DNA-BINDING PROPERTIES OF CIS-[PT(NH3)(C6H11NH2)CL2], A METABOLITE OF AN ORALLY ACTIVE PLATINUM ANTICANCER DRUG

The compound cis,trans,cis-[Pt(NH3)(C6H11NH2)(OC(O)C3H7)2Cl2] (1) is the prototypical member of a new class of orally active platinum anticancer drugs. A major metabolite of this compound, formed after ingestion, is cis-[Pt(NH3)(C6H11NH2)Cl2](2). We have used enzymatic digestion/HPLC analysis to investigate the spectrum of adducts formed by the reaction of this Pt(II) reduction product with calf thymus DNA. The major adduct (54%) formed is an intrastrand cross-link involving adjacent guanosine residues, followed in frequency by interstrand or long range intrastrand cross-links also involving guanosine nucleosides (18%). Unlike cisplatin, 2 forms d(ApG) intrastrand cross-links to only a minor extent (8%). The presence of the cyclohexylamine ligand gives rise to two orientational isomers of the platinated d(GpG) moiety, differing with respect to the positioning of the cyclohexyl group toward either the 3' or the 5' direction of the phosphodiester linkage. These isomers were observed for platination of calf thymus DNA (2:1 ratio) as well as shorter oligonucleotides. The coordination sites of platinum in the adducts were identified by studies of the reaction products of 2 with d(GpG). H-1 and Pt-195 NMR spectroscopy revealed that platinum is bonded to the N7 positions of the two guanine bases. The individual d(GpG)-2 orientational isomers were synthesized and isolated from reagents in which both the ammine ligand and the N7 position of the 3'-guanine base were labeled with N-15. Comparison with the N-15-N-15 coupling constants in the N-15{H-1} NMR spectrum of the two isomers allowed for determination of the stereochemistry at the platinum metal center. From this information the orientational isomer having the cyclohexyl group directed toward the 3' end of the platinated strand was identified as the more abundant of the two d(GpG)-2 isomers formed in the reaction of 2 with calf thymus DNA. This isomer is less disruptive to the hydrogen bonding between the NH3 ligand and the 5'-phosphate group, a structural feature previously identified as being important in the major cisplatin adducts with DNA. Two orientational isomers were also formed upon reaction of 2 with the dodecanucleotide d(TCTAGGCCTTCT), which contains a single d(GpG) platination site. Separation and purification of the two platinated dodecanucleotide orientational isomers allowed for construction of modified M13 genomes containing a single isomer of each of the two d(GpG)-2 adducts. Each purified isomer was incorporated into a gapped heteroduplex, providing the two corresponding isomers of the site-specifically platinated M13 genomes. Studies of the replication of these platinated genomes with T7 DNA polymerase revealed differences in the position within the genome at which DNA synthesis is inhibited. The cis-[Pt(NH3)(C6H11NH2){d(GpG)-N7(1), -N7(2)}] orientational isomers inhibited DNA replication less efficiently than the parent cisplatin complex, allowing more (10-15% versus 8% for cisplatin) translesion synthesis.