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...

Reported here is a detailed study of the kinetics and mechanism of formation of a 1,4 GG interstrand cross-link by [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(n)NH(2))](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of platinum antitumor complexes. The reaction of the self-complementary 12-mer duplex 5'-[d(ATATGTACATAT)(2)] with (15)N-1 has been studied at 298 K, pH 5.4, by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Initial electrostatic interactions with the duplex are observed for 1 and the monoaqua monochloro species (2). Aquation of 1 to yield 2 occurs with a pseudo-first-order rate constant of (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding to the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.47 +/- 0.06 M(-(1) s(-1). There is an electrostatic interaction between the unbound [PtN(3)Cl] group of 3 and the duplex, which is consistent with H-bonding interactions observed in the molecular model of the monofunctional (G/Cl) adduct. Closure of 3 to form the 1,4 GG interstrand cross-link (5) most likely proceeds via the aquated (G/H(2)O) intermediate (4) (pseudo-first-order rate constant = (3.62 +/- 0.04) x 10(-5) s(-1)) followed by closure of 4 to form 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 +/- 0.04) x 10(-5) s(-1). Closure is ca. 10-55-fold faster than that found for 1,2 GG intrastrand cross-link formation by the diaqua form of cisplatin. Changes in the (1)H and (15)N shifts of the interstrand cross-link 5 indicate that the initially formed conformer (5(i)) converts irreversibly into other product conformer(s) 5(f). The NMR data for 5(i) are consistent with a molecular model of the 1,4 GG interstrand cross-link on B-form DNA, which shows that the NH(2) protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH(2) environments indicative of interactions between the NH(2) protons and the DNA. HPLC characterization of the final product showed only one major product peak that was confirmed by ESI-FTICR mass spectroscopy to be a cross-linked adduct of (15)N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.

Abstract-In continuous chemical processes, variations of process variables usually travel along propagation paths in the direction of the control path and process flow. This paper describes a data-driven method for identifying the direction of propagation of disturbances using historical process data. The novel concept is the application of transfer entropy, a method based on the conditional probability density functions that measures directionality of variation. It is sensitive to directionality even in the absence of an observable time delay. Its performance is studied in detail and default settings for the parameters in the algorithm are derived so that it can be applied in a large scale setting. Two industrial case studies demonstrate the method.