Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage

Clinically used bleomycin A₅ has been employed in a study of double-strand cleavage of a library of 10 hairpin DNAs originally selected on the basis of their strong binding to bleomycin. Each of the DNAs underwent double-strand cleavage at more than one site, and all of the cleavage sites were within, or in close proximity to, an eight-base-pair region of the duplex that had been randomized to create the original library. A total of 31 double-strand cleavage sites were identified on the 10 DNAs, and 14 of these sites were found to represent coupled cleavage sites, that is, events in which one of the two strands was always cleaved first, followed by the associated site on the opposite strand. Most of these coupled sites underwent cleavage by a mechanism described previously by the Povirk laboratory and afforded cleavage patterns entirely analogous to those reported. However, at least one coupled cleavage event was noted that did not conform to the pattern of those described previously. More surprisingly, 17 double-strand cleavages were found not to result from coupled double-strand cleavage, and we posit that these cleavages resulted from a new mechanism not previously described. Enhanced double-strand cleavages at these sites appear to be a consequence of the dynamic nature of the interaction of Fe·BLM A₅ with the strongly bound hairpin DNAs

Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

A study of BLM A₅ was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal-free BLM. The ability of Fe­(II)·BLM to affect cleavage on both the 3′ and 5′ arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A₅-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates were apparent when examining the library of 10 hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe·BLM A₅. The existence of multiple sites of cleavage on both the 3′ and 5′ arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method and gave results consistent with earlier reports of double-strand DNA cleavage but with a sequence selectivity that was different from those reported previously

Selective Functionalization of Antimycin A Through an <i>N</i>‑Transacylation Reaction

Acylation of 3-(<i>N</i>-formylamino)­salicylic acids resulted in transacylation with loss of the formyl moiety. The reaction proceeds through a bis-<i>N</i>-acylated intermediate, which undergoes facile deformylation. This transacylation reaction has been employed for the site-specific functionalization of the mitochondrial poison antimycin A, affording several novel derivatives. The selective cytotoxicity of some of these derivatives toward cultured A549 human lung epithelial adenocarcinoma cells, in comparison with WI-38 normal human lung fibroblasts, illustrates one application of this transacylation reaction

Efficient Asymmetric Synthesis of Tryptophan Analogues Having Useful Photophysical Properties

Two new fluorescent probes of protein structure and dynamics have been prepared by concise asymmetric syntheses using the Schöllkopf chiral auxiliary. The site-specific incorporation of one probe into dihydrofolate reductase is reported. The utility of these tryptophan derivatives lies in their absorption and emission maxima which differ from those of tryptophan, as well as in their large Stokes shifts and high molar absorptivities

Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase β with Unnatural Lysine Analogues

DNA polymerase β (Pol β) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5′-deoxyribose phosphate (dRP) lyase and “gap-filling” DNA polymerase activities. The lyase reaction is believed to occur via a β-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the ε-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol β enzymes were produced by coupled <i>in vitro</i> transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNA_CUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol β having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of <i>N</i>-methyllysine (<b>4</b>) affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (<b>6</b>) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side-chain methylene group with a bridging S atom (thialysine, <b>2</b>). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an ε-amino group (homoarginine, <b>5</b>) or a sterically constrained secondary amine (piperidinylalanine, <b>3</b>) led to almost complete suppression of dRP excision activity and the ability of Pol β to support BER. These results help to define the tolerance of Pol β to subtle local structural and functional alterations

An Optimized Pyrimidinol Multifunctional Radical Quencher

A series of aza analogues (<b>4</b>–<b>9</b>) of the experimental neuroprotective drug idebenone (<b>1</b>) have been prepared and evaluated for their ability to attenuate oxidative stress induced by glutathione depletion and to compensate for the decrease in oxidative phosphorylation efficiency in cultured Friedreich’s ataxia (FRDA) fibroblasts and lymphocytes and also coenzyme Q₁₀-deficient lymphocytes. Modification of the redox core of the previously reported <b>3</b> improved its antioxidant and cytoprotective properties. Compounds <b>4</b>–<b>9</b>, having the same redox core, exhibited a range of antioxidant activities, reflecting side chain differences. Compounds having side chains extending 14–16 atoms from the pyrimidinol ring (<b>6</b>, <b>7</b>, and <b>9</b>) were potent antioxidants. They were superior to idebenone and more active than <b>3</b>, <b>4</b>, <b>5</b>, and <b>8</b>. Optimized analogue <b>7</b> and its acetate (<b>7a</b>) are of interest in defining potential therapeutic agents capable of blocking oxidative stress, maintaining mitochondrial membrane integrity, and augmenting ATP levels. Compounds with such properties may find utility in treating mitochondrial and neurodegenerative diseases such as FRDA and Alzheimer’s disease

Selective Functionalization of Antimycin A Through an <i>N</i>‑Transacylation Reaction

Acylation of 3-(<i>N</i>-formylamino)­salicylic acids resulted in transacylation with loss of the formyl moiety. The reaction proceeds through a bis-<i>N</i>-acylated intermediate, which undergoes facile deformylation. This transacylation reaction has been employed for the site-specific functionalization of the mitochondrial poison antimycin A, affording several novel derivatives. The selective cytotoxicity of some of these derivatives toward cultured A549 human lung epithelial adenocarcinoma cells, in comparison with WI-38 normal human lung fibroblasts, illustrates one application of this transacylation reaction

The Carbamoylmannose Moiety of Bleomycin Mediates Selective Tumor Cell Targeting

Recently, we reported that both bleomycin (BLM) and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Here, we demonstrate that the conjugate of the BLM carbamoylmannose moiety with Cy5** showed tumor cell selective binding and also enhanced cellular uptake in most cancer cell lines. The carbamoyl functionality was required for tumor cell targeting. A dye conjugate prepared from a trivalent cluster of carbamoylmannose exhibited levels of tumor cell binding and internalization significantly greater than those of the simple carbamoylmannose–dye conjugate, consistent with a possible multivalent receptor

DNA Methylation Reduces Binding and Cleavage by Bleomycin

In a recent study, we described the enhanced double-strand cleavage of hairpin DNAs by Fe·bleomycin (Fe·BLM) that accompanies increasingly strong binding of this antitumor agent and suggested that this effect may be relevant to the mechanism by which BLM mediates its antitumor effects. Because the DNA in tumor cells is known to be hypomethylated on cytidine relative to that in normal cells, it seemed of interest to study the possible effects of methylation status on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found to bind strongly to bleomycin, and their methylated counterparts, were used to study the effect of methylation on bleomycin-induced DNA degradation. Under conditions of limited DNA cleavage, there was a significant overall decrease in the cleavage of methylated hairpin DNAs. Cytidine methylation was found to result in decreased BLM-induced cleavage at the site of methylation and to result in enhanced cleavage at adjacent nonmethylated sites. For two of the three hairpin DNAs studied, methylation was accompanied by a dramatic decrease in the binding affinity for Fe·BLM, suggesting the likelihood of diminished double-strand cleavage. The source of the persistent binding of BLM by the third hairpin DNA was identified. Also identified was the probable molecular mechanism for diminished binding and cleavage of the methylated DNAs by BLM. The possible implications of these findings for the antitumor selectivity of bleomycin are discussed

Mitochondrial Nitroreductase Activity Enables Selective Imaging and Therapeutic Targeting

Nitroreductase (NTR) activities have been known for decades, studied extensively in bacteria and also in systems as diverse as yeast, trypanosomes, and hypoxic tumors. The putative bacterial origin of mito­chondria prompted us to explore the possible existence of NTR activity within this organelle and to probe its behavior in a cellular context. Presently, by using a profluorescent near-infrared (NIR) dye, we characterize the nature of NTR activity localized in mammalian cell mito­chondria. Further, we demonstrate that this mito­chondrially localized enzymatic activity can be exploited both for selective NIR imaging of mito­chondria and for mito­chondrial targeting by activating a mito­chondrial poison specifically within that organelle. This constitutes a new mechanism for mito­chondrial imaging and targeting. These findings represent the first use of mito­chondrial enzyme activity to unmask agents for mito­chondrial fluorescent imaging and therapy, which may prove to be more broadly applicable