Journal of Medicinal Chemistry
Article
(Top − Bottom)[CaCl2]nH
18F]7b‐hTGase 2
to the reaction temperature of 110 °C for 10 min. The reaction
solution was cooled on ice for a short time (2 min), and water (50
μL) was added. An aliquot of the reaction mixture (10 μL) was
withdrawn for analysis of the crude mixture by radio-TLC and
analytical radio-HPLC. The remaining reaction mixture was filtered
(PTFE filter, pore size of 0.22 μm), and the filtrate was purified by
semipreparative radio-HPLC. The peak representing the product was
collected (approximately 5 mL) and immediately diluted with H2O to
an overall volume of 50 mL. The resulting solution was subjected to
solid-phase extraction by using a 500 mg of LiChrolut RP-18 (40−63
μm) or Chromafix C-18 ec (s) cartridge (preconditioned with 2 mL
of ethanol and 10 mL of water). The cartridge was washed with water
(2 × 2 mL), and the product was eluted with ethanol (2 × 0.5 mL).
An aliquot (10 μL) of that solution was withdrawn for quality control
(determination of molar activity as well as (radio)chemical purity by
analytical radio-HPLC and radio-TLC). The product solution was
evaporated to a volume of <100 μL and cooled on ice for a short time
(2 min), and a defined volume was withdrawn for further experiments.
Enzymatic Radio-TLC Experiments. All measurements were
conducted at 30 °C in 0.5 mL Eppendorf tubes. The following buffer
systems were used: assay buffer pH 6.5/7.4/8.0 (100 mM MES for
pH 6.5 or MOPS pH 7.4 and 8.0, varying CaCl2 concentrations, 50
μM EDTA, adjusted to the respective pH values with 1 M NaOH)
and enzyme buffer (100 mM MES, 0 or 3 mM CaCl2, 10 mM TCEP,
20% (v/v) glycerol, adjusted to pH 6.5 with 1 M NaOH). The buffers
were stored at 0 °C for periods of up to 2 weeks and freshly prepared
after that period. For the radio-TLC experiments, a considerable
lower molar activity of 1 GBq/μmol for [18F]7b was assumed during
planning of experiment to ensure that [enzyme] ≫ [[18F]7b] (the
determined molar activities for these experiments were between 5 and
18 GBq/μmol resulting in a 5−18 times higher [enzyme] compared
to [[18F]7b]). For analysis of the reactions, an aliquot (5−10 μL) was
withdrawn at the distinct time point and added to the same volume of
1 M HCl (stop of the reaction); an amount of 2 μL of that solution
was finally spotted on a TLC plate. Further processing was conducted
as described in the chromatography section.
[
= Bottom +
nH
[CaCl2]nH + EC50
[
18F]7b
(II)
Influence of GTP-γ-S on [18F]7b Binding to hTGase 2 at pH 7.4. A
10 μM solution of [18F]7b in assay buffer (pH 7.4, 2 mM CaCl2) was
prepared. This stock solution (6 μL) was added to assay buffer (48
μL, CaCl2-free) containing distinct concentrations of GTP-γ-S (0,
1.25, 6.25, 12.5, 62.5, 125, 625, 1250, 6250, and 12 500 nM). The
reactions were initiated upon addition of the hTGase 2 stock solution
in enzyme buffer (6 μL, 1 mg/mL, CaCl2-free). After 30 min, an
aliquot of each solution was withdrawn. The fraction of complex
“[18F]7b-hTGase 2” was plotted against [GTP-γ-S] (in nM), and the
resulting curve was analyzed by nonlinear regression according to eq
II to obtain the IC50 value for GTP-γ-S and the Hill coefficient nH.
Using [18F]7b Binding to hTGase 2 for Competitive Inhibition
Experiments. A 10 μM solution of [18F]7b in assay buffer (pH 7.4, 2
mM CaCl2) was prepared. This stock solution (3 μL) was added to
12.5% DMSO/assay buffer (3 μL, pH 7.4, CaCl2-free) containing
distinct concentrations of compound 7b, C, or D (0, 2, 5, 10, 20, 100,
and 500 μM). To these mixtures, assay buffer (21 μL, pH 7.4, CaCl2-
free) was added. The reactions were initiated upon addition of the
hTGase 2 stock solution in enzyme buffer (3 μL, 1 mg/mL, CaCl2-
free). After 5 min, an aliquot of each solution was withdrawn. The
fraction of complex “[18F]7b-hTGase 2” was plotted against
[inhibitor] (in μM), and the resulting curve was analyzed by
nonlinear regression according to eq II to obtain the IC50 value for the
respective inhibitor and the Hill coefficient nH.
Determination of [18F]7b Binding to hTGase 2 at a Low Ca2+
Concentration (100 μM). A 10 μM solution of [18F]7b in assay buffer
(pH 7.4, 1 mM CaCl2) was prepared. This stock solution (6 μL) was
added to assay buffer (48 μL, pH 7.4, CaCl2-free). The reaction was
initiated upon addition of the hTGase 2 stock solution in enzyme
buffer (6 μL, 1 mg/mL, CaCl2-free). After distinct time points (0.5, 1,
2, 3, and 4 h) an aliquot (2 μL) of the solution was withdrawn. The
fraction of complex [18F]7b-hTGase 2 was plotted against the time.
To ensure that [18F]7b is stable under these conditions, a similar
reaction mixture without the presence of enzyme was analyzed after 4
h.
Radiometric Determination of the Inactivation Constants kinact
/
KI of [18F]7b toward hTGases at Different pH Values. A 100 μM
solution of [18F]7b in assay buffer (3 mM CaCl2) was prepared. This
stock solution (5 μL) was added to the respective assay buffer (445
μL, 3 mM CaCl2). The reactions were initiated upon addition of the
respective hTGase stock solution (50 μL, 1 mg/mL for hTGase 2 and
0.5 mg/mL for hTGase 6, which corresponds to 10 and 5 μM active
enzyme concentration, respectively). The time points for withdrawal
of aliquots for analysis were 0, 5, 10, 15, 20, 25, and 30 min. To
ensure that [18F]7b is stable under these conditions, a similar reaction
mixture without the presence of enzyme was analyzed after 30 min.
Within the obtained radioluminograms, the spots of [18F]7b and [18F]
7b-hTGase were integrated. The fraction of “free [18F]7b” was plotted
against the time (in s), and the resulting curve was analyzed by
nonlinear regression according to the equation of one-phase decay in
Prism. The obtained kobs value (in s−1) represents (kinact/KI)[enzyme]
as [enzyme] ≫ [18F]7b. Division by [enzyme] provided the
Radio-SDS−PAGE Experiments. Radio-SDS−PAGE was per-
formed as horizontal PAGE using the Multiphor II electrophoresis
system from GE Healthcare. Both the gel and the buffer strips were
obtained from GE Healthcare (ExcelGel SDS, gradient 8−18). The
following buffer systems were used: assay buffer pH 7.4/8.0 (100 mM
MOPS, varying CaCl2 concentrations, 50 μM EDTA, adjusted to the
respective pH value with 1 M NaOH), lysis buffer (modified RIPA
buffer; 150 mM NaCl, 50 mM Tris, pH 8.0, 1 μg/mL Leupeptin, 1
mM PMSF, 5 mM NaF, 1 mM NaVO4, 1 mM DTT), SDS−PAGE
sample buffer containing 312.5 mM Tris-HCl, pH 6.8, 10% (w/v)
SDS, 40% (w/v) glycerol, 5% (w/v) ß-mercaptoethanol, bromophe-
nol blue. Radioactive bands were visualized by using the Fujix
Bas2000 TR radioluminography system or the Amersham Typhoon 5
biomolecular imager. The radioluminograms were analyzed by the
software AIDA (Advanced Image Data Analyzer, version 5.1 SP4
Build 1244). For quantification of the radioactive bands, a TLC stripe
with spots of defined activity of [18F]7b was placed under the same
imaging plate as the gel. For the radio-SDS−PAGE experiments, the
molar activity of [18F]7b was assumed to be 5 GBq/μmol during
planning of experiment as the determination of molar activity was
delayed compared to start of the biological experiments. The
determined molar activities for these experiments were up to 100
GBq/μmol. However, the concentration of [18F]7b was still higher
than enzyme concentration in cell lysates or cells which ensures
labeling of all activatable TGase 2 molecules.
Determination of the Ca2+-Dependence of [18F]7b Binding to
hTGase 2 at pH 7.4. A 10 μM solution of [18F]7b in assay buffer (pH
7.4, CaCl2-free) was prepared. This stock solution (6 μL) was added
to assay buffer (48 μL, pH 7.4) containing distinct concentrations of
CaCl2 (0.0125, 0.125, 0.25, 0.375, 0.5, 0.625, 0.875, 1.25, 2.5, and
12.5 mM). The reactions were initiated upon addition of the hTGase
2 stock solution in enzyme buffer (6 μL, 1 mg/mL, CaCl2-free). After
30 min, an aliquot of each solution was withdrawn. The proportion of
complex “[18F]7b-hTGase 2” was plotted against [CaCl2] (in mM),
and the resulting curve was analyzed by nonlinear regression
according to eq II to obtain the EC50 value for Ca2+ and the Hill
coefficient nH.
Binding of [18F]7b to Recombinant hTGases. A 100 μM solution
of [18F]7b in assay buffer (pH 8.0, 4 mM CaCl2) was prepared. This
stock solution (5 μL) was added to assay buffer (445 μL, pH 8.0, 3
mM CaCl2). The reaction was initiated upon addition of the
respective hTGase stock solution (50 μL, 1 mg/mL for hTGase 2 and
0.5 mg/mL for hTGase 6), and the mixture was incubated for 20 min
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J. Med. Chem. 2021, 64, 3462−3478