Biology Reference
In-Depth Information
revealed that treatment resulted in intercalation into
DNA,
2333
an accumulation of AP sites,
234
and potentia-
tion of the cell killing effects of MMS and TMZ.
235
However, these effects do not seem to be only due to
inhibition of APE1 but also through TopoII. Neverthe-
less, this compound may have efficacy in combination
therapy with agents that generate lesions repaired
through BER.
The first reported APE1 DNA repair inhibitor is
CRT0044876 (7-nitroindole-2-carboxylic acid).
224
This
compound has conflicting reports of efficacy on APE1
inhibition,
196
and may have issues regarding cell perme-
ability as well as a nitro-aromatic ring that oftentimes
will result in toxicity. Due to these and other issues,
Madhusudan's group is using a virtual screening
approach to screen for more promising APE1 repair
inhibitors which has generated several hits.
196,230
Another class of compounds identified by HTS are the
arylstibonic acids (Compound 13755 and 13793) which
seem to have poor cell permeability and the potential
to be non-specific for APE1 but inhibit any enzyme
that interacts with DNA or RNA (
Figure 3.4
B).
236
Zawahir
et al.
utilized a pharmacophore-based
approach
226
to identify potential APE1 endonuclease
inhibitors. A pharmacophore modeling approach refers
to the exploitation of known functional groups or sites of
interaction between target of interest and macromole-
cule such as protein or DNA as the platform for
discovery of a molecule that disrupts that key interac-
tion.
237
In the case of APE1, Zawahir
et al.
utilized the
known interactions of APE1 with AP site-containing
DNA including components of hydrophobicity, H-
bond acceptor, and negatively ionizable features to
design a virtual screen. This approach predicts mole-
cules that would imitate an AP site to “trick” APE1
into binding to it and thereby block its action on DNA.
The result being that AP sites are left unrepaired and
single- and double-strand breaks accumulate resulting
in tumor cell death (Figure 3.3). Due to the inclusion of
bound Mg
2
þ
ion in determining the pharmacophore
model, all of the compounds identified are negatively
charged and specifically contain a carboxylate group.
IC
50
values for
in vitro
endonuclease inhibition for the
best of these compounds are in the 3
and may not be specific for APE1, therefore the search
for an APE1 repair inhibitor continues.
Kelley's group has also used a fluorescence-based
high-throughput assay to screen a library of 60,000 small
molecule compounds for ability to inhibit APE1 AP
endonuclease activity.
225
In addition to this published
report, they employed computer and computational
models to evaluate a library of chemical structures to
find additional chemical structures. Several libraries
were screened to assess the similarity of structures
including commercial vendor (Maybridge) and Drug
Discovery Center of the University of Cincinnati. Four
compounds with IC
50
values less than 10
m
M were iden-
tified, validated and characterized. The most promising
compounds were designated as APE1 Repair Inhibitor
AR01, 02, 03, and 06. AR03 is chemically distinct from
the previously reported small molecule inhibitors of
APE1, is cell-permeable, but does contain a planar
fused-ring structure that suggests potential DNA inter-
calating ability (Figure 3.3).
Inhibitors of APE1 Redox Activity
The redox activity of APE1 affects the DNA binding
activity of several crucial transcription factors in cancer
survival and growth signaling pathways. Therefore, tar-
geting this function of APE1 could lead to a decrease in
tumor cell signaling of angiogenesis, hypoxia, and
uncontrolled growth. The impact of APE1 redox activity
on transcription factors that regulate DNA repair
activity such as p53 and HIF-1
a
is unexplored. We spec-
ulate that a blockade of APE1 redox activity may blunt
the tumor's ability to respond to hypoxia and nutrient
deprivation and thereby sensitize the tumor to chemo-
therapeutic agents. Pancreatic cancer cells were more
sensitive to hypoxia when APE1 redox activity was
blocked,
214
supporting this idea that APE1 redox activity
can affect the cells' ability to respond to hypoxia and
perhaps the DNA repair capacity. While the human
body possesses two general reduction-oxidation
systems (thioredoxin and glutaredoxin/glutathione)
that help maintain intracellular homeostasis overall by
scavenging reactive oxygen species (ROS),
238
e
239
APE1
is distinct and functions differently from those
systems.
211
APE1 does not globally reduce transcription
factors;
240
rather, it selectively influences factors that
directly govern critical cellular functions, including
DNA repair, angiogenesis, and stress responses (e.g.,
inflammation, hypoxia). APE1 also indirectly affects
other “mission critical” cellular functions downstream
of its effectors. By inhibiting APE1's redox activity
multiple tumor signaling pathways are blocked
6
m
M range
(Compound 1, 17, and 21). However, these compounds
were not tested in cell-based assays and thus have not
been evaluated for cell permeability.
An HTS of the Library of Pharmacologically Active
Compounds (LOPAC) using a similar approach to quan-
titate the cutting ability of APE1 on AP site-containing
DNA resulted in three compounds: 6-hydroxy-DL-
DOPA, Reactive Blue 2, and myricetin.
205
In this screen,
two of three compounds identified as inhibitors were
also negatively charged compounds. The LOPAC
compounds are known to interact with other targets
e
as
APE1 regulates Egr-1, NF
k
B, AP-1, and HIF-1
a
.
241
e
242
The mechanism by which APE1 performs these redox
e
