DNA modifications by a novel bifunctional trinuclear platinum Phase I anticancer agent

The DNA-binding profile of a novel, trinuclear platinum Phase I clinical ag ent (BBR3464) is summarized. The structure of BBR3464 is best described as two trans-[PtCl(NH3)(2)] units linked by a tetra-amine [trans-Pt(NH3)(2){H2 N(CH2)(6)NH2}(2)](2+) unit. The +4 charge of BBR3464, the presence of at le ast two Pt coordination units capable of binding to DNA, and the consequenc es of such DNA binding are remarkable departures from the cisplatin structu ral paradigm. The chemical and biological features argue that the drug shou ld be considered the first clinical representative of an entirely new struc tural class of DNA-modifying anticancer agents. The high charge on BBR3464 facilitates rapid binding to DNA with a t(1/2) of similar to 40 min, signif icantly faster than the neutral cisplatin. The melting temperature of DNA a dducted by BBR3464 increased at low ionic strength but decreased in high sa lt for the same rb. This unusual behavior is in contrast to that of cisplat in. BBR3464 produces an unwinding angle of 14 degrees in negatively superco iled pSP73 plasmid DNA, indicative of bifunctional DNA binding. Quantitatio n of interstrand DNA-DNA cross-linking in plasmid pSP73 DNA linearized by E coRI indicated approximately 20% of the DNA to be interstrand cross-linked. While this is significantly higher than the value for cisplatin, it is, in terestingly, lower than that for dinuclear platinum compounds such as [{tra ns-PtCl(NH3)(2)}(2)H2N(CH2)(6)NH2](2+) (BBR3005) where interstrand cross-li nking efficiency may be as high as 70-90%. Either the presence of charge in the linker backbone or the increased distance between platinating moieties may contribute to this relatively decreased ability of BBR3464 to induce D NA interstrand cross-linking. Fluorescence experiments with ethidium bromid e were consistent with the formation of long-range delocalized lesions on D NA produced by BBR3464. The sequence preference for BBR3464 on plasmid DNA was determined to the exact base pair by assaying extension of the polynucl eotide by Vent(R)(exo(+)) DNA polymerase. Strong sequence preference for si ngle dG or d(GG) sites was suggested. The presence of relatively few blocks on DNA in comparison to either cisplatin or BBR3005 was indicative of high sequence selectivity. The following appropriate sequence where stop sites occur was chosen: 5'-T'(23) G'(24) A'(25) A'(26) T'(27) T'(28) C'(29) G'(30) A'(31) G'(32) C' (33) T'(34) C'(35) G'(36) G'(37) T'(38) A'(39) 3'- A(23) C-24 T-25 T-26 A(2 7) A(28) G(29) C-30 T-31 C-32 G(33) A(34) G(35) C-36 C-37 A(38) T-39 molecular modeling on 1,4 interstrand (G'(30) to G(33)) and 1,5 intrastrand (G(33) to G(29)) cross-links further confirmed the similarity in energy be tween the two forms of cross-link. Finally, immunochemical analysis confirm ed the unique nature of the DNA adducts formed by BBR3464. This analysis sh owed that antibodies raised to cisplatin-adducted DNA did not recognize DNA modified by BBR3464. In contrast, DNA modified by BBR3464 inhibited the bi nding of antibodies raised to transplatin-adducted DNA. Thus, the bifunctio nal binding of BBR3464 contains few similarities to that of cisplatin but m ay have a subset of adducts recognized as being similar to the transplatinu m species. In summary, the results point to a unique profile of DNA binding for BBR3464, strengthening the originial hypothesis that modification of D NA binding in manners distinct from that of cisplatin will also lead to a d istinct and unique profile of antitumor activity.