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 t1/2 of approximately 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 rb. This unusual behavior is in contrast to that of cisplatin. BBR3464 produces an unwinding angle of 14 degrees 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 VentR(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: [sequence: see text] 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 profi...

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements

“…Immediately before injection into the MT cell, the drugs were diluted in a 50 mM NaClO 4 buffer to a final concentration of 1 mM. This concentration greatly exceeds the basepair concentration (well below 1 nM) of the DNA present in the capillary after washing, and considering the reported binding kinetics (3,46,47), it is thus sufficient to almost saturate all cross-linking sites. Representative data at different drug concentrations are reported in Figs.…”

“…It is important to note that, as explained above, we follow protocols (3,46,47) that allow the drug to incubate in its most reactive form (in the absence of chloride ions). Other authors (9,19) have performed similar experiments with MT, but they used incubation buffers that inactivate the platinum drugs, and as a consequence, they needed larger drug concentrations to achieve saturation of the DNA binding sites.…”

“…On the basis of the literature, in our incubation conditions, such a half-period is 1.5 h for CIS and BBR3005 (48,49) and 15 min for BBR3464 (3). As a consequence, we assumed that for all the drugs, the reaction with DNA reached saturation within our incubation time.…”

“…The main cellular target of this drug is DNA. Similar to bifunctional alkylating agents, CIS interferes with DNA function by forming cross-links within DNA and between DNA and DNA-associated proteins (e.g., DNA polymerases, DNA topoisomerases, and histones) (3,4). Cellular studies have shown that the most frequent bivalent DNA adducts produced by CIS cross-link adjacent purines within one DNA strand, with a preference for guanines (5,6).…”

“…In addition to monofunctional Pt complexes, multinuclear Pt(II) agents have been developed. These derivatives form long-range (Pt, Pt) inter-and intrastrand cross-links that lead to the formation of a range of DNA adducts; thus, they may improve the spectrum of antitumor activity and confer the ability to overcome CIS resistance (3,12). A few nanomechanical methods have been used so far to analyze the interactions between DNA and platinum-based drugs.…”