly, subsequent homology modelling studies suggested that the heteroaryl substituent may occupy a putative hydrophobic pocket that could be further exploited. In this communication, we report studies designed to probe this region of the ATP-binding site of the kinase, through the synthesis and biological evaluation of a focused library of chromen-4-one DNA-PK inhibitors bearing -alkoxyphenyl substituents at the 8-position. Our previous studies have utilised the chromenone triflate , , or boronic ester as key intermediates for the preparation of 8-substituted chromenone libraries, employing Suzuki–Miyaura cross-coupling reactions. This strategy was also amenable for the synthesis of the target -alkoxyphenylchromen-4-ones (–, and –). Introduction of the 8-phenyl substituent was achieved by coupling of , formed in situ by reaction of with bis(pinacolato)diboron, with 4-bromo-2-fluoronitrobenzene to give the chromen-4-one in 58% yield (). The 3-alkoxy side chain was then introduced in good overall yield by a fluoro-displacement reaction, using an alkoxide generated through treatment of the respective alcohol with sodium hydride in DMF. Reduction of the nitro intermediates and to the required arylamines (, ) proceeded in excellent yields using zinc in acetic acid. Finally, in a one-pot two-step process, acylation of – with chloroacetyl chloride was followed by reaction with morpholine to provide inhibitors – (). In this series, the choice of a morpholine-substituted side chain was based upon previous studies which demonstrated that the parent biaryl derivative exhibited good inhibitory activity against DNA-PK (IC=14nM) and potentiated the DNA damage elicited by ionising radiation at 2Gy (HeLa cells, DMR (0.5μM)=8 and DMR (0.1μM)=11).