Biology Reference
In-Depth Information
APE1 INHIBITORS
invasion, and angiogenesis, therefore signaling can be
“switched off” for multiple tumor pathways.
The lethality of APE1 mouse knockouts at E3.5 to E9.5,
the lack of viable cell lines completely deficient for
APE1
194
and prior studies using targeted reduction in
APE1 in cancer cells establish APE1's importance in
normal and tumor cell survival. The rationale for APE1
as a target in cancer is well-justified based on the
following: (1) APE1 expression is upregulated or dysre-
gulated in many types of cancers including prostate,
pancreatic, ovarian, cervical, germ cell tumor, rhabdo-
myosarcoma, and colon.
195
e
196
Both the DNA repair
activity and redox regulatory activity of APE1 impact
upon multiple signaling pathways suggesting that
tumor cells may be “addicted” to its functions.
(2) Blockade of APE1 function potentiates the cytotoxicity
of many laboratory-based and clinically relevant
agents including methylmethane sulfonate (MMS),
H
2
O
2
, bleomycin, TMZ, melphalan, cisplatin, radiation,
and gemcitabine.
72,146,197
e
201
(3) High APE1 expression
has also been associated with resistance to chemo-
therapy and radiation, incomplete responses, shorter
time to progression, poorer survival and high
angiogenesis.
3,202
e
210
(4) APE1's regulation of HIF-1
a
plays an important role in the tumor and the tumor
microenvironment.
211
(5) APE1 plays a role in normal
and cancer-associated endothelial cell function and
survival.
211
e
212
Inhibition of the redox function of APE1
may exert antitumor effects based on APE1 function in
the tumor microenvironment which includes fibroblasts,
endothelial cells, pericytes, leukocytes, and extra-cellular
matrix.
213
Inhibition of APE1 redox activity can block the
proliferation and migration of pancreatic cancer cells
214
and similar decreases in proliferation were observed
with APE1 inhibition in pancreatic cancer-associated
endothelial cells (PCECs).
212
Other studies provide addi-
tional evidence that APE1 functions are supportive in
cells that affect tumor growth such as endothelial cells.
211
The importance of redox signaling through APE1 in both
the tumor cells as well as the tumor-associated endothe-
lium is becoming increasingly important and suggests
that animal models of cancer are essential to assess the
effects of APE1 inhibition on tumor growth. These
features of the APE1 protein provide the framework for
developing small molecules that can target its functions
as a cancer treatment strategy (
Figure 3.3
). Both the
DNA repair and redox activities of APE1 are important
for cellular survival and proliferation. Blockade of
APE1 DNA repair activity will result in an accumulation
of DNA damage (especially if paired with a chemothera-
peutic agent that generates DNA base damage) leading to
tumor cell death. Likewise, inhibition of the redox
activity of APE1 can affect expression of downstream
genes of critical transcription factors involved in survival,
Inhibitors of APE1 DNA Repair Function
Inhibitors of the DNA repair activity of APE1 are
under development. Currently two classes of molecules
reportedly block APE1 endonuclease (repair) activity:
molecules like methoxyamine (MX) which bind to the
DNA and stop APE1 from performing its endonuclease
activity and small molecules that would directly bind to
APE1 and inhibit its activity on AP site-containing DNA.
MX binds to the aldehyde in the AP site on the DNA, not
APE1 (
Figure 3.4
B)
215
e
216
and could be considered
a BER inhibitor. Because it is not specific for APE1 or
AP sites, MX could affect unintentional targets,
including any molecule with an aldehyde group and
all other enzymes that bind DNA substrates.
217
MX
potentiates the cytotoxicity of alkylating agents such as
TMZ in a wide variety of cancer cell lines both
in vitro
and in xenograft models and is currently in clinical trials
for patients with advanced refractory cancers.
218
e
221
MX
(trademark TRC102) is being studied in phase I clinical
trials in combination with pemetrexed or TMZ. The
cytotoxicity of pemetrexed, a folate anti-metabolite that
generates AP sites, can be potentiated by MX both
in
vitro
and
in vivo
.
222
In the phase I trial with pemetrexed,
TRC102 was well tolerated with favorable pharmacoki-
netic (PK) and pharmacodynamic (PD) properties.
Persistence of AP sites was observed in patients with
combination treatment of pemetrexed and TRC102.
223
Several studies have utilized high throughput screens
(HTS) to identify inhibitors of APE1 endonuclease
activity.
205,224
e
226
The basic principle of this assay relies
on APE1's ability to cleave a double-stranded deoxyoli-
gonucleotide substrate at a residue that mimics an AP
site (
Figure 3.4
A). Using fluorescence to monitor the
APE1 activity, thousands of compounds can be screened
in a relatively short amount of time. After identification
of the potential “hits,” several other assays are used to
validate and prove specificity for APE1 inhibition. A
gel-based endonuclease assay employs a similar strategy
as the HTS in that this assay also monitors APE1 cleavage
of a deoxyoligonucleotide at an AP site mimic, tetrahy-
drofur (THF).
227
Another important assay to demon-
strate the selectivity of the compound is to screen the
library for the compounds' ability to bind to DNA. The
screen could identify a false positive from one HTS that
screens for endonuclease activity if the compound non-
specifically binds to DNA and thereby inhibits APE1's
ability to cut the DNA but doesn't directly inhibit
APE1 endonuclease activity. A fluorescent intercalator
displacement (FID) assay can be used to estimate the
DNA binding capacity of selected APE1 inhibitors
.
228
e
229
