Cytotoxicity and DNA interactions of some platinum(II) complexes with substituted benzimidazole ligands.

In the present study, four Pt(II) complexes with 2-ethyl (1)/or benzyl (2)/or p-chlorobenzyl (3)/or 2-phenoxymethyl (4) benzimidazole carrier ligands were evaluated for their in vitro cytotoxic activities against the human HeLa cervix, oestrogen receptor-positive MCF-7 breast, and oestrogen receptor-negative MDA-MB 231 breast cancer cell lines. The plasmid DNA interactions and inhibition of the BamHI restriction enzyme activities of the complexes were also studied. Complex 3 was found to be more active than carboplatin for all examined cell lines and comparable with cisplatin, except for the HeLa cell line.

Intermolecular and intramolecular quencher based quantum dot nanoprobes for multiplexed detection of endonuclease activity and inhibition.

DNA cleavage by endonucleases plays an important role in many biological events such as DNA replication, recombination, and repair and is used as a powerful tool in medicinal chemistry. However, conventional methods for assaying endonuclease activity and inhibition by gel electrophoresis and chromatography techniques are time-consuming, laborious, not sensitive, or costly. Herein, we combine the high specificity of DNA cleavage reactions with the benefits of quantum dots (QDs) and ultrahigh quenching abilities of inter- and intramolecular quenchers to develop highly sensitive and specific nanoprobes for multiplexed detection of endonucleases. The nanoprobe was prepared by conjugating two sets of DNA substrates carrying quenchers onto the surface of aminated QDs through direct assembly and DNA hybridization. With this new design, the background fluorescence was significantly suppressed by introducing inter- and intramolecular quenchers. When these nanoprobes are exposed to the targeted endonucleases, specific DNA cleavages occur and pieces of DNA fragments are released from the QD surface along with the quenchers, resulting in fluorescence recovery. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The detection was accomplished with a single excitation light. Multiplexed detection was demonstrated by simultaneously assaying EcoRI and BamHI (as model analytes) using two different emissions of QDs. The limits of detection were 4.0 × 10(-4) U/mL for EcoRI and 8.0 × 10(-4) U/mL for BamHI, which were at least 100 times more sensitive than traditional gel electrophoresis and chromatography assays. Moreover, the potential application of the proposed method for screening endonuclease inhibitors has also been demonstrated. The assay protocol presented here proved to be simple, sensitive, effective, and easy to carry out.

Inhibition of restriction enzymes EcoRI, BamHI and HindIII by phenethylphenylphthalimides derived from thalidomide.

We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of compounds with a phenethylphenylphthalimide skeleton, based on α-glucosidase inhibitors and liver X receptor antagonists derived from thalidomide. Structural development afforded the potent restriction enzyme inhibitors 25 and 26.

Linker length modulates DNA cross-linking reactivity and cytotoxic potency of C8/C8' ether-linked C2-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers.

A C2/C2'-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 4b (DRG-16) with a C8-O(CH2)nO-C8' diether linkage (n = 5) has been synthesized that shows markedly superior in vitro cytotoxic potency (e.g., >3400-fold in IGROV1 ovarian cells) and interstrand DNA cross-linking reactivity (>10-fold) compared to the shorter homologue 4a (SJG-136; n = 3). In contrast, for the C-ring unsubstituted series, the corresponding n = 5 dimer (3c) is generally less cytotoxic and has a lower interstrand cross-linking reactivity compared to its shorter n = 3 homologue (3a). Dimer 4b cross-links DNA with >10-fold efficiency compared to 4a, and also inhibits the activity of the restriction endonuclease BamH1 more efficiently than either 3a or 4a. The C2-exo-unsaturated PBD dimers 4a,b are not only more effective than their C-ring saturated counterparts in terms of induced DeltaTm shift, but they also exert this effect more rapidly. Thus, while 3a and 3c exert 68 and 35% of their maximum effect immediately upon interaction with DNA, this level increases to 76 and 97% for 4a and 4b, respectively. Molecular modeling shows a rank order of 4b (n = 5) > 4a (n = 3) > 3a (n = 3) > 3c (n = 5) in terms of binding energy toward duplexes containing embedded target 5'-GAT(1-2)C cross-link sequences, reflecting the superior fit of the C2-exo-unsaturated rather than saturated C-rings of the PBD dimers. A novel synthesis of core synthetic building blocks for PBD dimers via stepwise Mitsunobu reaction and nitration with Cu(NO3)2 is also reported.

DNA (cytosine-N4-)- and -(adenine-N6-)-methyltransferases have different kinetic mechanisms but the same reaction route. A comparison of M.BamHI and T4 Dam.

We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N(4)-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K(m) for either DNA (100-fold higher) or S-adenosyl-l-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction. The DNA methylation rate was analyzed as a function of both substrate and product concentrations. It was found to exhibit product inhibition patterns consistent with a steady state random bi-bi mechanism in which the dominant order of substrate binding and product release (methylated DNA, DNA(Me), and S-adenosyl-l-homocysteine, AdoHcy) was Ado-Met DNA DNA(Me) AdoHcy. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine-N(6)-)-MTase. The two differed with respect to an effector action of substrates and in the rate-limiting step of the reaction (product inhibition patterns are the same for the both MTases). From this we conclude that the common chemical step in the methylation reaction, methyl transfer from AdoMet to a free exocyclic amino group, is not sufficient to dictate a common kinetic scheme even though both MTases follow the same reaction route.

Design, synthesis, and evaluation of new noncross-linking pyrrolobenzodiazepine dimers with efficient DNA binding ability and potent antitumor activity.

New sequence selective mixed imine-amide pyrrolobenzodiazepine (PBD) dimers have been developed that are comprised of DC-81 and dilactam of DC-81 subunits tethered to their C8 positions through alkanedioxy linkers (comprised of three to five and eight carbons). Thermal denaturation studies show that after 18 h of incubation with calf thymus DNA at a 5:1 DNA/ligand ratio, one of them (5c) increases the DeltaT(m) value by 17.0 degrees C. Therefore, these unsymmetrical molecules exhibit significant DNA minor groove binding affinity and 5c linked through the pentanedioxy chain exhibits efficient DNA binding ability that compares with the cross-linking DSB-120 PBD dimer (DeltaT(m) = 15.4 degrees C). Interestingly, this imine-amide PBD dimer has been linked with a five carbon chain linker unlike DSB-120, which has two DC-81 subunits with a three carbon chain linker, illustrating the effect of the noncross-linking aspect by introducing the noncovalent subunit. The binding affinity of the compounds has been measured by restriction endonuclease digestion assay based on inhibition of the restriction endonuclease BamHI. This study reveals the significance of noncovalent interactions in combination with covalent bonding aspects when two moieties of structural similarities are joined together. This allows the mixed imine-amide PBD dimer with a five carbon chain linker to achieve an isohelical fit within the DNA minor groove taking in to account both the covalent bonding and the noncovalent binding components. This has been supported by molecular modeling studies, which indicate that the PBD dimer with a five carbon chain linker gives rise to maximum stabilization of the complex with DNA at the minor groove as compared to the other PBD dimers with three, four, and eight carbon chain linkers. The energy of interaction in all of the complexes studied is correlated to the DeltaT(m) values. Furthermore, this dimer 5c has significant cytotoxicity in a number of human cancer cell lines.

Non-essential loci in the BamHI-I and -F fragments of the HVT FC126 genome.

The sequence of BamHI-I fragment of the herpesvirus of turkeys (HVT) FC126 strain DNA was analyzed for the presence of potential open reading frames (ORFs). Four complete (ORFs 2 to 5) and 2 partial ORFs (ORFs 1 and 6) were detected. ORFs 2 and 3 were homologous to the HSV-1 UL55 and the EHV-1 gene 3, respectively. The ORF 6 was already partially sequenced by Smith et al. (Virology 207, 205-216, 1995), and was homologous to a Marek's disease virus (MDV) ORF located in a similar position (ORF 21; Ross et al., Virus Genes 7, 33-51, 1993a). No significant homology was found for the other ORFs. ORF 4 was antisense to ORF 3. Two HVT recombinants having an expression cassette inserted into two intergenic sites were generated and tested for viremia in chickens. Results demonstrated that these 2 intergenic loci are non-essential for in vitro and in vivo HVT replication. A 650 bp deletion in the repeat region flanking UL (TRL and IRL (BamHI-F)) has been identified in some DNA molecules of HVT FC126 strain. This deletion covers the entire truncated pp38 homologous ORF and the N-terminus of a small ORF which has no detectable homology with any known gene. Our results indicate that (1) this genomic region including the HVT pp38 homologue was not essential for in vitro and in vivo growth of HVT, and (2) this deletion had no apparent effect on Marek's disease (MD) protection induced by HVT.

The berenil ligand directs the DNA binding of the cytotoxic drug Pt-berenil.

To determine the affinity towards DNA sequences of novel antitumor drugs in comparison with their parental compounds may lead to the design of new analogous drugs with improved antitumor activity. Thus, the affinities of Pt-berenil towards different DNA sites relative to cis-DDP and berenil drugs were analysed using DNase I footprinting and restriction endonuclease analysis. The data show that the Pt-berenil drug inhibits the cutting activity of Hind III enzyme to the same extent as the berenil ligand. In contrast, inhibition by Pt-berenil of the cutting activity of Bam HI enzyme is significantly lower than that of cis-DDP. These results indicate that although the cis-Pt(II) centres of Pt-berenil maintain certain affinity toward G + C regions, which are the main binding sequences of cis-DDP, however, the berenil ligand seems to direct the Pt-berenil molecule towards A + T regions, which are the binding sequences preferred by berenil. In fact, 1H- and 195Pt-NMR spectra of Pt-berenil:nucleoside complexes show that Pt-berenil not only covalently binds to N7 of guanosine but also to N1/N7 of adenosine.

A quantitative assay to measure the relative DNA-binding affinity of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics based on the inhibition of restriction endonuclease BamHI.

An assay has been developed (restriction endonuclease digestion assay--RED100) based on inhibition of the restriction endonuclease BamHI that is capable of quantitative evaluation of the relative DNA-binding affinity of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics. This method provides comparable results to those obtained from thermal denaturation and ethidium bromide displacement assays but is much more sensitive, discriminating between molecules of similar structure such as DC-81, iso-DC-81 and neothramycin. The results reveal a trend between relative DNA-binding affinity and in vitro cytotoxicity for the PBDs in two tumour cell lines studied.

Sequence specific inhibition of DNA restriction enzyme cleavage by PNA.

Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned into pUC 19 were flanked by BamH1, Sal1 or Pstl sites, respectively. In all cases it was found that complete inhibition of restriction enzyme cleavage was obtained with the complementary PNA, a significantly reduced effect was seen with a PNA having one mismatch, and no effect was seen with a PNA having two mismatches. These results show that PNA can be used as sequence specific blockers of DNA recognizing proteins.

In vitro inhibition of bacterial DNA gyrase by cinodine, a glycocinnamoylspermidine antibiotic.

Cinodine, a broad-spectrum glycocinnamoylspermidine antibiotic, binds to DNA and irreversibly inhibits bacterial and phase DNA synthesis. Cinodine was found to inhibit the activity of Micrococcus luteus DNA gyrase in vitro, but it did not inhibit the activities of two other DNA-binding enzymes, namely, topoisomerase I and BamHI. Although we cannot yet conclude that DNA gyrase is an intracellular target of the drug, in vitro inhibition of the enzyme by cinodine appears to be specific.

B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs.

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.