Abstract

O4-Alkyl lesions are a form of DNA damage that occurs due to exposure to some alkylating agents, which can be repaired by O6-Alkylguanine DNA alkyltransferases (AGTs). AGTs from different species differ in their substrate specificity and proficiency towards repair of these lesions. O4-alkyl-pyrimidines with C5-modifications were prepared by using a combination of small molecule and solid phase synthesis in order to investigate their repair by different AGT homologues such as human AGT (hAGT), E. coli OGT and Ada-C and a hAGT/OGT chimera (hOGT). Studies have shown that hAGT is less efficient at removing O4 lesions than the E. coli variants. Previous work from our group revealed that replacing the C5-methyl group with hydrogen results in an increase in the proficiency of repair of O4-alkyl lesions observed by the human homologue. It was observed that the presence of a C5-fluorine atom resulted in a futher increase in repair of a O4-methyl lesion, particularly by hAGT and the chimera. Repair of the O4-methyl lesion increased about 20-fold after 1 min with 5 equivalents of AGT when fluorine rather than hydrogen was at the C5-position. Repair of larger lesions at the O4 position such as ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl were also evaluated for analogs with fluorine at the C5 position. Interestingly, all substrates were repaired efficiently. Ada-C was able to repair larger adducts such as O4-benzyl and O4-4-hydroxybutyl. Human AGT showed a preference for the benzyl adduct, as observed previously in the literature. Smaller adducts (such as O4-ethyl) were repaired faster than the larger ones by the various AGTs with O4-7-hydroxyheptyl repair observed to occur more efficiently relative to its 4 carbon analogue presumably due to a more flexible chain allowing for a more optimal accommodation for the removal of the lesion. Other halogens such as chlorine and bromine at the C5 position were also analyzed to observed their influence relative to fluorine. Repair of O4-methyl lesions with chlorine at the C5-position occurred faster relative to bromine. The larger C5-bromine did influence repair with a maximum of 55% removal of the O4-methyl lesion by the AGTs observed. The presence of chlorine at the C5-position had a positive influence on removal of the O4-methyl group by human AGT with slightly faster repair observed compared to the C5-fluorine analog. Overall, the presence of the C5-fluorine group was shown to have a positive influence on O4-methyl repair by AGT, distinct from the influence of chlorine or bromine at the C5-position. These studies have expanded our knowledge of the range of substrates that can be repaired by AGTs and may be useful in guiding the design of hAGT inhibitors as therapeutics against uncontrolled cellular proliferation seen in cancer.