Intercalation Model for DNA-Cross Linking in a 1-Nitro-9-aminoacridine Derivative,an Analog of the Antitumor Agent “Ledakrin” (Nitracrine)

Abstract

Ledakrin (nitracrine), C-283, is a 1-nitro-9-aminoacridine derivative that is used in Poland as an antitumor agent. In order to investigate the basis of the activity of this compound the structure of another analog, [9-(3-dimethyl-l-methylpropylimino)-l-nitro-9, 10-dihydroacridine], C-829, that has similar activity, was determined by X-ray crystallographic techniques and was compared with that of ledakrin, already reported in the literature. In both molecules the proximity of the 1-nitro to the substituted 9-aminoacridine group causes extensive distortions. These compounds are believed to act, after metabolic “activation”, by cross-linking DNA. Such cross-linking does not occur in the absence of the 1-nitro group or if the nitro group is moved to the 2-, 3- or 4-position. Computer-assisted model-building has been used to test possible intercalative models. It has shown that functional groups on C-829 or C-283 are, when the acridine portion of the molecule is intercalated as in a proflavine dinucleoside phosphate complex, in positions suitable for DNA cross-linking by activated 1-nitro- 9-aminoacridine derivatives.     

Chromosome aberrations induced by 9-aminoacridine derivatives

The induction of chromosomal aberrations by 5 derivatives of nitro-9-aminoacridine in V79 Chinese hamster cells was observed. The clastogenic activity of the compounds tested depended on the position of the NO2 group in the acridine ring. The strongest clastogens were derivatives with NO2 in position 1. The remaining derivatives placed in decreasing order of clastogenic activity were: 3-nitro, 4-nitro and 2-nitro. In addition, 2-nitro and 3-nitro derivatives produced hyperdiploid/polyploid metaphases.     

The Development of A-ring Modified Analogues of Oestrone-3-O-sulphamate as Potent Steroid Sulphatase Inhibitors with Reduced Oestrogenicity

Steroid sulphatases regulate the formation of oestrogenic steroids which can support the growth of endocrine-dependent breast tumours. The development of potent steroid sulphatase inhibitors could therefore have considerable therapeutic potential. Several such inhibitors have now been developed of which the most potent to date is oestrone-3-O-sulphamate (EMATE). Unexpectedly, this inhibitor proved to be a potent oestrogen. In an attempt to reduce the oestrogenicity, whilst retaining the potent sulphatase inhibitory properties associated with this type of molecule, a number of A-ring modified derivatives were designed and synthesized. A-ring modified compounds included the 2-methoxy, 2/4-nitro, 2/4-n-propyl and 2/4-allyl EMATE analogues. The ability of these derivatives to inhibit oestrone sulphatase activity was examined using placental microsomes. The allyl-substituted EMATE derivatives were more potent inhibitors than the propyl analogues but were all considerably less potent than EMATE. In contrast, the 2-methoxy and 2/4-nitro analogues were potent sulphatase inhibitors with 4-nitro EMATE being 5 times more active than EMATE. The 4-nitro, 2-methoxy, 4-n-propyl and 4-allyl derivatives were also tested in vivo for their oestrogenicity and ability to inhibit sulphatase activity. While both 4-nitro and 2-methoxy EMATE were potent inhibitors in vivo, 2-methoxy EMATE had no stimulatory effect on uterine growth in ovariectomized rats. The identification of a potent steroid sulphatase inhibitor lacking any oestrogenicity, such as 2-methoxy EMATE, should be of considerable value in evaluating the potential of steroid sulphatase inhibition for breast cancer therapy.     

Role of the functional groups of the sibiromycin molecule in DNA binding]

Biological activity of 2 derivatives of sibiromycin, an antibiotic close by its chemical structure to antramycin and their capacity for formation of complexes with DNA was studied. Anhydrosibiromycin like sibiromycin formed a complex with DNA. The antibiotic increased the DNA melting point but to a less extent than sibiromycin. Anhydrosibiromycin had a low activity in the system of DNA-dependent RNA-polymerase. The low biological activity of anhydrosibiromycin must be due to instability of the antibiotic complex with DNA. Methyl ether of sibiromycin by the phenol hydroxyl, the other derivative of sibiromycin had no biological activity and did not interact with DNA. On the basis of experimental data it was suggested that definite functional groups of the sibiromycin participated in DNA binding.     

Peroxidase catalyzed nitration of tryptophan derivatives. Mechanism, products and comparison with chemical nitrating agents.

The enzymatic nitration of tryptophan derivatives by oxidation of nitrite has been studied using lactoperoxidase and horseradish peroxidase, and compared with the chemical nitration produced by nitrogen dioxide and peroxynitrite. HPLC, mass spectra and NMR analysis of the mixture of products clearly show that nitration occurs at position 4-, 6-, 7-, and N1 of the indole ring, and nitrosation at position N1. Kinetic studies performed on peroxidase/NO2-/H2O2 systems showed substrate saturation behavior with all the tryptophan derivatives employed. The rate dependence on nitrite concentration was found to be linear with horseradish peroxidase while it exhibited saturation behavior with lactoperoxidase. The composition of the product mixture depends on the nitrating agent. While the production of 4-nitro, 6-nitro, 7-nitro and N1-nitro derivatives follows a similar trend, indicating that they are formed according to a similar mechanism, the ratio between the N1-nitroso derivative and other derivatives depends markedly on the nitrite concentration when tryptophan modification is performed by the peroxidase/H2O2/nitrite systems. Analysis of the data indicates that at low nitrite concentration the enzymatic reaction occurs through the classical peroxidase cycle. At high nitrite concentration the reaction proceeds through a different intermediate that we assume to be a protein bound peroxynitrite species.     

Complexes of derivatives of 1-nitro-9-aminoacridine with DNA.

Substituted 1-nitro-9-aminoacridine derivatives were shown to inhibit RNA and to a lesser extent protein synthesis in cultured human cells. Complex formation between the compounds studied and DNA were considered to be responsible for their cytostatic action. Two types of complexes differing in their binding forces were found. The biological activity of the studied compounds seems not to be dependent on the existence of a positive charge on the acridine ring.     

Synthesis and biological evaluation of rhodanine derivatives as PRL-3 inhibitors

A series of rhodanine derivatives was synthesized and evaluated for their ability to inhibit PRL-3. Benzylidene rhodanine derivative showed good biological activity, while compound 5e was the most active in this series exhibiting an IC50 value of 0.9 microM in vitro and showed a reduced invasion in cell-based assay.     

New derivative of 2-(2,4-dihydroxyphenyl)thieno-1,3-thiazin-4-one (BChTT) elicits antiproliferative effect via p38-mediated cell cycle arrest in cancer cells

2-(2,4-Dihydroxyphenyl)thieno-1,3-thiazin-4-ones are a group of new compounds with potential anticancer activity. This type of derivatives was poorly investigated in the area of synthesis and biological activities. In the present study the antiproliferative action of the most active derivative BChTT was described. The aim of biological evaluation was to investigate the ability of the compound to inhibit cancer cell proliferation and identify mechanism involved in its action on the molecular level. BChTT inhibited the proliferation of lung cancer A549, colon cancer HT-29 and glioma C6 cells in the concentration-dependent manner. It was not toxic to normal cells including skin fibroblasts, hepatocytes and oligodendrocytes in the antiproliferative concentrations. BChTT decreased the DNA synthesis in the treated cancer cells and induced cell cycle arrest in the G0/G1 phase. Moreover, the ability of the compound to activate p38 kinase and decrease cyclin D1 expression was estimated. Participation of p38 kinase in the antiproliferative action of the compound was confirmed by the analysis of BChTT activity in the cells with the p38 silenced gene. The obtained results may suggest the ability of the tested derivative to inhibit cancer cells proliferation by induction of p38-mediated cyclin D1 downregulation.     

Functions and regulation of the APOBEC family of proteins

APOBEC1 is a cytidine deaminase that edits messenger RNAs and was the first enzyme in the APOBEC family to be functionally characterized. Under appropriate conditions APOBEC1 also deaminates deoxycytidine in single-stranded DNA (ssDNA). The other ten members of the APOBEC family have not been fully characterized however several have deoxycytidine deaminase activity on ssDNAs. Despite the nucleic acid substrate preferences of different APOBEC proteins, a common feature appears to be their intrinsic ability to bind to RNA as well as to ssDNA. RNA binding to APOBEC proteins together with protein-protein interactions, post-translation modifications and subcellular localization serve as biological modulators controlling the DNA mutagenic activity of these potentially genotoxic proteins.     

Interactions of antitumor triazoloacridinones with DNA

Triazoloacridinones (TA) are a new group of potent antitumor compounds, from which the most active derivative, C-1305, has been selected for extended preclinical trials. This study investigated the mechanism of TA binding to DNA. Initially, for selected six TA derivatives differing in chemical structures as well as cytotoxicity and antitumor activity, the capability of noncovalent DNA binding was analyzed. We showed that all triazoloacridinones studied stabilized the DNA duplex at a low-concentration buffer but not at a salt concentration corresponding to that in cells. DNA viscometric studies suggested that intercalation to DNA did not play a major role in the mechanism of the cytotoxic action of TA. Studies involving cultured cells revealed that triazoloacridinone C-1305 after previous metabolic activation induced the formation of interstrand crosslinks in DNA of some tumor and fibroblast cells in a dose dependent manner. However, the detection of crosslink formation was possible only when the activity of topoisomerase II in cells was lowered. Furthermore, it was impossible to validate the relevance of the ability to crosslink DNA to biological activity of TA derivatives.     

A-ring and E-ring modifications of the cytotoxic alkaloid Luotonin A: Synthesis,computational and biological studies

A series of new Luotonin A derivatives with substituents at rings A and E was synthesized, together with some E-ring-unsubstituted derivatives. Subsequently, the compound library was examined in silico for their binding into a previously proposed site in the DNA/topoisomerase I binary complex. Whereas no convincing correlation between docking scores and biological data from in vitro assays could be found, one novel 4,9-diamino Luotonin A derivative had strong antiproliferative activity based on massive G2/M phase arrest. As this biological activity clearly differs from the reference compound Camptothecin, this strongly indicates that at least some Luotonin A derivatives may be potent antiproliferative agents, however with a different mode of action.     

Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.     

The number and distances of positive charges of polyamine side chains in a series of perylene diimides significantly influence their ability to induce G-quadruplex structures and inhibit human telomerase

We have synthesized eight polyamine perylene diimides to conjugate the efficiency of perylene derivatives in stabilizing G-quadruplex structures and the polyamines' biological activity, due to specific interactions with different DNA domains. Our study was carried out by investigating the ability of these derivatives to induce inter- and intramolecular G-quadruplex structures by polyacrylamide gel electrophoresis (PAGE) and to inhibit telomerase in a modified TRAP assay. The two properties appear to be satisfactorily correlated and they show that the number and distances of positive charges in the side chains dramatically influence both these features. Although our previous studies on perylene derivatives with mono-positively charged side chains indicated that self assembly in aqueous solution leads to a major efficiency, the result observed with the spermine derivative suggests that a too strong aggregation is unfavourable, because it determines a lower solubility of the compounds.     

Synthesis and biological evaluation of new N-(2-hydroxy-4(or 5)-nitro/aminophenyl)benzamides and phenylacetamides as antimicrobial agents

A new series of N-(2-hydroxy-4(or 5)-nitro/aminophenyl)benzamide and phenylacetamide derivatives (1a-1n, 2a-2n) were synthesized and evaluated for antibacterial and antifungal activities against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and their drug-resistant isolate. Microbiological results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms at MIC values between 500 and 1.95 microg/ml. Benzamide derivative 1d exhibited the greatest activity with MIC values of 1.95, 3.9, and 7.8 microg/ml against drug-resistant B. subtilis, B. subtilis, and S. aureus, respectively.     

Preparation and characterization of biotinylated psoralen.

Biotinylated psoralen (BPsor), a psoralen derivative containing a biotin moiety attached via a long-chain, positively charged linker, has been synthesized and its interactions with DNA and avidin have been studied. As do other psoralen derivatives, BPsor photoreacts with DNA to form interstrand crosslinks. The biotin binds to streptavidin after the reaction of BPsor with DNA, and this property has been used to measure low levels of BPsor modified DNA by ELISA with streptavidin and biotinylated alkaline phosphatase. In addition, BPsor retains the biological activity of psoralen, as shown by its ability to inhibit lymphocyte proliferation at a level of 10 ng/ml.     

Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2'-fluoronucleoside and arabinofuranosylcytosine]

A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.     

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their β1,4-GalT inhibitory and anti-cancer properties

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit β-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.