Journal of Medicinal Chemistry
Article
with an IC50 value of 0.12 μM. We further tested a hydrazide
compound, 8Br-Hz, but found that hydroxamic acid is optimal.
Next, we examined the cellular function of the inhibitors as
apoptosis inducers. The more potent inhibitors, such as 8Br-
HA, can more effectively induce growth inhibition together
contrast, 8Br-Hz did not suppress the viability of these cell
lines (Figure S14G). In addition, FI-15 did not suppress
inhibitors or agonists of AP3Aase activity, might be promising
anticancer agents. It will be important to identify the allosteric
sites at which these inhibitors bind to cause a structural change
that induces apoptosis signaling. If we can clarify them, we can
not only elucidate the structural basis of how Ap3A binding
turns on the apoptosis signaling of FHIT but also realize
efficient design and development of non-nucleotide and
nonhydrolyzable apoptosis inducers targeting FHIT.
viability effectively or selectively under our conditions (GI50
>
EXPERIMENTAL SECTION
10 μM in both cell lines, Figure S14H). Note that Marx and
co-workers used HEK293T and H1299 cells as FHIT-positive
and FHIT-negative cells, respectively.25 We further evaluated
the inhibition mechanism of 8Br-HA and compared this
compound with the reported inhibitors, suramin and FI-15,
whose inhibition mechanisms were not reported. All of the
inhibitors evaluated showed noncompetitive inhibition as
judged from Lineweaver−Burk plots, in accordance with the
with the result of Ap3Aase inhibition assay because Ap3Aase
activity was more effectively suppressed by 8Me-HA, 8Br-HA,
and FI-15 than in TG-CMP assay even though the
concentration was higher (100 vs 1 μM) and the affinity was
higher (Km = ∼2 vs 37 μM).24 These results support
noncompetitive inhibition mechanisms of our inhibitors
(Figures 7B,C and S17, Table 1). These results imply that
the FHIT inhibitor-induced allosteric structural change of
FHIT protein has a greater effect on Ap3A hydrolysis than on
TG-CMP hydrolysis. To exclude the possibility that 8Br-HA is
an irreversible inhibitor, in addition, we set out to measure the
FHIT activity using FHIT eluents purified by gel filtration after
an incubation of FHIT with or without 8Br-HA and found that
FHIT activity was not suppressed in the eluent incubated with
8Br-HA, suggesting that 8Br-HA is a reversible inhibitor
(Figure S18). It was also found that 8Br-HA inhibited Ap3A
activity more potently than FI-15 (∼40-fold). Thus, we
consider that 8Br-HA is a practical and potent apoptosis
inducer targeting the FHIT tumor-suppressor protein (Figures
■
General Information. Proton nuclear magnetic resonance spectra
(1H NMR) and carbon nuclear magnetic resonance spectra (13C
NMR) were recorded on a JEOL JNM-ECZ500R, JEOL JNM-LA500
or Varian VNMRS 500 spectrometer in the indicated solvent.
Chemical shifts (δ) are reported in parts per million relative to the
internal standard, tetramethylsilane (TMS). Electrospray ionization
(ESI) mass spectra were recorded on a JEOL JMS-T100LC mass
spectrometer equipped with a nanospray ion source. Ultraviolet−
visible-light absorption spectra were recorded on an UV-1800
spectrophotometer (Shimadzu, Japan). Fluorescence spectra were
recorded on an RF-5300PC fluorometer (Hitachi, Japan). Analytical
HPLC was performed with a Shimadzu pump system equipped with a
reversed-phase ODS column (Inertsil ODS-3 4.6 × 150 mm2, GL
Sciences) at a flow rate of 1.0 mL/min. Semipreparative HPLC
purification was performed with a JASCO PU-2086 pump system
equipped with a reversed-phase ODS column (Inertsil ODS-3 20 ×
250 mm2, GL Sciences) at a flow rate of 10 mL/min. Microplate
fluorescence assay was performed on an ARVO X5 plate reader
(PerkinElmer). For chemical screening, reagents were dispensed with
a Multidrop Combi (Thermo Scientific) into 384-well plates. For
western blot analysis, the immunoblots were visualized by an
LAS3000 (FUJIFILM, Japan). Confocal fluorescence images were
taken with an IX-71 (Olympus) equipped with a disc scanning unit.
Half-area 96-well microplates (#3694) were purchased from Corning.
Glass-bottomed dishes (D11130H) were purchased from Matsunami.
PLL-coated 96-well microplates (4860-040) were purchased from
IWAKI. TaKaRa BCA Protein Assay Kit was purchased from Takara
Bio Inc. Recombinant human FHIT protein was purchased from
ATGEN. Recombinant ENPP1, 3, and 4 were prepared as previously
described.33,40 Anti-FHIT (rabbit, 1/1000), anti-α-tubulin (mouse, 1/
1000), goat antirabbit IgG-HRP conjugates (1/1000), and antimouse
IgG-HRP conjugate (1/1000) antibodies were purchased from Cell
Signaling Technology or Sigma-Aldrich. HCC827-luc and H460-luc
cells were purchased from JCRB Cell Bank. Staurosporin and Z-VAD-
fmk were purchased from Abcam and Cayman, respectively. CCK-8
reagent, Hoechst, and propidium iodide were purchased from
Dojindo. CellEvent caspase-3/7 Green Detection Reagent was
purchased from Invitrogen. PD-10 column was purchased from GE
Healthcare. All other reagents and solvents were purchased from
Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd. (TCI), Wako Pure
Chemical Industries, Kanto Kagaku, Junsei Kagaku, or Nacalai
Tesque, and used without further purification. Flash column
chromatography was performed using silica gel 60 (particle size
0.032−0.075) supplied by Taikoh-shoji. The purity of all synthesized
compounds was assessed by HPLC and was ≥95%.
CONCLUSIONS
■
FHIT is well established to function as a tumor-suppressor
protein that induces apoptosis in an AP3A-binding-dependent
manner. Since its expression is downregulated in precancerous
lesions, and FHIT agonists act as apoptosis inducers, FHIT is
considered to be not only a useful biomarker for very early
diagnosis of various cancers but also potential drug target in
FHIT-expressing cancer cells. Here, we designed and
synthesized a series of FHIT fluorescent probes that utilize
the AP3Aase activity of FHIT. We optimized the probe
structure for high and specific reactivity with FHIT and
developed an optimized screening assay for FHIT inhibitors.
We confirmed that the developed probes show high selectivity
for FHIT over other phosphodiesterases, including the ENPP
family. Screening of a compound library with this assay
identified several hits. Structural development of a hit
compound afforded potent FHIT inhibitors. Among them,
the inhibitors containing hydroxamic acid induce apoptosis of
FHIT-expressing cancer cells via caspase-3/7 activation, and
are more potent than the previously reported FI-15. The
mechanism of the inhibitors was noncompetitive and they
inhibited the hydrolysis of the natural substrate, Ap3A, more
efficiently than the synthetic substrate, TG-CMP. Our results
support the idea that FHIT binders, no matter whether
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
■
sı
Synthetic schemes and procedures of TG-NMP, FI-15,
HCM-CMP, TG-AMP derivatives, and FHIT inhibitors
(Schemes S1−S7); absorption and fluorescence spectra
and properties of TG-NMP, HCM-CMP, and 6-
substituted TG-AMP derivatives, Michaelis−Menten
plots of TG-NMP, HCM-CMP, and 6-substituted TG-
AMP derivatives, HPLC analyses of FHIT reaction of
9574
J. Med. Chem. 2021, 64, 9567−9576