DNA Modifications by a Novel Bifunctional Trinuclear Platinum Phase I Anticancer Agent

Abstract

The DNA-binding profile of a novel, trinuclear platinum Phase I clinical agent (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{H₂N(CH₂)₆NH₂}₂]²⁺ unit. The +4 charge of BBR3464, the presence of at least two Pt coordination units capable of binding to DNA, and the consequences of such DNA binding are remarkable departures from the cisplatin structural paradigm. The chemical and biological features argue that the drug should be considered the first clinical representative of an entirely new structural class of DNA-modifying anticancer agents. The high charge on BBR3464 facilitates rapid binding to DNA with a t_1/2 of ∼40 min, significantly faster than the neutral cisplatin. The melting temperature of DNA adducted by BBR3464 increased at low ionic strength but decreased in high salt for the same r_b. This unusual behavior is in contrast to that of cisplatin. BBR3464 produces an unwinding angle of 14° in negatively supercoiled pSP73 plasmid DNA, indicative of bifunctional DNA binding. Quantitation of interstrand DNA−DNA cross-linking in plasmid pSP73 DNA linearized by EcoRI indicated approximately 20% of the DNA to be interstrand cross-linked. While this is significantly higher than the value for cisplatin, it is, interestingly, lower than that for dinuclear platinum compounds such as [{trans-PtCl(NH₃)₂}₂H₂N(CH₂)₆NH₂]²⁺ (BBR3005) where interstrand cross-linking 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 DNA interstrand cross-linking. Fluorescence experiments with ethidium bromide were consistent with the formation of long-range delocalized lesions on DNA produced by BBR3464. The sequence preference for BBR3464 on plasmid DNA was determined to the exact base pair by assaying extension of the polynucleotide by Vent_R(exo⁺) DNA polymerase. Strong sequence preference for single 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: molecular modeling on 1,4 interstrand (G‘₃₀ to G₃₃) and 1,5 intrastrand (G₃₃ to G₂₉) cross-links further confirmed the similarity in energy between the two forms of cross-link. Finally, immunochemical analysis confirmed the unique nature of the DNA adducts formed by BBR3464. This analysis showed that antibodies raised to cisplatin-adducted DNA did not recognize DNA modified by BBR3464. In contrast, DNA modified by BBR3464 inhibited the binding of antibodies raised to transplatin-adducted DNA. Thus, the bifunctional binding of BBR3464 contains few similarities to that of cisplatin but may have a subset of adducts recognized as being similar to the transplatinum species. In summary, the results point to a unique profile of DNA binding for BBR3464, strengthening the originial hypothesis that modification of DNA binding in manners distinct from that of cisplatin will also lead to a distinct and unique profile of antitumor activity.