Effect of intercalating and groove-binding ligands on formation of covalent complexes between nitracrine (Ledakrin, C-283) or 8-methoxypsoralen and DNA.

The effect of ethidium bromide, actinomycin D, distamycin A and netropsin on covalent binding of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine, Ledakrin, C-283) and 8-methoxypsoralen to DNA was examined. The competition was assayed either directly with [3H]- and [14C]nitracrine or indirectly by estimation of transcriptional template activity of nitracrine-DNA and 8-methoxypsoralen-DNA complexes formed in the presence of the ligands. A higher protective effect of ethidium bromide and distamycin on the photo-binding of 8-methoxypsoralen than on the dithiothreitol-dependent attachement of nitracrine to DNA assayed at 0.15 M KCl or NaCl was observed. The non-intercalating antibiotics showed lower competitive effect on 8-methoxypsoralen binding than ethidium bromide. Actinomycin D showed relatively low competition for both drugs with DNA. In contrast to the reaction of 8-methoxypsoralen, the decrease of nitracrine binding in the presence of competing ligands considerably depends on ionic strength. Particularly high inhibition of the adduct formation in the presence of ethidium at 1 M KCl was shown, while the amount of nitracrine bound in the presence of distamycin increases at elevated ionic strength. The results may indicate steric demands of the reaction between nitracrine and DNA.

Inhibition of DNA-dependent RNA synthesis by 8-methoxypsoralen.

The effect of the photobinding of 8-methoxypsoralen to phage T7 DNA on different steps of RNA synthesis in vitro was assayed. Total RNA synthesis is reduced to a few percent and the transcript size is decreased, as shown by means of gel filtration on a Sepharose 4B column when DNA of the adduct content of six drug molecules per 10(3) nucleotides is used. The initiation of RNA chains seems to be less affected, as inferred from an abortive initiation assay. Synthesis of pppApU on DNA of the same adduct content is inhibited to 34% of the corresponding controls, while the overall RNA synthesis is inhibited to 6%. The amount of the enzyme needed for maximal retention of DNA, the kinetics of its binding and the decay of the polymerase-DNA complex at high ionic strength (or on decrease of the temperature) are similar with DNA either irradiated in the absence of the drug or DNA bearing six 8-methoxypsoralen molecules per 10(3) nucleotides. It is concluded from this study that 8-methoxypsoralen partially inhibits initiation and blocks movement of RNA polymerase along the template, inducing premature termination. It does not appear to influence the binding of the enzyme to DNA.

Thiol-dependent inhibition of RNA synthesis in vitro by acridines: structure-inhibition relationships.

In the presence of sulfhydryl compounds an anticancer drug, 1-nitro-9-aminoalkylacridine derivative, forms with DNA irreversible, probably covalent, complexes of decreased template properties. Five 9-substituted 1-nitro-9-aminoacridine derivatives of cytostatic activity show irreversible thiol-dependent inhibitory effects on the RNA synthesis in vitro system while equal inhibition is observed both in the presence and in the absence of dithiothreitol with biologically inactive analogues of nitrocrine. In the absence of sulfhydryl compounds the inhibition depends on the planarity of the acridine ring. Hence, both 1-nitro-9-aminoalkylacridine and tetrahydroacridine derivatives show low inhibitory effect.

Cytotoxic and DNA-damaging properties of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and its analogues.

An antitumor drug N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and its three close structural analogs N-[2-(hydroxyethylamino)ethyl]acridine-4-carboxamide (DACAH), N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (amino-DACA), and N-[2-(hydroxyethylamino)ethyl]-9-aminoacridine-4-carboxamide (amino-DACAH) were studied for their ability to inhibit RNA synthesis in vitro and to form topoisomerase II-mediated DNA lesions in relation to cell-killing activity. All tested compounds induced chromatin lesions characteristic of topoisomerase II-blocking drugs (DNA breaks and DNA-protein cross-links) in treated cells, but were much less active than reference antileukemic acridine m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide). The ability to form these lesions was dependent on the structure of the 4-carboxamide side-chain, which seems to be an important factor affecting the drug transport rate through cell membrane. A 4-carboxamide chain with an N-2-(dimethylamino)ethyl moiety resulted in more efficient transport through cell membranes, higher cytotoxicity, and DNA-damaging activity. The mode of action of acridine-4-carboxamides was further elucidated by their incubation with cells in the presence of antitopoisomerase II agents of a known mechanism of inhibition. These were: bisdioxopiperazine (ICRF-187), a catalytic inhibitor of topoisomerase II, and etoposide (VP-16), an inducer of a cleavable complex of the enzyme with DNA. The cytotoxicity of DACA and its analogs was not antagonized by preincubating cells with ICRF-187. All tested acridines protected cells against DNA breakage induced by VP-16, but the extent of protection varied significantly. Amino-DACA, which easily penetrates cell membrane, fully inhibited DNA break formation, whereas other analogs exhibited a low degree of protection when used at high concentration. Our results suggest that the acridine-4-carboxamides discussed here are poor topoisomerase II poisons and that this enzyme is not their main target.

Actinomycin D specifically inhibits the interaction between transcription factor Sp1 and its binding site.

The mode of action of many anticancer drugs involves DNA interactions. We here examine the ability of actinomycin D to alter the specific binding of transcription factors Spl and NFkappaB to their DNA sequences. Employing an electrophoretic mobility shift assay, it is shown that actinomycin D inhibits complex formation between nuclear proteins present in the extracts from stimulated human umbilical vein endothelial cells and the Sp1-binding site. Actinomycin D is also able to induce disruption of preformed DNA-protein complexes, pointing to the importance of an equilibrium of three components: actinomycin D, protein and DNA for drug action. The effect of actinomycin D is sequence-specific, since no inhibition is observed for interaction of nuclear proteins with the NFkappaB binding site. The results support the view that DNA-binding drugs displaying high sequence-selectivity can exhibit distinct effects on the interaction between DNA and different DNA-binding proteins.

Transcription factors as targets of anticancer drugs.

Several general and gene- and cell-selective transcription factors are required for specific transcription to occur. Many of them exert their functions through specific contacts either in the promoter region or at distant sequences regulating the initiation. These contacts may be altered by anticancer drugs which form non-covalent complexes with DNA. Covalent modifications of DNA by alkylating agents may prevent transcription factors from recognizing their specific sequences or may constitute multiple "unnatural" binding sites in DNA which attract the factors thus decreasing their availability in the cell. The anticancer drug-transcription factor interplay which is based on specific interactions with DNA may contribute to pharmacological properties of the former and provide a basis for the search for new drugs.

Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents. Effects on overall RNA synthesis and synthesis of the initiating dinucleotide.

A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system. Escherichia coli DNA-dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses. By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes.

Effect of DNA-interacting drugs on phage T7 RNA polymerase.

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Structure of acridines and their effect on RNA synthesis in vitro.

1. Ledakrin (C-283), a 1-nitro-9-aminopropylacridine derivative, inhibits RNA synthesis in vitro if the complex with DNA is formed in the presence of thiol. 2. Using several analogues of Ledakrin, it has been found that the 1-nitro group is essential for enhancement of the inhibition by thiol; the length of 9-aminoalkyl side chain also plays a role in the reaction between DNA and the dye. 3. It is suggested that the low inhibitory effect of Ledakrin in the absence of thiol compounds is due to a steric hindrance between neighbouring 1-nitro and 9-aminoalkyl groups. This hypothesis has been confirmed by assaying inhibition of RNA synthesis by several analogues of Ledakrin.

Some properties of the irreversible complexes of nitracrine (ledakrin, C-283) with polynucleotides.

In the presence of sulfhydryl compounds an anticancer drug nitracrine (NA), 1-nitro-9-aminoalkylacridine derivative forms strong, probably covalent complexes with DNA. It has been found that it binds with similar efficiency to RNA and DNA exhibiting a certain preference for single-stranded structure. At NA/polynucleotide ratio of 0.15 and nucleic acids concentration 100 microgram/ml the numbers of drug molecules bound per 10(3) nucleotides were about 10--13 for native calf thymus DNA, 19--28 for denatured DNA and 23--36 for RNA. Some base specificity to guanine is observed both in polydeoxyribo- and polyribonucleotides. The complexes of NA with DNA and double-stranded synthetic polynucleotides exhibit decreased transcriptional template activity in bacterial RNA synthesis in vitro system except poly(A) synthesis on poly(dA) x poly(dT) which is insensitive to the drug. The drug binding in vitro leads to cross-link formation in DNA as shown by means of ultraviolet spectrophotometry and hydroxylapatite chromatography of heat-denatured NA-DNA complexes. The amount of the double bonds introduced by the drug is however relatively low as compared with cross-linking of irradiated 8-methoxy-psoralen-DNA (MOP-DNA) complexes.

DNA binding, cytotoxicity and inhibitory effect on RNA synthesis of two new 1-nitro-9-aminoacridine dimers.

Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different.

Transcriptional template activity of covalently modified DNA.

The transcriptional template activity of covalent modified DNA is compared. 8-Methoxypsoralen (MOP), 3,4'dimethyl-8-methoxypsoralen (DMMOP) and benzopsoralen (BP) forming with DNA covalent complexes upon UV irradiation and exhibiting preference to pyrimidines, mostly thymines, differ in their cross-linking potency. MOP and DMMOP form both monoadducts and diadducts while no cross-links are formed by BP. Nitracrine (NC) forms covalent complexes with DNA upon reductive activation with dithiothreitol exhibiting a preference to purines and low cross-linking potency. Semilogarithmic plots of the relative template activity against the number of the drugs molecules covalently bound per 10(3) DNA nucleotides fit to regression lines corresponding to one-hit inactivation characteristics. The number of drug molecules decreasing RNA synthesis to 37% differ from 0.25 to 1.26 depending on the template used and the base preference but no dependence on the cross-linking potency was found.

Crosslinking of cellular DNA by nitracrine and furocoumarin derivatives.

The anticancer drug, nitracrine, a 1-nitro-9-aminoalkyl derivative of acridine exhibits potent cytotoxic effects which are due to its metabolic activation, followed by covalent binding to macromolecules--DNA being the target for the drug. The renaturable fraction of DNA from L-1210 cells pretreated with nitracrine is assayed by means of ethidium bromide fluorescence assay and chromatography on hydroxyapatite column. The effect of the drug was compared with furocoumarins of different DNA crosslinking potencies. The existence of crosslinks in DNA upon incubation of cells with nitracrine (1-4 microM) have been confirmed with two different methods under the conditions where 8-methoxypsoralen, a classic crosslinking agent induced the renaturation. The DNA preparation isolated from the drug pretreated cells exhibited decreased transcriptional template activity with E. coli DNA-dependent RNA polymerase.

Inhibition of RNA synthesis in vitro and cell growth by anthracycline antibiotics.

New derivatives of doxorubicin and daunorubicin with amidine group bonded to daunosamine at C-3' atom and bearing the morpholine ring attached to the amidine group have been recently synthesized. Their cytotoxic activities and effects on RNA synthesis in vitro were assayed. The drug concentrations inhibiting mouse leukaemia L1210 cell growth to 50% were about two- and three fold higher for the derivatives compared to doxorubicin and daunorubicin respectively. Inhibition of phage T7 RNA polymerase by the non-covalently interacting derivatives was also slightly lower than that by the parent compounds. As doxorubicin and daunorubicin, their amidine derivatives in the presence of dithiothreitol and Fe(III) ions are activated and covalently bind to DNA. The adducts formed affect RNA polymerase activity. Several bands corresponding to prematurely terminated RNA chains are observed by means of polyacrylamide gel electrophoresis. The patterns of bands are virtually identical for all the anthracyclines studied here and are similar to the terminations induced by actinomycin D. This observation is consistent with a notion that the adducts are formed at guanine in GpC sequences which are also binding sites of actinomycin D. A substantial difference between daunorubicin and its amidine derivative is shown by means of high performance liquid chromatography. The derivative undergoes rapid rearrangements in the presence of dithiothreitol and Fe(III) ions, while daunorubicin is stable for several hours under these conditions. The results presented here indicate that the amidine derivatives despite bulky morpholine substitution exhibit biological activity in the systems used here.

Photoreaction of furocoumarins with DNA is similarly inhibited by minor and major groove-interacting ligands.

It was found that methyl green, a major groove binding ligand and the minor groove binding ligands, netropsin and 2,7-di-tert-butylproflavine inhibit, to a similar extend a monoadduct forming benzopsoralen and monoadduct and diadduct forming derivatives of psoralen (8-methoxypsoralen and 3,4'-dimethyl-8-methoxypsoralen). Caffeine exhibits an inhibitory effect on furocoumarin photobinding to DNA at 10(3) fold higher concentration. Together with the previously published results it is concluded that both occupancy of the major and minor groove as well as intercalation hinder photobinding of furocoumarins to DNA.

In vitro binding of nitracrine to DNA in chromatin.

In the presence of sulfhydryl compounds nitracrine, an anticancer drug, binds covalently to DNA. The accessibility of DNA in chromatin both to nitracrine and to 8-methoxypsoralen, which was used as a reference compound in this study, when assayed in NaCl concentrations from 0 to 2 M show similar characteristics. The initial decrease reaches a minimum at 0.15 M NaCl above which dissociation of non-histone proteins and histones at higher ionic strengths is demonstrated by an increase in accessible sites. The relative accessibility of DNA in chromatin to nitracrine is, however, lower than that found for 8-methoxypsoralen. Partial dissociation of chromatin with 0.7 M NaCl increases the accessibility of DNA in chromatin when assayed in the absence of NaCl but has no apparent influence when estimated at ionic strength close to physiological conditions.

Effects of anticancer drugs on transcription in vitro.

The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E. coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E. coli RNA polymerase ("abortive initiation"); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter phi 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G x C) specificity (actinomycin D), the non-intercalating, d(A x T) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.

Inhibition of RNA synthesis in vitro by acridines--relation between structure and activity.

The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied. The primary mechanism of action is related to inhibition of the enzyme binding to DNA. The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA. Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives. No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here. The aniline ring seems to decrease the inhibitory potency of a ligand. The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison.