Conformation, recognition by high mobility group domain proteins, and nucleotide excision repair of DNA intrastrand cross-links of novel antitumor trinuclear platinum complex BBR3464

The new antitumor trinuclear platinum compound [(trans-PtCl(NH(3))(2))(2)mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)](4+) (designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is characterized by the rapid formation of long range intra- and interstrand cross-links. We examined how the structures of the various types of the intrastrand cross-links of BBR3464 affect conformational properties of DNA, and how these adducts are recognized by high mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have revealed that intrastrand cross-links of BBR3464 create a local conformational distortion, but none of these cross-links results in a stable curvature. In addition, we have observed no recognition of these cross-links by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.     

Circular dichroism study of the irreversibility of conformational changes induced by polyamine-linked dinuclear platinum compounds

In this work, the reversibility of both the B-->Z and B-->A conformational change in polymer DNA induced by polynuclear platinum compounds was studied. The compounds examined were: [[trans-PtCl(NH(3))(2)](2)[NH(2) (CH(2))(6)NH(2)]](2+) (BBR3005); [[trans-PtCl(NH(3))(2)](2)[mu-spermine-N1,N12]](4+) (BBR3535); [[trans-PtCl(NH(3))(2)](2)[mu-spermidine-N1,N8]](3+) (BBR3571); [[trans-PtCl(NH(3))(2)](2)[mu-BOC-spermidine]](2+) (BBR3537); and [[trans-PtCl(NH(3))(2)](2)[mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+) (BBR3464). The conformational changes were assessed by circular dichroism and the reversibility of the transitions was tested by subsequent titration with the DNA intercalator ethidium bromide (EtBr). Fluorescent quenching was also used to assess the ability of ethidium bromide to intercalate into A and/or Z-DNA induced by the compounds. The results were compared with those produced by the simple hexamminecobalt cation [Co(NH(3))(6)](3+). The data suggest that while conformational changes induced by electrostatic interactions are confirmed to be reversible, covalent binding induces irreversible changes in both the A and Z conformation. The relevance of these changes to the novel biological action of polynuclear platinum compounds is discussed.     

Comparison of structural effects in 1,4 DNA-DNA interstrand cross-links formed by dinuclear and trinuclear platinum complexes

The novel anticancer drug ([[trans-PtCl(NH(3))(2)](2)-mu-[trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2)]](NO(3))(4)) (BBR3464, 1,0,1/t,t,t, TPC) forms a 1,4-interstrand cross-linked adduct with the self-complementary DNA octamer 5'-d(ATG*TACAT)(2)-3', with the two platinum atoms coordinated in the major groove at N7 positions of guanines four base pairs apart on opposite DNA strands [Y. Qu, N.J. Scarsdale, M.-C. Tran, N. Farrell, J. Biol. Inorg. Chem. 8 (2003) 19-28]. The structure of the identical cross-link formed by the dinuclear [[trans-PtCl(NH(3))(2)](2)-mu-NH(2)(CH(2))(6)NH(2)]](NO(3))(2) (BBR3005, 1,1/t,t, DPC) was examined for comparison. The adduct was characterized and analyzed by MS, UV and NMR spectroscopy. NMR analysis of the adduct shows platination of the unique guanine residues. The strong H8/H1' intraresidue cross-peaks observed for all purine residues (A1, G3, A5 and A7) are consistent with a syn-conformation of the nucleoside unit in all cases. Thus, the structure resembles closely that formed by the trinuclear compound. Further confirmation of this similarity comes from the increase in melting temperature (66 degrees for DPC, 60 degrees for TPC, 22 degrees for free oligonucleotide). Since DNA is the principal target in vivo for these Pt cross-linking agents, the unique structural perturbations induced by these cross-links may be related to the increased cytotoxicity and antitumor activity of polynuclear platinum compounds as compared to cisplatin (cis-DDP). The similarity in the structures suggests opportunities to "deliver" the cross-link in a more efficient manner than the current clinically tested drug.     

Cellular pharmacology of polynuclear platinum anti-cancer agents

Study of the cellular pharmacology of the dinuclear platinum complexes, BBR3005 ([?trans-PtCl(NH3)2?2H2N(CH2)6NH2]2+), BBR3171 ([?cis-PtCl(NH3)2?2H2N(CH2)6NH2]2+) and the trinuclear platinum complex, BBR3464 ([?trans-PtCl(NH3)2?2 mu-?trans-Pt(NH3)2(H2N(CH2)6NH2)2?]4+) was undertaken in wild type and cisplatin-resistant L1210 murine leukemia cell lines. All complexes are potent cytotoxic agents against the wild type cell line. Only BBR3464 shows enhanced activity against the cisplatin-resistant cell line following a brief exposure. This enhanced activity is attributable, in part, to preserved accumulation, which contrasts with diminished accumulation of cisplatin and both dinuclear platinum complexes. The cisplatin-resistant cell line is relatively tolerant of DNA adducts induced by both cisplatin and BBR3464, but BBR3464 is much less affected. All complexes induce DNA interstrand cross-links. Di/trinuclear complex-induced interstrand cross-linking peaks early, suggesting rapid genomic access and interaction. Subsequent decay suggests susceptibility to DNA repair mechanisms. Peak and area-under-the-curve values for interstrand cross-linking among the complexes correlate poorly with cytotoxic effects, especially in the cisplatin-resistant cell line. This suggests that all interstrand cross-linking adducts are not equal in their cytotoxic effect, or other, non-interstrand cross-linking adducts are significant. BBR3464 has been selected for clinical development largely on the basis of results from in vivo activity and toxicity studies. These results show BBR3464 to have unique properties in the context of acquired cisplatin-resistance that enhance its candidacy as a potential anticancer agent.     

DNA interstrand cross-links of the novel antitumor trinuclear platinum complex BBR3464. Conformation,recognition by high mobility group domain proteins,and nucleotide excision repair

The novel phase II antitumor polynuclear platinum drug BBR3464 ([(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](NO(3))(4)) forms intra- and interstrand cross-links (CLs) on DNA (which is the pharmacological target of platinum drugs). We examined first in our recent work how various intrastrand CLs of BBR3464 affect the conformation of DNA and its recognition by cellular components (Zehnulova, J., Kasparkova, J., Farrell, N., and Brabec, V. (2001) J. Biol. Chem. 276, 22191-22199). In the present work, we have extended the studies on the DNA interstrand CLs of this drug. The results have revealed that the interstrand CLs are preferentially formed between guanine residues separated by 2 base pairs in both the 3' --> 3' and 5' --> 5' directions. The major 1,4-interstrand CLs distort DNA, inducing a directional bending of the helix axis and local unwinding of the duplex. Although such distortions represent a potential structural motif for recognition by high mobility group proteins, these proteins do not recognize 1,4-interstrand CLs of BBR3464. On the other hand, in contrast to intrastrand adducts of BBR3464, 1,4-interstrand CLs are not removed from DNA by nucleotide excision repair. It has been suggested that interstrand CLs of BBR3464 could persist considerably longer in cells compared with intrastrand adducts, which would potentiate the toxicity of the interstrand lesions to tumors sensitive to this polynuclear drug.     

DNA modifications by a novel bifunctional trinuclear platinum phase I anticancer agent.

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 profile of DNA binding for BBR3464, strengthening the original 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.     

Consequences of nucleic acid conformation on the binding of a trinuclear platinum drug.

BBR3464, a charged trinuclear platinum compound, is the first representative of a new class of anticancer drugs to enter phase I clinical trials. The structure of BBR3464 is characterized by two [trans-PtCl(NH(3))(2)] units linked by a tetraamine [trans-Pt(NH(3))(2)?H(2)N(CH(2))(6)NH(2)?(2)] unit. The +4 charge of BBR3464 and the separation of the platinating units indicate that the mode of DNA binding will be distinctly different from those of classical mononuclear drugs such as cisplatin, cis-[PtCl(2)(NH(3))(2)]. The reaction of BBR3464 with three different nucleic acid conformations was assessed by gel electrophoresis. Comparison of single-stranded DNA, RNA, and double-stranded DNA indicated that the reaction of BBR3464 with single-stranded DNA and RNA was faster than that with duplex DNA, and produced more drug-DNA and drug-RNA adducts. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry was used to further characterize the binding modes of BBR3464 with the DNA substrates. BBR3464 binding to different nucleic acid conformations raises the possibility that the adducts of single-stranded DNA and RNA may play a role in the different antitumor efficacies of this novel drug as compared with cisplatin.     

Sequence specificity, conformation, and recognition by HMG1 protein of major DNA interstrand cross-links of antitumor dinuclear platinum complexes

Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [?trans-PtCl(NH(3))(2)?H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site-specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues.     

Kinetic study of azole-bridged dinuclear platinum(II) complexes reacting with a hairpin-stabilized double-stranded oligonucleotide

The cytotoxic dinuclear platinum(II) complexes [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-pz)](NO(3))(2) (pz=pyrazolate) (1) and [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-1,2,3-ta-N1,N2)](NO(3))(2) (1,2,3-ta=1,2,3-triazolate) (2), were allowed to react with the hairpin-stabilized double-stranded oligonucleotide d(TATGGCATT(4)ATGCCATA), to determine the amounts of intrastrand and interstrand DNA adducts. The reaction kinetics was investigated by reversed-phase HPLC, and the resulting products were analyzed using mass spectroscopy combined with enzymatic digestion, and Maxam-Gilbert sequencing. The reaction of 1 results in the formation of the 1,2-intrastrand d(GG) adduct as the major final product. The two most abundant products of 2 were identified as isomeric 1,2-intrastrand d(GG) adducts differing probably in platinum coordination to the triazole ring. No GG-interstrand crosslinks were detected with either compound. d(GGC)-d(GCC) sequences of DNA do thus not appear to represent significant targets for forming interstrand crosslinks with either 1 or 2.     

Sequence-dependent conformational changes in DNA induced by polynuclear platinum complexes

In this work, the B-->Z transition of poly(dG-dC).poly(dG-dC) and the B-->A transition of poly(dG).poly(dC) and of calf thymus (CT) DNA fragments modified by antitumor bifunctional polynuclear platinum complexes were investigated by circular dichroism (CD). The transition from the B- to Z-form of DNA was inducible with all three compounds studied, as indicated by an inversion of the B-form spectra. The B-->A transition in poly(dG).poly(dC) was induced easily by platinum complex binding alone, while the B-->A transition in CT DNA was induced by ethanol but inhibited by coordination of all polynuclear platinum compounds used here. It was shown that the compound [?cis-PtCl(NH3)2?2 mu-?H2N(CH2)6NH2?] (NO3)2 (1,1/c,c) was most effective at inhibiting the B-->A transition in CT DNA, and [?trans-PtCl(NH3)2?2 mu-?trans-Pt(NH3)2(H2N(CH2)6NH2)2?] (NO3)4 (1,0,1/t,t,t) was least effective, while the effectiveness of [?trans-PtCl(NH3)2?2 mu-?H2N(CH2)6NH2?] (NO3)2 (1,1/t,t) fell between the two. This corresponded to the relative amounts of interstrand crosslinks in double-stranded DNA caused by each compound.     

Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes

Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.     

Cooperative effects in long-range 1,4 DNA-DNA interstrand cross-links formed by polynuclear platinum complexes: an unexpected syn orientation of adenine bases outside the binding sites

The novel phase II anticancer drug BBR3464 ([[ trans-PtCl(NH(3))(2)](2)- micro -[ trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2)]](NO(3))(4)) forms a 1,4-interstrand cross-link adduct with the self-complementary DNA octamer 5'-d(ATG*TACAT)(2)-3', with the two platinum atoms coordinated in the major groove at the N7 positions of guanines that are four base pairs apart on opposite DNA strands. The "central" tetraamine linker [ trans-H(2)N(CH(2))(6)NH(2)Pt(NH(3))(2)NH(2)(CH(2))(6)NH(2)] was located in or close to the minor groove. The adduct was characterized and analyzed by MS, UV and NMR spectroscopy. NMR analysis of the adduct shows strong H8/H1' intraresidue crosspeaks observed for the A1 and A7 resonances, consistent with a syn conformation for these bases which is usually not observed for adenine residues and bases not directly involved in the cross-link in oligonucleotides. The strong intraresidue H8/H1' crosspeak is also observed for G3. Examination of the structure thus reveals unusual cooperative effects unique to this class of anticancer drugs and is the first demonstration of cooperative effects in solution for an anticancer drug. The significant characteristic of the structure is the lack of severe DNA distortion such as a kink, directed bend or significant unwinding of the helices which are characteristic for DNA adducts of mononuclear complexes. This may contribute to the lack of protein recognition of the cross-link by HMG-domain proteins, a biological consequence significantly different from that of mononuclear complexes such as cisplatin. Since DNA is the principal target in vivo for these Pt cross-linking agents, the unique structural perturbations induced by BBR3464 cross-links are likely related to its increased cytotoxicity and antitumor activity as compared to cisplatin ( cis-DDP).     

Comparison of cytotoxicity and cellular accumulation of polynuclear platinum complexes in L1210 murine leukemia cell lines

The antitumor activity of the trinuclear Phase I clinical agent, BBR3464, is matched by that of polyamine-linked dinuclear complexes. The cytotoxicity and cellular accumulation of three polynuclear platinum complexes: [?trans-PtCl(NH3)2?2 mu-?trans-Pt(NH3)2(H2N(CH2)6-NH2)2?]4+ (BBR3464), [?trans-PtCl(NH3)2?2(H2N(CH2)3NH2(CH2)4NH2)]3+ (BBR3571), and [?trans-PtCl(NH3)2?2(H2N(CH2)6-NH2)]2+ (BBR3005), were studied in a series of murine L1210 cell lines and compared with cisplatin. Besides murine L1210 cell lines sensitive (/0) and resistant (/DDP) to cisplatin, the efficacy of the compounds in a cell line rendered resistant to BBR3464 (/3464) was examined. Finally, to examine possible uptake pathways of these novel charged complexes, cytotoxicity in a cell line resistant to the polyamine synthesis inhibitor, methylglyoxal-bis(guanylhydrazone) (/MGBG), was studied. Cytotoxicity profiles of BBR3571 most closely matched that of BBR3464. Both agents showed significantly reduced cytotoxicity in L1210/ BBR3464. The cytotoxicity of neither agent was affected by the polyamine uptake-deficient cell line and indeed both complexes showed significantly enhanced cytotoxicity in L1210/MGBG relative to wild-type L1210/0. The cellular uptake of both BBR3464 and BBR3571 was enhanced in L1210/DDP. These studies suggest that the chemical feature of a diamine linker containing an internal charge contributes significantly to the anticancer profiles of both the trinuclear platinum complex, BBR3464, which incorporates a charged platinum into a diamine linker, and the dinuclear platinum complex, BBR3571, which incorporates only a naturally occurring polyamine as diamine linker.     

Studies on activities, cell up take and DNA binding of four multinuclear complexes of the form: [[trans-PtCl(NH(3))(2)](2)mu-[trans-Pd(NH(3))(2)-(H(2)N(CH(2))(n)NH(2))(2)]]Cl(4) where n=4-7

The activity against human cancer cell lines including ovarian: A2780, A2780(cisR), cell up take, DNA-binding and nature of interaction with pBR322 plasmid DNA have been studied for four multinuclear complexes code named DH4Cl, DH5Cl, DH6Cl and DH7Cl, having the general formula: [[trans-PtCl(NH(3))(2)](2)mu-[trans-Pd(NH(3))(2)-(H(2)N(CH(2))(n)NH(2))(2)]]Cl(4) where n=4, 5, 6 and 7 for DH4Cl, DH5Cl, DH6Cl and DH7Cl, respectively. The compounds are found to exhibit significant anticancer activity against ovarian cancer cell lines: A2780, A2780(cisR) and A2780(ZD0473R). DH6Cl in which the linking diamine has six carbon atoms is found to be the most active compound. As the number of carbon atoms in the linking diamine is decreased below six and increased above six, the activity is found to decrease, illustrating structure-activity relationship. All the multinuclear compounds are believed to form a plethora of long-range interstrand GG adducts with DNA dictated by the sequence of bases in the DNA strands. Increasing prevention of BamH1 digestion with the increase in concentration of the compounds is due to global changes in DNA conformation brought about by interstrand long-range binding of the compounds with DNA.     

Antitumor bifunctional dinuclear PtII complex BBR3535 forms interduplex DNA cross-links under molecular crowding conditions

When antitumor platinum drugs react with DNA they form various types of intrastrand and interstrand cross-links (CLs). One class of new antitumor platinum compounds comprises bifunctional Pt^II compounds based on the dinuclear or trinuclear geometry of leaving ligands. It has been shown that the DNA-binding modes of dinuclear or trinuclear bifunctional Pt^II agents are distinct from those of mononuclear cisplatin, forming markedly more intramolecular interstrand CLs. However, at least two types of DNA interstrand cross-linking by bifunctional Pt^II complexes can be envisaged, depending on whether the platinum complex coordinates to the bases in one DNA molecule (intramolecular interstrand CLs) or in two different DNA duplexes (interduplex CLs). We hypothesized that at least some antitumor bifunctional poly(di/tri)nuclear complexes could fulfill the requirements placed on interduplex DNA cross-linkers. To test this hypothesis we studied the interduplex cross-linking capability of a representative of antitumor polynuclear agents, namely, dinuclear Pt^II complex [{trans-PtCl(NH₃)₂}₂-μ-{trans-(H₂N(CH₂)₆NH₂(CH₂)₂NH₂(CH₂)₆NH₂)}]⁴⁺ (BBR3535). The investigations were conducted under molecular crowding conditions mimicking environmental conditions in the cellular nucleus, namely, in medium containing ethanol, which is a commonly used crowding agent. We found with the aid of native agarose gel electrophoresis that the DNA interduplex cross-linking efficiency of BBR3535 under molecular crowding conditions was remarkable: the frequency of these CLs was 54%. In contrast, the interduplex cross-linking efficiency of mononuclear cisplatin or transplatin was markedly lower (approximately 40-fold or 18-fold, respectively). We suggest that the production of interduplex CLs in addition to other DNA intramolecular adducts may provide polynuclear Pt^II compounds with a wider spectrum of cytotoxicity.