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redox inhibition of APE1 than the proliferation. This is
the first time that this role of APE1 has been clearly
demonstrated. These data suggest that the redox func-
tion of APE1 plays a distinct functional role in angiogen-
esis. 46 In acute myeloid leukemia cells (HL-60 and PLB),
inhibition of APE1's redox activity with E3330 results in
hypersensitivity to retinoic acid (RA); RA induces cell
proliferation and apoptosis through a complex mecha-
nism. 160 E3330 has also been shown to inhibit growth
of pancreatic cancer cell lines, an effect that is enhanced
under hypoxic conditions, 161 as well as pancreatic
cancer-associated endothelial and endothelial progen-
itor cells. 159
In an effort to improve the efficacy and druggability
of E3330, a series of benzoquinone and naphthoquinone
derivatives of E3330 ( Figure 11.9 ) were synthesized and
characterized for redox inhibition and cell killing. 162 The
benzoquinone derivatives showed little improvement
over the parent compound E3330 in terms of IC 50 values
for redox inhibition and 50% growth inhibition values.
Substitution of the C 9 H 19 substituent with a shorter alkyl
chain C 4 H 9 resulted in a significant increase in the
concentration required for 50% cell growth inhibition.
E3330 inhibits the redox activity with an IC 50 of 10 m M
and inhibits 50% of cell growth at a concentration of
35 m M. However, several of the derivatives, especially
those with electron withdrawing substituents including
chloro, bromo, or methoxy substituents at the 3 position
of the naphthoquinone ring, had significantly lower IC 50
values for redox inhibition (1 m M as compared to 10 m M
for E3330) and 50% growth inhibition values of ~ 10 m M
vs. 35 m M. The least active of the naphthoquinone series
were those with a methyl group on the 3 position of the
naphthoquinone ring. No redox activity was observed
for a dimethoxynaphthyl derivative, which lacks the
quinone (unpublished results). Functionalization of the
carboxylic acid as a hydroxyethyl amide in the naphtho-
quinone series had no effect on redox or cell-based
activity of the compound suggesting that the carboxylic
acid moiety is not required for activity. 162
Three of the most promising naphthoquinone
compounds ( Figure 11.9 ) and E3330 were further charac-
terized. 163 The interaction of these compounds with
APE1 was tested by global ESI-QTOF and LC-MS/MS
analysis and compared to E3330. In a 30 minute reaction
including all four compounds, covalent adducts were
observed only for the chloro-containing compounds
7-60 and 10-52. Both 7-60 and 10-52 were found to
form
7 covalent adducts of APE1 with loss of
the chloride. For these two compounds, a Michael addi-
tion resulting from nucleophilic attack by a sulfhydryl of
APE1 on the compound results in loss of the chloride in
an irreversible modification. Sites of modification were
verified by LC-MS/MS analysis. The compound
8-51 was shown to react reversibly with the solvent-
accessible Cys residues 99 and 138 in a global ESI-QTOF
þ
1to
þ
FIGURE 11.9 The chemical structures of the redox inhibitor (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4- benzoquninoyl)]2-nonyl-2-propenoic
acid (E3330) along with naphthoquinone analogs are shown. The numbers shown in parentheses are those previously reported. 162 The analogs
shown were selected for further characterization in Matrigel and TUNEL assays, and the methoxy analog 8-51 was found to have the most
promising properties.
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