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Differential Selection of Acridine Resistance Mutations in Human DNA Topoisomerase II Is Dependent on the Acridine Structure

The Institute for Cell and Molecular Biosciences, the Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom

Type II DNA topoisomerases are targets of acridine drugs. Nine mutations conferring resistance to acridines were obtained by forced molecular evolution, using methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA), methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and N-[2-(dimethylamino)ethyl]acridines-4-carboxamide (DACA) as selection agents. Mutations H514Y, E522K, G550R, A596T, Y606C, R651C, and D661N were in the B' domain, and G465D and P732L were not. With AMCA, four mutations were selected (E522K, G550R, A596T, and D661N). Two mutations were selected with mAMCA (Y606C and R651C) and two with mAMSA (G465D and P732L). It is interesting that there was no overlap between mutation selection with AMCA and mAMSA or mAMCA. AMCA lacks the methoxy substituent present in mAMCA and mAMSA, suggesting that this motif determines the mutations selected. With the fourth acridine DACA, five mutations were selected for resistance (G465D, H514Y, G550R, A596T, and D661N). G465D was selected with both DACA and mAMSA, and G550R, A596T, and D661N were selected with both DACA and AMCA. DACA lacks the anilino motif of the other three drugs but retains the acridine ring motif. The overlap in selection with DACA and mAMSA or AMCA suggests that altered recognition of the acridine moiety may be involved in these mutations. We used restriction fragment length polymorphisms and heteroduplex analysis to demonstrate that some mutations were selected multiple times (G465D, E522K, G550R, A596T, and D661N), whereas others were selected only once (H514Y, Y606C, R651C, and P732L). Here, we compare the drug resistance profile of all nine mutations and report the biochemical characterization of three, G550R, Y606C, and D661N.

Address correspondence to: Dr. Caroline A. Austin, The Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH, United Kingdom. E-mail: caroline.austin{at}ncl.ac.uk