title: Differential selection of acridine resistance mutations in human DNA topoisomerase IIbeta is dependent on the acridine structure.
abstract: 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 betaH514Y, betaE522K, betaG550R, betaA596T, betaY606C, betaR651C, and betaD661N were in the B' domain, and betaG465D and betaP732L were not. With AMCA, four mutations were selected (betaE522K, betaG550R, betaA596T, and betaD661N). Two mutations were selected with mAMCA (betaY606C and betaR651C) and two with mAMSA (betaG465D and betaP732L). 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 (betaG465D, betaH514Y, betaG550R, betaA596T, and betaD661N). betaG465D was selected with both DACA and mAMSA, and betaG550R, betaA596T, and betaD661N 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 (betaG465D, betaE522K, betaG550R, betaA596T, and betaD661N), whereas others were selected only once (betaH514Y, betaY606C, betaR651C, and betaP732L). Here, we compare the drug resistance profile of all nine mutations and report the biochemical characterization of three, betaG550R, betaY606C, and betaD661N.
authors.affiliation: The Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH, United Kingdom.
authors.forename: Chrysoula
authors.initials: C
authors.lastname: Leontiou
authors.forename: Gary P
authors.initials: GP
authors.lastname: Watters
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Gilroy
authors.forename: Pauline
authors.initials: P
authors.lastname: Heslop
authors.forename: Ian G
authors.initials: IG
authors.lastname: Cowell
authors.forename: Kate
authors.initials: K
authors.lastname: Craig
authors.forename: Robert N
authors.initials: RN
authors.lastname: Lightowlers
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Austin
mesh_terms: D000166:Acridines / Q000737:chemistry / Q000494:pharmacology*; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D019020:Directed Molecular Evolution; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D015394:Molecular Structure; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D000166:Acridines; D004268:DNA-Binding Proteins; D004250:DNA Topoisomerases, Type II