DNA-protein cross-linking by trans-[PtCl(2)(E-iminoether)(2)]. A concept for activation of the trans geometry in platinum antitumor complexes

The structure-pharmacological activity relationships generally accepted for antitumor platinum compounds stressed the necessity for the cis-[PtX(2)(amine)(2)] structure while the trans-[PtX(2)(amine)(2)] structure was considered inactive. However, more recently, several trans-platinum complexes have been identified which are potently toxic, antitumor-active and demonstrate activity distinct from that of conventional cisplatin (cis-[PtCl(2)(NH(3))(2)]). We have shown in the previous report that the replacement of ammine ligands by iminoether in transplatin (trans-[PtCl(2)(NH(3))(2)]) results in a marked enhancement of its cytotoxicity so that it is more cytotoxic than its cis congener and exhibits significant antitumor activity, including activity in cisplatin-resistant tumor cells. In addition, we have also shown previously that this new trans compound (trans-[PtCl(2)(E-iminoether)(2)]) forms mainly monofunctional adducts at guanine residues on DNA, which is generally accepted to be the cellular target of platinum drugs. In order to shed light on the mechanism underlying the antitumor activity of trans-[PtCl(2)(E-iminoether)(2)] we examined oligodeoxyribonucleotide duplexes containing a single, site-specific, monofunctional adduct of this transplatin analog by the methods of molecular biophysics. The results indicate that major monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] locally distort DNA, bend the DNA axis by 21 degrees toward the minor groove, are not recognized by HMGB1 proteins and are readily removed from DNA by nucleotide excision repair (NER). In addition, the monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] readily cross-link proteins, which markedly enhances the efficiency of this adduct to terminate DNA polymerization by DNA polymerases in vitro and to inhibit removal of this adduct from DNA by NER. It is suggested that DNA-protein ternary cross-links produced by trans-[PtCl(2)(E-iminoether)(2)] could persist considerably longer than the non-cross-linked monofunctional adducts, which would potentiate toxicity of this antitumor platinum compound toward tumor cells sensitive to this drug. Thus, trans-[PtCl(2)(E-iminoether)(2)] represents a quite new class of platinum antitumor drugs in which activation of trans geometry is associated with an increased efficiency to form DNA-protein ternary cross-links thereby acting by a different mechanism from 'classical' cisplatin and its analogs.     

DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.     

Preparation, cytotoxicity and interactions with nucleophiles of three isomeric transplatinum complexes containing methylpiperidine ligands

Three isomeric complexes, trans-[PtCl2(NH3)(2-methylpiperidine)], trans-[PtCl2(NH3)(3-methylpiperidine)] and trans-[PtCl2(NH3)(4-methylpiperidine)], were prepared and their cytotoxicities against six ovarian cancer cell lines, three sensitive and three resistant to cisplatin, were measured. There were no significant differences in the cytotoxicities of the three isomers against these cell lines. The interactions of the three complexes with reduced glutathione (GSH) and with ubiquitin (Ub), as a model protein, were studied. The trans-[PtCl2(NH3)(2-methylpiperidine)] reacted approximately twice as slowly with GSH as did the other two isomers. In the 1:1 interactions of the three complexes with ubiquitin (Mr = 8565 amu), trans-[PtCl2(NH3)(3-methylpiperidine)] and trans-[PtCl2(NH3)(4-methylpiperidine)] attained 100% modification while trans-[PtCl2(NH3)(2-methylpiperidine)] reached only less than 50% modification. Trans-[PtCl2(NH3)(2-methylpiperidine)] reacts significantly less efficiently with GSH and proteins than the other two isomers yet this is not reflected in the cytotoxicity values. These results indicate that for these complexes, in these cell lines, cytosolic detoxification probably does not play a dominant role in determining the cytotoxicity of the complexes.     

Chiral differentiation of DNA adducts formed by enantiomeric analogues of antitumor cisplatin is sequence-dependent

1,2-GG intrastrand cross-links formed in DNA by the enantiomeric complexes [PtCl(2)(R,R-2,3-diaminobutane (DAB))] and [PtCl(2)(S,S-DAB)] were studied by biophysical methods. Molecular modeling revealed that structure of the cross-links formed at the TGGT sequence was affected by repulsion between the 5'-directed methyl group of the DAB ligand and the methyl group of the 5'-thymine of the TGGT fragment. Molecular dynamics simulations of the solvated platinated duplexes and our recent structural data indicated that the adduct of [PtCl(2)(R,R-DAB)] alleviated this repulsion by unwinding the TpG step, whereas the adduct of [PtCl(2)(S,S-DAB)] avoided the unfavorable methyl-methyl interaction by decreasing the kink angle. Electrophoretic retardation measurements on DNA duplexes containing 1,2-GG intrastrand cross-links of Pt(R,R-DAB)(2+) or Pt(S,S-DAB)(2+) at a CGGA site showed that in this sequence both enantiomers distorted the double helix to the identical extent similar to that found previously for the same sequence containing the cross-links of the parent antitumor cis-Pt(NH(3))(2)(2+) (cisplatin). In addition, the adducts showed similar affinities toward the high-mobility-group box 1 proteins. Hence, whereas the structural perturbation induced in DNA by 1,2-GG intrastrand cross-links of cisplatin does not depend largely on the bases flanking the cross-links, the perturbation related to GG cross-linking by bulkier platinum diamine derivatives does.     

Conformation, protein recognition and repair of DNA interstrand and intrastrand cross-links of antitumor trans-[PtCl2(NH3)(thiazole)

Replacement of one ammine in clinically ineffective trans-[PtCl2(NH3)2] (transplatin) by a planar N-heterocycle, thiazole, results in significantly enhanced cytotoxicity. Unlike 'classical' cisplatin {cis-[PtCl2(NH3)2]} or transplatin, modification of DNA by this prototypical cytotoxic transplatinum complex trans-[PtCl2(NH3)(thiazole)] (trans-PtTz) leads to monofunctional and bifunctional intra or interstrand adducts in roughly equal proportions. DNA fragments containing site-specific bifunctional DNA adducts of trans-PtTz were prepared. The structural distortions induced in DNA by these adducts and their consequences for high-mobility group protein recognition, DNA polymerization and nucleotide excision repair were assessed in cell-free media by biochemical methods. Whereas monofunctional adducts of trans-PtTz behave similar to the major intrastrand adduct of cisplatin [J. Kasparkova, O. Novakova, N. Farrell and V. Brabec (2003) Biochemistry, 42, 792-800], bifunctional cross-links behave distinctly differently. The results suggest that the multiple DNA lesions available to trans-planaramine complexes may all contribute substantially to their cytotoxicity so that the overall drug cytotoxicity could be the sum of the contributions of each of these adducts. However, acquisition of drug resistance could be a relatively rare event, since it would have to entail resistance to or tolerance of multiple, structurally dissimilar DNA lesions.     

The fate of platinum(II) and platinum(IV) anti-cancer agents in cancer cells and tumours

SRIXE mapping has been used to gain insight into the fate of platinum(II) and platinum(IV) complexes in cells and tumours treated with anticancer active complexes to facilitate the development of improved drugs. SRIXE maps were collected of thin sections of human ovarian (A2780) cancer cells treated with bromine containing platinum complexes, cis-[PtCl(2)(3-Brpyr)(NH(3))] (3-Brpyr=3-bromopyridine) and cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] (OAcBr=bromoacetate), or a platinum complex with an intercalator attached cis-[PtCl(2)(2-[(3-aminopropyl)amino]-9,10-anthracenedione)(NH(3))]. After 24h the complexes appear to be localised in the cell nucleus with a lower concentration in the surrounding cytoplasm. In cells treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] the concentration of bromine was substantially higher than in control cells and the bromine was co-localised with the platinum consistent with the 3-bromopyridine ligand remaining bound to the platinum. The cells treated with cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] also showed an increased level of bromine, but to a much lesser extent than for those treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] suggestive of substantial reduction of the platinum(IV) complex. Maps were also collected from thin sections of a 4T1.2 neo 1 mammary tumour xenograft removed from a mouse 3h after treatment with cis,trans,cis-[PtCl(2)(OH)(2)(NH(3))(2)] and revealed selective uptake of platinum by one cell.     

Comparison of the electronic properties,and thermodynamic and kinetic parameters of the aquation of selected platinum(II) derivatives with their anticancer IC<Subscript>50</Subscript> indexes

Three potential anticancer agents {trans-[PtCl(2)(NH(3))(thiazole)], cis-[PtCl(2)(NH(3))(piperidine)], and PtCl(2)(NH(3))(cyclohexylamine) (JM118)} were explored and compared with cisplatin and the inactive [PtCl(dien)](+) complex. Basic electronic properties, bonding and stabilization energies were determined, and thermodynamic and kinetic parameters for the aquation reaction were estimated at the B3LYP/6-311++G(2df,2pd) level of theory. Since the aquation process represents activation of these agents, the obtained rate constants were compared with the experimental IC(50) values for several tumor cells. Despite the fact that the processes in which these drugs are involved and the way in which they affect cells are very complex, some correlations can be deduced.     

Modulation of the chemical and biological properties of trans platinum complexes: monofunctional platinum complexes containing one nucleobase as potential antiviral chemotypes

Replacement of one of the chloride leaving groups in trans-[PtCl2(NH3)(L)] by the nucleobase 9-ethylguanine gives the nucleobase cations [SP-4-2]-[PtCl(9-ethylguanine)(NH3)(L)]+ (L = NH3, 1; L = quinoline, 3), which are models for the monofunctional adduct on DNA. Displacement of Cl- in 1 and 3 by either 5'-guanosine monophosphate (5'-GMP) or N-acetyl-L-methionine (N-AcMet) showed clear kinetic preference for the sulfur (estimated half-lives of 1.5 and 4 h with N-AcMet against 7 and 17 h for 5'-GMP for 1 and 3, respectively). To further examine the kinetic preference, 1-methylcytosine (1-MeCyt) analogs were prepared, [SP-4-2]-[PtCl(1-Me-Cyt)(NH3)(L)]+ (L=NH3, 2; L=quinoline, 4). The -MeCyt compounds, 2 and 4, resulted in slower rates of substitution by both 5'-GMP and N-AcMet in comparison to 1 and 3 (estimated half-lives for N-AcMet of 5 and 13.5 h and for 5'-GMP of 6 and 14 h for 2 and 4, respectively). Interestingly in this case, however, no selectivity for the sulfur site was observed, a possible explanation being that molecular recognition across the square plane enhances the rate of reaction with 5'-GMP. The affinity of 3 towards S-donor ligands was exploited to remove zinc from the zinc-finger site of the C-terminal finger of the HIV-nucleocapsid protein, NCp7. The ability to eject zinc further suggested the biological antiviral application of [SP-4-2]-[PtCl(nucleobase)(NH3)(L)]+. A preliminary survey against HIV and herpes viruses indeed showed encouraging results with some antiviral specificity, dependent on the exact nature of the compound. The initial results suggest consideration of [SP-4-2]-[PtCl(nucleobase)(NH3)(L)]+ as a novel antiviral chemotype.     

Increased targeting of adenine-rich sequences by (2-amino-2-methyl-3-butanone oxime)dichloroplatinum(II) and investigations into its low cytotoxicity

Using assays based on the inhibition of restriction enzyme cleavage of plasmid and synthetic DNA, the complex (2-amino-2-methyl-3-butanone oxime)dichloroplatinum(II), [PtCl2(ambo)], has been shown to have an increased tendency for binding to adenine-rich sequences when compared to cis[PtCl2(NH3)2] (cisplatin). [PtCl2(ambo)] was found to form substantially fewer interstrand adducts than does cisplatin. The in vitro cytotoxicity of [PtCl2(ambo)] against a human bladder cancer cell line was determined and found to be more than two orders of magnitude lower than that of cisplatin, yet it was also found to be equally effective at passing into cells and binding to isolated DNA.     

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.     

Preparation of platinum(IV) complexes with dipeptide and diimine. X-ray crystal structure and 195Pt NMR spectra

We prepared platinum(IV) complexes containing dipeptide and diimine or diamine, the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complex, where -N,N,O means dipeptide coordinated as a tridentate chelate, dipeptide=glycylglycine (NH(2)CH(2)CON(-)CH(2)COO(-), digly, where two protons of dipeptide are detached when the dipeptide coordinates to metal ion as a tridentate chelate), glycyl-L-alanine (NH(2)CH(2)CON(-)CHCH(3)COO(-), gly-L-ala), L-alanylglycine (NH(2)CH CH(3)CON(-)CH(2)COO(-), L-alagly), or L-alanyl-L-alanine (NH(2)CHCH(3)CON(-)CHCH(3)COO(-), dil-ala), and diimine or diamine=bipyridine (bpy), ethylenediamine (en), N-methylethylenediamine (N-Me-en), or N,N'-dimethylethylenediamine (N,N'-diMe-en). In the complexes containing gly-L-ala or dil-ala, two separate peaks of the (195)Pt NMR spectra of the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complexes appeared in, but in the complexes containing digly or L-alagly, one peak which contained two overlapped signals appeared. One of the two complexes containing gly-L-ala and bpy, [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3), crystallized and was analyzed. This complex has the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions of a=9.7906(3)A, b=11.1847(2)A, c=16.6796(2)A, Z=4. The crystal data revealed that this [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex has the near- (Cl, CH(3)) configuration of two possible isomers. Based on elemental analysis, the other complex must have the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) configuration. The (195)Pt NMR chemical shifts of the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex and the far- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex are 0 ppm and -19 ppm, respectively (0 ppm for the Na(2)[PtCl(6)] signal). The additive property of the (195)Pt NMR chemical shift is discussed. The (195)Pt NMR chemical shifts of [PtCl(dipeptide-N,N,O)(bpy)]Cl appeared at a higher field when the H attached to the dipeptide carbon atom was replaced with a methyl group. On the other hand, the (195)Pt NMR chemicals shifts of [PtCl(dipeptide-N,N,O)(diamine)] appeared at a lower field when the H attached to the diamine nitrogen atom was replaced with a methyl group, in the order of [PtCl(digly-N,N,O)(en)]Cl, [PtCl(digly-N,N,O)(N-Me-en)]Cl, and [PtCl(digly-N,N,O)(N,N'-diMe-en)]Cl.     

The mechanism of action of platinum(IV) complexes in ovarian cancer cell lines

The reduction potentials, lipophilicities, cellular uptake and cytotoxicity have been examined for two series of platinum(IV) complexes that yield common platinum(II) complexes on reduction: cis-[PtCl(4)(NH(3))(2)], cis,trans,cis-[PtCl(2)(OAc)(2)(NH(3))(2)], cis,trans,cis-[PtCl(2)(OH)(2)(NH(3))(2)], [PtCl(4)(en)], cis,trans-[PtCl(2)(OAc)(2)(en)] and cis,trans-[PtCl(2)(OH)(2)(en)] (en=ethane-1,2-diamine, OAc=acetate). As previously reported, the reduction occurs most readily when the axial ligand is chloride and least readily when it is hydroxide. The en series of complexes are marginally more lipophilic than their ammine analogues. The presence of axial chloride or acetate ligands results in a slighter higher lipophilicity compared with the platinum(II) analogue whereas hydroxide ligands lead to a substantially lower lipophilicity. The cellular uptake is similar for the platinum(II) species and their analogous tetrachloro complexes, but is substantially lower for the acetato and hydroxo complexes, resulting in a correlation with the reduction potential. The activities are also correlated with the reduction potentials with the tetrachloro complexes being the most active of the platinum(IV) series and the hydroxo being the least active. These results are interpreted in terms of reduction, followed by aquation reducing the amount of efflux from the cells resulting in an increase in net uptake.     

Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.     

Bending studies of DNA site-specifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-[Pt(NH3)2(N3-cytosine)Cl]+

Duplex oligonucleotides containing a single intrastrand [Pt(NH3)2]2+ cross-link or monofunctional adduct and either 15 or 22 bp in length were synthesized and chemically characterized. The platinum-modified and unmodified control DNAs were polymerized in the presence of DNA ligase and the products studied on 8% native polyacrylamide gels. The extent of DNA bending caused by the various platinum-DNA adducts was revealed by their gel mobility shifts relative to unplatinated controls. The bifunctional adducts cis-[Pt(NH3)2[d(GpG)]]+, cis-[Pt(NH3)2[d(ApG)]]+, and cis-[Pt(NH3)2[d(G*pTpG*)]], where the asterisks denote the sites of platinum binding, all bend the double helix, whereas the adduct trans-[Pt(NH3)2[d(G*pTpG*)]] imparts a degree of flexibility to the duplex. When modified by the monofunctional adduct cis-[Pt(NH3)2(N3-cytosine)(dG)]Cl the helix remains rod-like. These results reveal important structural differences in DNAs modified by the antitumor drug cisplatin and its analogs that could be important in the biological processing of the various adducts in vivo.     

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).     

Steric control of stereoselective interactions between the platinum(II) complex [PtCl2(1,4-diazacycloheptane)] and DNA: comparison with cis-[PtCl2(NH3)2] and [PtCl2(ethane-1,2-diamine)] using DNA binding and molecular modeling studies

The rate and extent of binding of [PtCl2(hpip)] (hpip=homopiperazine-1,4-diazacycloheptane) and cis-[PtCl2(NH3)2] to calf thymus DNA was measured using atomic absorption spectroscopy and it was found that [PtCl2(hpip)] bound both more rapidly and to a greater extent than did cis-[PtCl2(NH3)2]. The binding of [PtCl2(hpip)] and [PtCl2(en)] (en=ethane-1,2-diamine) to salmon sperm DNA and to synthetic, self-complementary 10-base-pair and 52-base-pair oligonucleotides was studied using enzymatic digestion and HPLC analysis of the products. [PtCl2(hpip)] forms approximately two-fold fewer GpG and ApG intrastrand adducts and concomitantly more monofunctional adducts than does [PtCl2(en)]. In the case of [PtCl2(hpip)], two GpG adducts, corresponding to the different orientations of the hpip ligand with respect to the DNA, were observed in a 1:3.3 ratio. The minor product corresponds to the orientation in which the bulkier propylene chain of the hpip ligand is adjacent to, and makes close contacts with, the floor of the major groove. When the reaction was repeated with a synthetic oligonucleotide decamer duplex, the ratio of the two forms was approximately 1:1.9 and with the 52-mer duplex it was 1:2.4, revealing an apparent systematic dependence of stereoselectivity on nucleotide size. Computer modeling of the two adducts formed by [PtCl2(hpip)] and those formed by [PtCl2(en)] and cis-[PtCl2(NH3)2] revealed that non-bonded interactions between the hpip ligand and the DNA were probably responsible for both the decreased proportion of GpG adducts formed by [PtCl2(hpip)] and the stereoselectivity exhibited in the formation of these adducts. This is the first case in which the stereoselectivity can be ascribed to steric factors alone.     

Sterically hindered cisplatin derivatives with multiple carboxylate auxiliary arms: synthesis and reactions with guanosine-5'-monophosphate and plasmid DNA.

Two novel sterically hindered cisplatin derivatives with the ligand L=NH(2)C(CH(2)CH(2)COOH)(3) were prepared: cis-PtCl(2)L(2) and cis-PtCl(2)L(NH(3)). The starting compound for the syntheses was NH(2)C(CH(2)CH(2)COOtBu)(3), also known as a building block for dendrimers. cis-PtCl(2)L(2) was prepared from K(2)PtCl(4) in an unusual two-phase reaction in water-chloroform, followed by deprotection of the tert-butyl protective groups with formic acid to yield a water-soluble complex. The mixed-ligand compound cis-PtCl(2)L(NH(3)) was prepared from [PPh(4)][PtCl(3)(NH(3))] in methanol, with subsequent deprotection in formic acid. DNA-binding properties of the two compounds were investigated using the model base guanosine-5'-monophosphate (5'-GMP) and pBR322 plasmid DNA. While cisplatin [cis-PtCl(2)(NH(3))(2)] induced an unwinding of 12 degrees in pBR322 plasmid DNA, cis-PtCl(2)L(NH(3)) induced only 3 degrees unwinding, which is indicative of a monofunctional binding mode. Remarkably, cis-PtCl(2)L(2) did not induce any distortion in plasmid DNA, which strongly suggests that the compound does not bind to DNA. Test reactions with 5'-GMP, monitored by 1H and 195Pt NMR, confirmed that cis-PtCl(2)L(2) is unable to bind to DNA, whereas cis-PtCl(2)L(NH(3)) binds only one nucleotide. Apparently, binding of platinum to nucleotides at the coordination site cis with respect to the ligand L is prevented by steric crowding. Thus, cis-PtCl(2)L(NH(3)) must bind DNA monofunctionally at the trans position. Besides, both compounds have a chloride replaced by one of the carboxylate arms, forming a a seven-membered chelate ring. In theory, cis-PtCl(2)L(2) could also form a second chelate ring, but this was not observed.     

Platinum complexes with one radiosensitizing ligand [PtCl2(NH3) (sensitizer)]: radiosensitization and toxicity studies in vitro

Complexes of general formula [PtCl2(NH3)L] with one radiosensitizing ligand per platinum are compared with ligand L alone, complexes with two radiosensitizers per platinum [PtCl2L2], and their analogs with NH3 ligands, with respect to radiosensitizing properties and toxicity in CHO cells. Radiosensitizing ligands, L, were misonidazole, metronidazole, 4(5)-nitroimidazole, and 2-amino-5-nitrothiazole, and the ammine analogs were cis- and trans-DDP [diamminedichloroplatinum(II)] and the monoammine, K[PtCl3(NH3)]. Results are related to a previous study on plasmid DNA binding by these series. The toxicity of the mono series [PtCl2(NH3)L], attributable to DNA binding, is much higher than the corresponding bis complexes, [PtCl2L2]. For L = misonidazole, toxicity is similar to the monoammine, but higher in hypoxic than in aerobic cells. trans-[PtCl2(NH3)-(misonidazole)] is more toxic than the cis isomer. Except for L = 4(5)-nitroimidazole, the complexes [PtCl2(NH3)L] are more toxic than L in air and hypoxia. Hypoxic radiosensitization by the mono complexes is comparable to the monoammine and is not better than free sensitizers, again except for L = 4(5)-nitroimidazole. Significantly lower sensitization is observed in oxic cells. The bis complexes [PtCl2L2], which do not bind to DNA as well as the mono complexes, are less effective radiosensitizers and less toxic than the [PtCl2(NH3)L] series.     

Reaction of cis- and trans-[PtCl2(NH3)2] with reduced glutathione inside human red blood cells, studied by 1H and 15N-[1H] DEPT NMR

Reactions of cis- and trans-[PtCl2(NH3)2] with glutathione (GSH) inside intact red blood cells have been studied by 1H spin-echo nuclear magnetic resonance (NMR). Upon addition of trans-[PtCl2(NH3)2] to a suspension of red cells, there was a gradual decrease in the intensity of the resonances for free GSH, and new peaks were observed that were assignable to coordinated GSH protons in trans-[Pt(SG)Cl(NH3)2], trans-[Pt(SG)2(NH3)2], and possibly the S-bridged complex trans-[[NH3)2PtCl)2SG]+. Formation of trans-[Pt(SG)2(NH3)2] inside the cell was confirmed from the 1H NMR spectrum of hemolyzed cells, which were ultrafiltered to remove large protein molecules; the ABM multiplet of the coordinated GSH cys-beta CH2 protons was resolved using selective-decoupling experiments. Seventy percent of the total intracellular GSH was retained by the ultrafiltration membrane, suggesting that the mixed complex trans-[Pt(SG)(S-hemoglobin)(NH3)2] also is a major metabolite of trans-[PtCl2(NH3)2] inside red cells. The reaction of cis-[PtCl2(NH3)2] with intracellular GSH was slower; only 35% of the GSH had been complexed after a 4-hr incubation compared to 70% for the trans isomer. There was a gradual decrease in the intensity of the GSH 1H spin-echo NMR resonances, but no new peaks were resolved. This was interpreted as formation of high-molecular weight Pt:GSH and mixed GS-Pt-S(hemoglobin) polymers. By using a 15N-[1H] DEPT pulse sequence, we were able to study the reaction of cis-[PtCl2(15NH3)2] with red cells at concentrations as low as 1 mM. 15NH3 ligands were released, and no resonances assignable to Pt-15NH3 species were observed after a 12-hr incubation.