Unique Base-Step Recognition by a Platinum-Acridinylthiourea Conjugate Leads to a DNA Damage Profile Complementary to That of the Anticancer Drug Cisplatin

摘要

The sequence specificity and time course of covalent DNA adduct formation of the novel platinum-acridine conjugate [PtCl(en)(ACRAMTU)](NO_3)_2 [PT-ACRAMTU,2;en=ethane-1,2-diamine,ACRAMTU=l-[2-(acridin-9-ylamino)ethyl]-l,3-dimethylthiourea] have been investigated using restriction enzyme cleavage and transcription footprinting assays and compared to the damage produced by the clinical agent cis-diamminedichloroplatinum(II) (cisplatin,1).The rate of DNA binding of 1 and 2 was also monitored by atomic emission spectrometry.Restriction enzymes were chosen that cleave the phosphodiester linkage at,or adjacent to,the predicted damage sites.While conjugate 2 selectively protected supercoiled plasmid from cleavage by EcoRI and DraI enzymes at their respective restriction sites,Garrow doamAATTC and TTTarrow downAAA,1 inhibited DNA hydrolysis by HindIII and PspOMI at Aarrow dowmAGCTT and Garrow dowmGGCCC (arrows mark cleavage sites) more efficiently.Transcription footprinting using T7 RNA polymerase revealed major single-base damage sites for 2 at adenine in 5'-TA and 5'-GA sequences.In addition,the enzyme is efficiently stalled at guanine bases,primarily in the sequence 5'-CGA where the damaged nucleobase is flanked by two high-affinity intercalation sites of ACRAMTU.While 1 targets poly(G) sequences,the binding of 2 appears to be dominated by the groove and sequence recognition of the intercalator.The biochemical assays used confirm previous structural information extracted from mass spectra of DNA fragments modified by 2 isolated from enzymatic digests [Barry,C.G.,et al.(2003) J.Am.Chem.Soc.725,9629-9637].Possible DNA-binding mechanisms and biological consequences of the unprecedented modification of alternating TA sequences by 2,which occurred at a faster rate than binding to G,are discussed.