29 research outputs found
Hairpin DNA Sequences Bound Strongly by Bleomycin Exhibit Enhanced Double-Strand Cleavage
Clinically
used bleomycin A<sub>5</sub> 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 A<sub>5</sub> with the strongly bound hairpin DNAs
Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library
A study of BLM A<sub>5</sub> 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 A<sub>5</sub>-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 A<sub>5</sub>. 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<sub>CUA</sub> 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 Q<sub>10</sub>-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