Unique base-step recognition by a platinum-acridinylthiourea conjugate leads to a DNA damage profile complementary to that of the anticancer drug cisplatin.
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 = 1-[2-(acridin-9-ylamino)ethyl]-1,3-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, G downward arrow AATTC and TTT downward arrow AAA, 1 inhibited DNA hydrolysis by HindIII and PspOMI at A downward arrow AGCTT and G downward arrow GGCCC (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. 125, 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.
Budiman
Michael E
ME
Department of Chemistry, Wake Forest University, P.O. Box 7486 Reynolda Station, Winston-Salem, North Carolina 27109, USA.
Alexander
Rebecca W
RW
Bierbach
Ulrich
U
D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D010984:Platinum / Q000737:chemistry*; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013054:Spectrophotometry, Atomic; D013890:Thiourea / Q000737:chemistry*; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014508:Urea / Q000031:analogs & derivatives / Q000737:chemistry*; D014764:Viral Proteins
C471910:1-(2-(acridin-9-ylamino)ethyl)-1,3-dimethylthiourea; D014157:Transcription Factors; D014764:Viral Proteins; D010984:Platinum; D012313:RNA; D014508:Urea; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D013890:Thiourea; D002945:Cisplatin