Olga Novakova

Induced-fit recognition of DNA by organometallic complexes with dynamic stereogenic centers

Organometallic chemistry offers novel concepts in structural diversity and molecular recognition that can be used in drug design. Here, we consider DNA recognition by η6-arene Ru(II) anticancer complexes by an induced-fit mechanism. The stereochemistry of the dinuclear complex [((η6-biphenyl)RuCl(en))2-(CH2)6]2 + (3, en = ethylenediamine) was elucidated by studies of the half unit [(η6-biphenyl)RuCl(Et-en)]+ (2, where Et-en is Et(H)NCH2CH2NH2). The structures of the separated \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}R_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}S_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} diastereomers of 2 were determined by x-ray crystallography; their slow interconversion in water (t½ ≈ 2 h, 298 K, pH 6.2) was observed by NMR spectroscopy. For 2 and 3 the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}R_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} configurations are more stable than \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}S_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} (73:27). X-ray and NMR studies showed that reactions of 2 and 3 with 9-ethylguanine gave rise selectively to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}S_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} diastereomers. Dynamic chiral recognition of guanine can lead to high diastereoselectivity of DNA binding. The dinuclear complex 3 induced a large unwinding (31°) of plasmid DNA, twice that of mononuclear 2 (14°), and effectively inhibited DNA-directed RNA synthesis in vitro. This dinuclear complex gave rise to interstrand cross-links on a 213-bp plasmid fragment with efficiency similar to bifunctional cisplatin, and to 1,3-GG interstrand and 1,2-GG and 1,3-GTG intrastrand cross-links on site-specifically ruthenated 20-mers. Complex 3 blocked intercalation of ethidium considerably more than mononuclear 2. The concept of induced-fit recognition of DNA by organometallic complexes containing dynamic stereogenic centers via dynamic epimerization, intercalation, and cross-linking may be useful in the design of anticancer drugs.

Biophysical analysis of natural, double-helical DNA modified by anticancer heterocyclic complexes of ruthenium(III) in cell-free media

Modifications of natural DNA by three anticancer heterocyclic ruthenium(III) compounds were studied by methods of molecular biophysics. These methods included DNA binding studies using atomic absorption spectrophotometry, inhibition of restriction endonucleases, mapping of DNA adducts by transcription assay, interstrand cross-linking employing gel electrophoresis under denaturing conditions, DNA unwinding studied by gel electrophoresis, circular dichroism analysis of the B→Z transition in DNA, and DNA melting curves measured by absorption spectrophotometry. The results indicate that the complexes HIm[trans-Cl4Im2RuIII], HInd[trans-Cl4Ind2RuIII], and Na[trans-Cl4Im(Me2SO)RuIII] (Im and Ind stand for imidazole and indazole, respectively) coordinate irreversibly to DNA. Their DNA binding mode is, however, different from that of cisplatin. Interestingly, Na[trans-Cl4Im(Me2SO)RuIII] binds to DNA considerably faster than the other two ruthenium compounds and cisplatin. In addition, when Na[trans-Cl4Im(Me2SO)RuIII] binds to DNA it exhibits an enhanced base sequence specificity in comparison with the other two ruthenium complexes. Na[trans-Cl4Im(Me2SO)RuIII] also forms bifunctional intrastrand adducts on double-helical DNA which are capable of terminating RNA synthesis in vitro, while the capability of the other two ruthenium compounds to form such adducts is markedly lower. This observation has been interpreted to mean that the bifunctional adducts of HInd[trans-Cl4Ind2RuIII] and Na[trans-Cl4Im2RuIII] formed on rigid double-helical DNA are sterically more crowded by their octahedral geometry than those of Na[trans-Cl4Im(Me2SO)RuIII]. In addition, the adducts of all three ruthenium compounds affect the conformation of DNA, Na[trans-Cl4Im(Me2SO)RuIII] being most effective. It has been suggested that the altered DNA binding mode of ruthenium compounds in comparison with cisplatin might be an important factor responsible for the altered cytostatic activity of this class of ruthenium compounds in tumor cells.

DNA interactions of dinuclear RuII arene antitumor complexes in cell-free media

We recently synthesized and characterized water-soluble dinuclear Ru(II) arene complexes, in which two {(eta(6)-p-isopropyltoluene)RuCl[3-(oxo-kappaO)-2-methyl-4-pyridinonato-kappaO(4)]} units were linked by flexible chains of different length [(CH(2))(n) (n=4, 6, 8, 12)]. These new dinuclear ruthenium drugs were found to exert promising cytotoxic effects in human cancer cells. In the present work DNA modifications by these new dinuclear Ru(II) arene compounds, which differed in the length of the linker between the two Ru(II) centers, were examined by biochemical and biophysical methods. The complexes bind DNA forming intrastrand and interstrand cross-links in one DNA molecule in the absence of proteins. An intriguing aspect of the DNA-binding mode of these dinuclear Ru(II) compounds is that they can cross-link two DNA duplexes and also proteins to DNA--a feature not observed for other antitumor ruthenium complexes. Thus, the concept for the design of interhelical and DNA-protein cross-linking agents based on dinuclear Ru(II) arene complexes with sufficiently long linkers between two Ru centers may result in new compounds which exhibit a variety of biological effects and can be also useful in nucleic acids research.

DNA Interactions of Monofunctional Organometallic Osmium(II) Antitumor Complexes in Cell-Free Media

This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. We investigated the interactions between the potential biological target DNA and four osmium(II) arene complexes, of the type [(eta 6-arene)Os(LL)Cl]n+, where arene = biphenyl or p-cymene and LL = ethylenediamine, picolinate, or oxinate in an effort to understand their mechanism of action. Most notably we show that these complexes bind to DNA. DNA adducts of the OsII complexes that exhibit promising cytotoxic effects in ovarian tumor cell lines largely distort its conformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involves combined coordination to guanine residues and noncovalent interactions between the arene ligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin.

Activation of trans geometry in bifunctional mononuclear platinum complexes by a piperidine ligand: Mechanistic studies on antitumor action

A paradigm for the structure-pharmacological activity relationship of bifunctional platinum antitumor drugs is that the trans isomer of antitumor cisplatin (transplatin) is clinically ineffective. To this end, however, several new complexes of the trans structure have been identified that exhibit cytotoxicity in tumor cells that is even better than that of the analogous cis isomers. We reported recently (Kasparkova, J., Marini, V., Najajreh, Y., Gibson, D., and Brabec, V. (2003) Biochemistry 42, 6321–6332) that the replacement of one ammine ligand by the heterocyclic ligand, such as piperidine, piperazine, or 4-picoline in the molecule of transplatin resulted in a radical enhancement of its cytotoxicity. We examined oligodeoxyribonucleotide duplexes bearing a site-specific cross-link of the transplatin analogue containing the piperidine ligand by biochemical methods. The results indicate that in contrast to transplatin, trans-(PtCl2(NH3)(piperidine)) forms stable 1,3-intrastrand cross-links in double-helical DNA that distort DNA and are not readily removed from DNA by nucleotide excision repair system. Hence, the intrastrand cross-links of trans-(PtCl2(NH3)(piperidine)) could persist for a sufficiently long time, potentiating its toxicity toward tumor cells. trans-(PtCl2(NH3)(piperidine)) also forms in DNA minor interstrand cross-links that are similar to those of transplatin so that these adducts appear less likely candidates for genotoxic lesion responsible for antitumor effects of trans-(PtCl2(NH3)(piperidine)). Hence, the role of structurally unique intrastrand cross-links in the anti-tumor effects of transplatin analogues in which one ammine group is replaced by a heterocyclic ligand may predominate.

Diazido mixed-amine platinum(IV) anticancer complexes activatable by visible-light form novel DNA adducts

Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side-effects. Photoactivatable PtIV prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X-ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans-[Pt(N3)2(OH)2(MA)(Py)] (1; MA=methylamine, Py=pyridine) and trans,trans,trans-[Pt(N3)2(OH)2(MA)(Tz)] (2; Tz=thiazole), and interpret their photophysical properties by TD-DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1 p and 1 q. Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin-resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf-thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono- and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross-links, with evidence for DNA strand cross-linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin-type lesions. The photo-induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the PtII compounds trans-[PtCl2(MA)(Py)] (5) and trans-[PtCl2(MA)(Tz)] (6). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1, whereas the dark reactions of 5 and 6 with DNA were comparatively slow. Complexes 1 and 2 can therefore give rapid potent photocytotoxicity and novel DNA lesions in cancer cells, with no activity in the absence of irradiation.

mer-[Ru(terpy)Cl3] (terpy = 2,2′:6′,2″-terpyridine) shows biological activity, forms interstrand cross-links in DNA and binds two guanine derivatives in a trans configuration

Abstract The compound mer-[Ru(terpy)Cl3] was found to be active as a cytostatic in L1210 leukemia cells, with an activity in between cisplatin and carboplatin. It was shown that the Ru complex covalently binds to DNA and this binding results in the formation of ∼2% interstrand cross-links. In addition, no Ru-promoted nicks are formed. The parent compound has also been reacted with the DNA model bases 9-methylhypoxanthine (9mhyp) and 9-ethylguanine (9egua). The solid complexes trans-[Ru(terpy)(B-κN7)2(H2O)](PF6)2, where B = 9mhyp or 9egua, were isolated and characterized in acetone solution by proton NMR. The bases are symmetrically arranged around the metal center, and involved in hydrogen bonding with the water ligand, most likely via their O6 atoms. The water ligand has been substituted by acetonitrile. The complexes trans-[Ru(terpy)(B-κN7)2(CH3CN)](PF6)2, where B = 9mhyp or 9egua, have been prepared in situ. Compared to the aqua complexes, the hydrogen bond donor molecule is lost and a rearrangement of the guanine bases is observed.

Molecular Aspects of Antitumor Effects of a New Platinum(IV) Drug

The new platinum(IV) complex cis,trans,cis-[PtCl2(CH3COO)2-(NH3)(1-adamantylamine)] [adamplatin(IV)] seems promising for the perspective application in therapy of corresponding tumors. It is therefore of great interest to understand details of mechanisms underlying its biological efficacy. Cellular uptake of the drug, alterations in the target DNA induced by platinum drugs along with processing of platinum-induced damage to DNA and drug inactivation by sulfur-containing compounds belong to major pharmacological factors affecting antitumor effects of platinum compounds. We examined in the present work the significance of these factors in the mechanism of antitumor effects of adamplatin(IV) and compared the results with those of the parallel studies performed with “conventional” cisplatin. The results show that deactivation of adamplatin(IV) by sulfur-containing compounds (such as glutathione or metallothioneins) is likely to play a less significant role in the mechanism of resistance of tumor cells to adamplatin(IV) in contrast to the role of these reactions in the effects of cisplatin. Moreover, the treatment of tumor cells with adamplatin(IV) does not result in DNA modifications that would be markedly different from those produced by cisplatin. In contrast, the effects of other factors, such as enhanced accumulation of the drug in cells, strong inhibition of DNA polymerization by these adducts, lowered DNA repair, and DNA-protein cross-linking are different from the effects of these factors in the mechanism underlying activity of cisplatin. Hence, the differences between effects of adamplatin(IV) and cisplatin observed in the present work on molecular level may help understand the unique activity of adamplatin(IV).

Interactions of DNA with a new platinum(IV) azide dipyridine complex activated by UVA and visible light: relationship to toxicity in tumor cells.

The Pt(IV) diazido complex trans,trans,trans-[Pt(N(3))(2)(OH)(2)(pyridine)(2)] (1) is unreactive in the dark but is cytotoxic when photoactivated by UVA and visible light. We have shown that 1 when photoactivated accumulates in tumor cells and binds strongly to nuclear DNA under conditions in which it is toxic to tumor cells. The nature of the DNA adducts, including conformational alterations, induced by photoactivated 1 are distinctly different from those produced in DNA by conventional cisplatin or transplatin. In addition, the observation that major DNA adducts of photoactivated 1 are able to efficiently stall RNA polymerase II more efficiently than cisplatin suggests that transcription inhibition may contribute to the cytotoxicity levels observed for photoactivated 1. Hence, DNA adducts of 1 could trigger a number of downstream cellular effects different from those triggered in cancer cells by DNA adducts of cisplatin. This might lead to the therapeutic effects that could radically improve chemotherapy by platinum complexes. The findings of the present work help to explain the different cytotoxic effects of photoactivated 1 and conventional cisplatin and thereby provide new insights into mechanisms associated with the antitumor effects of platinum complexes photoactivated by UVA and visible light.

A comparison of DNA binding profiles of dinuclear platinum compounds with polyamine linkers and the trinuclear platinum phase II clinical agent BBR3464

Abstract. The DNA binding profiles of three bis Pt(II) polyamine-linked compounds, [{trans-PtCl(NH3)2}2{µ-spermine-N1,N12}]4+, [{trans-PtCl(NH3)2}2{µ-spermidine-N1,N8}]3+, and [{trans-PtCl(NH3)2}2{µ-BOC-spermidine}]2+, were compared with that of a novel trinuclear phase II clinical agent, [{trans-PtCl(NH3)2}2{µ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+. All of the compounds bind preferentially in a bifunctional manner, according to unwinding of supercoiled DNA circles. The kinetics of binding of these compounds corresponds to their relative charge (2+ to 4+). The preference for the formation of interstrand crosslinks, however, does not follow a charge-based pattern. By studying the results of DNA polymerase extension products on a DNA template modified by the compounds, and by incorporating the complementary method of RNA transcription mapping, it was possible to determine the nucleotide bases that are preferred sites of binding. The stop sites due to platinum adducts were determined, and some preliminary observations concerning the range and type of crosslinks were established. It can be concluded that dinuclear Pt compounds are similar to their trinuclear counterpart, and that charge differences do not contribute solely to the variances between the compounds.