Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
DOI 10.1007/s00210-011-0688-9
ORIGINAL ARTICLE
Inhibition of the adenylyl cyclase toxin, edema factor,
from Bacillus anthracis by a series of 18 monoand bis-(M)ANT-substituted nucleoside 5′-triphosphates
Hesham Taha & Stefan Dove & Jens Geduhn &
Burkhard König & Yuequan Shen & Wei-Jen Tang &
Roland Seifert
Received: 16 June 2011 / Accepted: 24 August 2011 / Published online: 24 September 2011
# Springer-Verlag 2011
W.-J. Tang
Ben May Cancer Institute, University of Chicago,
Chicago, IL, USA
tides modified with N-methylanthraniloyl (MANT)- or
anthraniloyl (ANT) groups at the 2′(3′)-O-ribosyl position,
with the unique preference for the base cytosine (Taha et
al., Mol Pharmacol 75:693 (2009)). MANT-CTP was the
most potent EF inhibitor (K i , 100 nM) among 16
compounds studied. Here, we examined the interaction of
EF with a series of 18 2′,3′-O-mono- and bis-(M)ANTsubstituted nucleotides, recently shown to be very potent
inhibitors of the AC toxin from Bordetella pertussis, CyaA
(Geduhn et al., J Pharmacol Exp Ther 336:104 (2011)). We
analysed purified EF and EF mutants in radiometric AC
assays and in fluorescence spectroscopy studies and
conducted molecular modelling studies. Bis-MANT nucleotides inhibited EF competitively. Propyl-ANT-ATP was the
most potent EF inhibitor (Ki, 80 nM). In contrast to the
observations made for CyaA, introduction of a second (M)
ANT-group decreased rather than increased inhibitor potency at EF. Activation of EF by calmodulin resulted in
effective fluorescence resonance energy transfer (FRET)
from tryptophan and tyrosine residues located in the
vicinity of the catalytic site to bis-MANT-ATP, but FRET
to bis-MANT-CTP was only small. Mutations N583Q,
K353A and K353R differentially altered the inhibitory
potencies of bis-MANT-ATP and bis-MANT-CTP. The
nucleotide binding site of EF accommodates bulky bis(M)ANT-substituted purine and pyrimidine nucleotides, but
the fit is suboptimal compared to CyaA. These data provide
a basis for future studies aiming at the development of
potent EF inhibitors with high selectivity relative to
mammalian ACs.
R. Seifert (*)
Institute of Pharmacology, Medical School of Hannover,
30625 Hannover, Germany
e-mail: seifert.roland@mh-hannover.de
Keywords Adenylyl cyclase . Bacillus anthracis . Edema
factor . MANT nucleotide . Molecular modelling .
Fluorescence spectroscopy
Abstract Bacillus anthracis causes anthrax disease and
exerts its deleterious effects by the release of three
exotoxins, i.e. lethal factor, protective antigen and edema
factor (EF), a highly active calmodulin-dependent adenylyl
cyclase (AC). Conventional antibiotic treatment is ineffective against either toxaemia or antibiotic-resistant strains.
Thus, more effective drugs for anthrax treatment are
needed. Our previous studies showed that EF is differentially inhibited by various purine and pyrimidine nucleoElectronic supplementary material The online version of this article
(doi:10.1007/s00210-011-0688-9) contains supplementary material,
which is available to authorized users.
H. Taha
Department of Pharmacology and Toxicology,
University of Regensburg,
90430 Regensburg, Germany
S. Dove
Department of Pharmaceutical and Medicinal Chemistry II,
University of Regensburg,
90430 Regensburg, Germany
J. Geduhn : B. König
Institute of Organic Chemistry, University of Regensburg,
90430 Regensburg, Germany
Y. Shen
College of Life Sciences, Nankai University,
Tianjin, People’s Republic of China
58
Abbreviations
AC
Adenylyl cyclase
mAC
Membranous adenylyl cyclase
ANT
Anthraniloyl
CaM
Calmodulin
CyaA
Bordetella pertussis adenylyl cyclase toxin
DMSO
Dimethyl sulphoxide
MANT
Methylanthraniloyl
EF
Edema factor adenylyl cyclase toxin from
Bacillus anthracis
EF3
Catalytic domain of edema factor adenylyl
cyclase toxin (amino acids 291–800)
FRET
Fluorescence resonance energy transfer
PMEApp 9-[2-(phosphomonomethoxy)ethyl]adenine
diphosphate
Pr
Propyl
Introduction
The spore-forming Bacillus anthracis secretes the exotoxins EF and lethal factor. As an AC, EF raises the
concentration of the second messenger cyclic AMP (cAMP)
inside host cells to supraphysiological levels (Tang and Guo
2009). EF is a key virulence factor for anthrax pathogenesis. An inactivating mutation in EF results in reduced
survival of germinated anthrax spores in macrophages
(Guidi-Rontani et al. 2001), and a strain of anthrax with a
defective EF gene has 100-fold reduced lethality in mice
(Brossier et al. 2000). EF enters host cells via a complex
with protective antigen, which is a pH-dependent protein
transporter (Tang and Guo 2009). The combination of
toxaemia caused by anthrax toxins and bacteremia due to
the rapid growth of anthrax bacteria in vital organs can
result in sepsis, pulmonary oedema and/or meningitis
within few days. Natural isolates of B. anthracis are
sensitive to a broad spectrum of antibiotics; thus, antibiotics
have been the primary therapy (Dixon et al. 1999).
However, antibiotics are ineffective against toxaemia and
antibiotic-resistant anthrax strains. The antibiotic treatment
for victims of the 2001 bioterrorism-related anthrax attack
in the USA resulted in a survival rate of slightly better than
50% for cases of inhalational anthrax (Atlas 2002). Some
survivors have experienced fatigue, shortness of breath,
chest pain and memory loss. This situation highlights an
urgent need for a more effective treatment to improve the
survival rate and quality of life of anthrax patients.
Previous studies resolved several crystal structures of
nucleotide–EF-CaM complexes and characterised the amino acids that are important for binding of the substrate ATP
and catalysis (Drum et al. 2002; Shen et al. 2002, 2005).
mAC and bacterial AC toxins are potently inhibited by
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
MANT-substituted nucleoside 5′-triphosphates (Gille et al.
2004; Mou et al. 2005, 2006; Göttle et al. 2007; Hübner et
al. 2011). (M)ANT nucleotides are environmentally sensitive fluorescence probes that show an increase in fluorescence upon interaction with a hydrophobic environment
(Hiratsuka 1983; Jameson and Eccleston 1997). We
exploited this property to monitor conformational changes
associated with activation in purified catalytic subunits of
mAC (Mou et al. 2005, 2006; Hübner et al. 2011),
Bordetella pertussis AC toxin, CyaA (Göttle et al. 2007)
and EF (Taha et al. 2009). By combining crystallographic
and molecular modelling approaches, we developed a threesite pharmacophore model for mAC, CyaA, and EF with
binding domains for the base, the MANT-substituted ribose
and the polyphosphate chain (Gille et al. 2004; Mou et al.
2006; Göttle et al. 2007; Wang et al. 2007; Hübner et al.
2011).
In our recent study, we systematically examined the
interactions of natural purine and pyrimidine nucleotides
and several (M)ANT-substituted analog with EF in terms of
catalysis, fluorescence changes and molecular modelling
(Taha et al. 2009). This study revealed that the structure–
activity relationships of (MANT)-nucleotides at EF, CyaA
and mAC are different; indicating that, in principle, the
development of potent and specific EF inhibitors is feasible.
Additionally, EF exhibited a unique preference for the base
cytosine. MANT-CTP was the most potent EF inhibitor
among the studied nucleotides, 5–10-fold more potent than
MANT-ATP (Taha et al. 2009). In this context, it is
intriguing to note that CTP is also a substrate for EF
(Göttle et al. 2010). MANT-CTP and MANT-ATP are
competitive EF inhibitors. Kinetic FRET competition
experiments with the non-fluorescent ATP analog PMEApp
revealed that both MANT-ATP and MANT-CTP reversibly
bind to the catalytic site. Mutagenesis studies showed that
F586 is crucial for FRET to MANT-ATP and MANT-CTP
and that the mutations N583Q, K353A and K353R
differentially alter the inhibitory potencies of MANT-ATP
and MANT-CTP (Taha et al. 2009).
Bis-(M)ANT-substituted nucleotides are more potent
inhibitors of CyaA than the mono-MANT-substituted
nucleotides (Geduhn et al. 2011). These data prompted us
to study systematically the interactions of this series of 18
mono- and bis-substituted (M)ANT nucleotides (Fig. 1)
with EF and several EF mutants in terms of catalysis,
fluorescence changes and molecular modelling.
Materials and methods
Materials Expression and purification of EF3, EF3 mutants
and CaM was performed as described (Taha et al. 2009).
PMEApp was supplied by Gilead Sciences (Foster City,
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
59
Fig. 1 Structures of mono- and bis-(M)ANT-substituted nucleoside 5′-triphosphates. Nucleotides differ from each other in the base and
substitution of the 2′- and 3′-O-ribosyl group
CA, USA). [α−32P]ATP (800 Ci/mmol) was purchased
from PerkinElmer (Rodgau Jügesheim, Germany). Aluminium oxide 90 active, (neutral, activity 1; particle size, 0.06–
0.2 mm) was purchased from Biomedicals (Eschwege,
Germany). Bovine serum albumin (fraction V, highest
quality) was bought from Sigma-Aldrich (Steinheim,
Germany). CaCl2, MnCl2 tetrahydrate and MgCl2 hexahydrate (highest quality) were purchased from Merck
(Darmstadt, Germany). Mono- and bis-(M)ANT nucleotides were synthesised as described (Taha et al. 2009;
Geduhn et al. 2011).
AC activity For the determination of the potency of AC
toxin inhibitors, assay tubes contained 10 μl of (bis)-(M)
ANT nucleotides at final concentrations from 10 nM to
100 μM as appropriate to obtain saturated inhibition curves
and 20 μl of EF3 or EF3(F586A) (10 pM final concentration) in 75 mM Tris/HCl, pH 7.4, containing 0.1% (m/v)
bovine serum albumin. Tubes were preincubated for 2 min
at 25°C, and reactions were initiated by the addition of
20 μL of reaction mixture consisting of the following
components to yield the given final concentrations:
100 mM KCl, 10 μM free Ca2+, 5 mM free Mn2+,
100 μM EGTA, 100 μM cAMP and 100 nM CaM. ATP
was added as non-labelled substrate at a final concentration
of 40 μM and as radioactive tracer [α-32P]ATP (0.2 μCi/
tube). Km and Vmax values were reported before (Taha et al.
2009). Tubes were incubated for 10 min at 25°C, and
reactions were stopped by the addition of 20 μL of 2.2 N
HCl. Denaturated protein was sedimented by a 1-min
centrifugation at 13,000×g. [32P]cAMP was separated from
[α-32P]ATP by transferring the samples to columns containing 1.4 g of neutral alumina. [32P]cAMP was eluted by
the addition of 4 ml of 0.1 M ammonium acetate solution,
pH 7.0. Blank values were about 0.02% of the total amount
of [α-32P]ATP added; substrate turnover was <3% of the
total amount of [α-32P]ATP added. Samples collected in
scintillation vials were filled up with 10 ml of doubledistilled water, and Čerenkov radiation was measured in a
PerkinElmer Tricarb 2800TR liquid scintillation counter.
Free concentrations of divalent cations were calculated with
WinMaxC (http://www.stanford.edu/∼cpatton/maxc.html).
For the determination of the potency of AC toxin inhibitors
at various EF3 mutants (H577A, N583A, N583Q, N583H,
K353A and K353R), the experiments were essentially
performed as described for EF3 with some modifications.
Specifically, the final enzyme concentrations were increased up to 2 nM in order to account for the lower
catalytic activity of the mutants. Moreover, the reaction
time was prolonged to 20 min at 30°C. For studying of the
inhibition mechanism of EF3 by bis-MANT nucleotides,
enzyme saturation experiments were performed in the
presence of bis-MANT nucleotides at final concentrations
from 0.5 to 10 μM as appropriate according to the potency
of the inhibitor (Fig. 2). For the basal saturation curve, 5 μl
of double-distilled water was added instead of the inhibitor.
Next, 5 μl of 50 μM to 600 μM ATP/Mn2+, plus 20 μl of
10 pM EF3 in 75 mM Tris/HCl, pH 7.4, containing 0.1%
(m/v) bovine serum albumin were added. Tubes were
preincubated for 2 min at 25°C, and reactions were initiated
by the addition of 20 μL of reaction mixture consisting of
the following components to yield the given final concentrations: 100 mM KCl, 10 μM free Ca2+, 5 mM free Mn2+,
100 μM EGTA, 100 μM cAMP, 100 nM CaM, and [α-32P]
ATP (0.2 μCi/tube).
Fluorescence studies Fluorescence experiments were performed using quartz UV ultra-microcuvettes from Hellma
60
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
A
Bis-MANT-ATP
B
Bis-MANT-CTP
0.200
0.175
0.175
0.150
(0 µM)
0.150
(0.5 µM)
(2.5 µM)
0.100
1/Velocity
1/Velocity
0.125
(5 µM)
0.075
(0 µM)
(0.5 µM)
(2.5 µM)
(5 µM)
(10 µM)
0.125
0.100
0.075
0.050
0.050
0.025
-0.0095
0.025
0.0005
0.0105
1/[ATP/Mn
+2
0.0205
--1
] (µM )
-0.015
-0.005
1/[ATP/Mn
0.005
2+
] (µM
0.015
--1
)
Fig. 2 Lineweaver–Burk analysis of the inhibition of EF3 AC activity
by bis-MANT-ATP and bis-MANT-CTP. AC activities were determined as described under “Materials and methods” with the indicated
concentrations of bis-MANT-ATP (0, 0.5, 2.5 and 5 μM) (a) and
MANT-CTP (0, 0.5, 2.5, 5 and 10 μM) (b). Reaction mixtures
contained 10 pM EF3, 100 mM KCl, 10 μM free Ca2+, 5 mM free
Mn2+, 100 μM EGTA, 100 μM cAMP, 100 nM calmodulin, 0.2 μCi/
tube [α−32P]ATP and unlabelled ATP/Mn2+ concentrations indicated
in the graph. Data were plotted reciprocally and analysed by linear
regression according to Lineweaver–Burk. Velocities on the y-axis are
given per second. Shown are the results of a representative experiment
performed in triplicates. SD values were <5% of the mean values.
Similar results were obtained in two independent experiments
(Müllheim, Germany, type 105.251-QS, light path of
length, 3×3 mm; centre, 15 mm; total volume, 70 μl; and
type 105.250-QS, light path length, 10×2 mm; centre,
15 mm; total volume, 150 μl) in a thermostated multicell
holder at 25°C in a Varian Cary Eclipse fluorescence
spectrometer (Varian, Darmstadt, Germany). In case of 150μl cuvettes, 140 μl of buffer consisting of 100 mM KCl,
100 μM CaCl2, 10 mM MnCl2 and 25 mM HEPES/NaOH,
pH 7.4, was added into the cuvette. Five microlitres of
10 μM EF3/EF3 mutants (final concentration, 300 nM),
5 μl of 10 μM CaM (final concentration, 300 nM) and bisMANT-ATP or bis-MANT-CTP (300 nM each) were
added. In case of experiments with 70-μl cuvettes, volumes
were adjusted stoichiometrically. The results obtained with
70- and 150-μl cuvettes were identical, with the 70-μl
cuvettes offering an opportunity to save EF3/EF3 mutant
proteins. Steady-state fluorescence emission spectra of
nucleotides were recorded at low speed in the scan mode
from λem 300 nm to 550 nm with λex 280 nm. Fluorescence
recordings were analysed with the spectrum package of the
Varian Cary Eclipse software version 1.1. Baseline fluorescence (buffer alone) and the baseline-corrected nucleotidedependent emission of each concentration of the ligand
(buffer+ nucleotide) were subtracted from the spectra
shown in Figs. 3 and S1–S7. In the competition experiments shown in Fig. 4, bis-MANT nucleotides were
displaced from the EF3 catalytic site using PMEApp. In
direct fluorescence experiments, bis-MANT nucleotides
were excited at λex 350 nm, and emission spectra were
recorded from 380 to 550 nm. In direct fluorescence
experiments, bis-MANT nucleotides were excited at λex
350 nm, and emission spectra were recorded from 380 to
550 nm. For an estimation of the hydrophobic properties of
the binding site interacting with the MANT-group, direct
fluorescence control experiments of the bis-MANT nucleotides were conducted in the presence of DMSO ranging from
0% to 100% (v/v) (Fig. S8).
Modelling of MANT- and bis-MANT nucleotide binding
modes to EF Docking studies were performed with the
molecular modelling package SYBYL 7.3 (Tripos L.P., St.
Louis, MO, USA) on a Silicon Graphics Octane workstation. For illustration of the interaction of 3′-MANT-CTP
with EF, our recently published model (Taha et al. 2009)
based on the crystal structures of EF-CaM in complex with
2′-deoxy-3′-ANT-ATP, PDB 1lvc and chain C (Shen et al.
2002) (Yb2+ replaced by Mg2+) was used.
For docking of bis-MANT-ATP, an initial computer
model was generated from the crystal structure of EFCaM in complex with 3′-deoxy-ATP, PDB 1xfv (Shen et al.
2005) since the nucleotide binding site of this structure is
very similar to that of CyaA in complex with PMEApp,
PDB 1zot (Guo et al. 2005), enabling a better comparison
with the binding mode of bis-(M)ANT nucleotides at CyaA
(Geduhn et al. 2011). An initial docking position of bisMANT-ATP resulted from superposition of the EF-CaM
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
λex = 280
Bis-MANT-ATP
A
61
50
Intensity (a.u.)
Intensity (a.u.)
50
EF3
CaM
40
30
20
EF3
CaM
40
30
20
10
0
300
10
350
400
450
500
0
300
550
350
Wavelength (nm)
C
Bis-MANT-ATP
-
λex = 350
D
450
500
550
λex = 350
Bis-MANT-CTP
2.5
Bis-MANT-ATP
EF3
CaM
5.0
2.5
405
430
455
480
505
530
Bis-MANT-CTP
EF3
CaM
2.0
Intensity (a.u.)
Intensity (a.u.)
400
Wavelength (nm)
7.5
0.0
380
λex = 280
Bis-MANT-CTP
B
1.5
1.0
0.5
0.0
380
405
430
455
480
505
530
Wavelength (nm)
Wavelength (nm)
Fig. 3 Analysis of the interaction of EF3 with bis-MANT-ATP and
bis-MANT-CTP in fluorescence experiments. FRET and direct
fluorescence experiments were performed as described under “Materials and methods”. The assay buffer consisted of 75 mM HEPES/
NaOH, 100 μM CaCl2, 100 mM KCl and 5 mM MnCl2, pH 7.4.
Nucleotides were added to the buffer to yield 300 nM final
concentrations. EF3 (300 nM final concentration) was added followed
by the addition of CaM (1 μM final concentration). Steady state
emission spectra were recorded. In FRET studies (a, b) emission was
scanned at an excitation wavelength of 280 nm after each addition. In
direct fluorescence studies (c, d) emission was scanned at an
excitation wavelength of 350 nm after each addition. In a and b, the
buffer and the MANT nucleotide basal fluorescence were subtracted
from the fluorescence after addition of EF3 (green line) and CaM
(blue line). Shown are superimposed recordings of a representative
experiment. Similar data were obtained in five independent experiments. a.u. arbitrary unit. Note the different scales of the y-axis in c
and d
structure with the model of CyaA in complex with bis-BrANT-ATP (Geduhn et al. 2011), allowing the modification
of rotatable bonds. Hydrogens were added, and charges
were assigned to the model (proteins and water molecules,
AMBER_FF99; bis-MANT-ATP, Gasteiger-Hueckel). The
Mg2+ ions received formal charges of 2. The complex was
refined in a stepwise approach. First, ∼50 minimisation
cycles with fixed ligand using the AMBER_FF99 force
field (Cornell et al. 1995) (steepest descent method);
second, ∼100 minimisation cycles of the ligand and the
surrounding (distance up to 6 Å) protein residues (Tripos
force field) (Clark et al. 1989); and, third, ∼100 minimisation cycles with fixed ligand (AMBER_FF99 force field,
Powell conjugate gradient) were performed. The second
and third steps were repeated with larger number of cycles
until a root mean square force of 0.01 kcal/mol× Å−1 was
approached. To avoid overestimation of electrostatic interactions, a distance-dependent dielectric constant of 4 was
applied. Molecular surfaces and lipophilic potentials (protein variant with the new Crippen parameter table) (Heiden
et al. 1993; Ghose et al. 1998) were calculated and
visualised by the program MOLCAD (MOLCAD, Darmstadt,
Germany) contained within SYBYL.
Data analysis All inhibition and saturation curves were
analysed by non-linear regression using the Prism 4.0
software (Graphpad, San Diego, CA, USA). Fluorescence
spectra were analysed using the spectrum package of the
Varian Cary Eclipse 1.1 software.
Results
Table 1 summarises the Ki values of bis-MANT-ATP and
bis-MANT-CTP at EF3 and EF3 mutants (H577A, N583A,
N583Q, N583H, K353A and K353R) in the presence of
Mn2+. bis-MANT-ATP and bis-MANT-CTP exhibited the
same inhibitory potencies at EF3. The F586A mutation
reduced the inhibitory potencies of bis-MANT-ATP and
bis-MANT-CTP by 5–6-fold, whereas the H577A mutation
did not decrease inhibitor potency. The N583A mutation
decreased inhibitor potency by 6–12-fold. The N583Q
62
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
A
2.5
1: Buffer
2: EF3
3: CaM
4: Bis-MANT-ATP
5: PMEApp
Intensity (a.u.)
2.0
4
1.5
1.0
5
3
0.5
2
1
0.0
0
5
10
15
20
Time (min)
B
2.0
1: Buffer
2: EF3
3: CaM
4: Bis-MANT-CTP
Bis-MANT5: PMEApp
Intensity (a.u.)
1.5
1.0
4
5
3
2
0.5
1
0.0
0
5
10
15
20
Time (min)
Fig. 4 Kinetic analysis of the interaction of EF3 with bis-MANT
nucleotides and CaM in FRET experiments. FRET kinetic experiments
were performed as described under “Materials and methods”. The
excitation wavelength was 280 nm and emission was detected at
430 nm over time. Successively, buffer (1), 300 nM EF3 (2), 1 μM
CaM (3), nucleotide (a bis-MANT-ATP, b bis-MANT-CTP, 300 nM
each) (4) and PMEApp (1 μM) (5) were added. A recording of a
representative experiment is shown. Similar data were obtained in four
independent experiments. a.u. arbitrary unit
substitution reduced the potency of bis-MANT-ATP by 60fold and the potency of bis-MANT-CTP by 120-fold. For
the N583H mutant, a 130–230-fold decrease in potency of
bis-MANT-ATP and bis-MANT-CTP was observed. The
K353A substitution reduced the potency of bis-MANT-ATP
and bis-MANT-CTP by 40–50-fold. The Ki value of bisMANT-ATP and bis-MANT-CTP at EF3(K353R) increased
about 3-fold.
Figure 2 shows the double-reciprocal analysis of EF3
inhibition kinetics by bis-MANT-ATP and bis-MANT-CTP
according to Lineweaver–Burk. The linear regression lines
intersected at the y-axis, i.e. Vmax remained constant,
whereas Km increased with increasing inhibitor concentration. These data reflect competitive inhibition of EF3 by
bis-MANT-ATP and bis-MANT-CTP.
Table 2 summarises the Ki values of mono- and bispropyl-ANT-NTPs and various mono-and bis-ANT-NTPs
substituted at position 5 of the anthraniloyl ring at EF3 in
the presence of Mn2+. In general, ATP analog were about 2fold more potent than the corresponding ITP analog.
Moreover, the inhibitory potencies of 5-substituted-MANT
nucleotides were slightly higher than those of the
corresponding bis-MANT nucleotides. Propyl-ANTnucleotides were more potent than the corresponding bispropyl-ANT analog. Among all compounds studies, propylANT-ATP was the most potent EF3 inhibitor.
Tryptophan (and tyrosine) residues in proteins are
excited at an excitation wavelength of 280 nm (Mou et al.
2005, 2006; Taha et al. 2009), resulting in substantial
endogenous fluorescence of EF3 with an emission maximum λem of 350 nm, which can then excite the (M)ANT
group of nucleotides, provided sufficient proximity between
donor and acceptor. Such energy transfer results in
increased fluorescence of the MANT-group at 420–
450 nm. Previous studies with mAC, CyaA and EF showed
that in the presence of Mn2+, FRET signals were much
larger than in the presence of Mg2+ (Mou et al. 2005, 2006;
Göttle et al. 2007; Taha et al. 2009). Therefore, all
fluorescence studies with EF3 were conducted in the
presence of Mn2+.
At a λex of 280 and 350 nm, bis-MANT nucleotides
exhibited only minimal endogenous fluorescence, providing an excellent signal/noise ratio for FRET studies. In the
absence of CaM, EF3 exhibited a strong emission peak at
350 nm when excited at 280 nm (Fig. 3a, b). Following the
addition of CaM, new fluorescence peaks with a maximum
λem of 425–430 nm became apparent. These new peaks
reflect FRET from tryptophan and tyrosine residues to
the MANT group and were the result of the substantial
CaM-induced conformational change in EF (Taha et al.
2009).
In FRET experiments with various EF3 mutants and bisMANT nucleotides, the signal intensities of endogenous
tryptophan and tyrosine fluorescence were similar. The
mutations F586A, H577A, N586A, N583Q, N583H and
K353A differentially altered FRET signal intensities of bisMANT-ATP and bis-MANT-CTP (Figs. S1–S7, panels A
and B). In EF3(F586A), the FRET signal was reduced by
23% with bis-MANT-ATP and 17% with bis-MANT-CTP.
The FRET signal of bis-MANT-ATP and bis-MANT-CTP
was reduced by 41% and 15% in EF3(N583A). In EF3
(N583Q) and EF3(K353A), the FRET signals with bisMANT-ATP were reduced by 45% and 27%, while with
bis-MANT-CTP no FRET signal was observed. The
analysis of the EF3 mutants H577A and N583H with bisMANT-ATP and bis-MANT-CTP revealed no FRET at all,
while in the K353R mutant, the FRET signal was similar to
that in EF3.
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
63
Table 1 Inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP at EF3 and EF3 mutants in the presence of Mn2+
Toxin
EF3
EF3
EF3
EF3
EF3
EF3
EF3
EF3
Bis-MANT-ATP Ki (μM)
Bis-MANT-ATP rel. pot.
Bis-MANT-CTP Ki (μM)
Bis-MANT-CTP rel. pot.
0.21±0.02
1.65±0.08
0.19±0.02
1.29±0.02
16.38±0.14
46.30±17.95
9.90±0.24
0.51±0.001
100
12.73
110.53
16.28
1.28
0.45
2.12
41.18
0.21±0.005
1.19±0.001
0.17±0.026
2.57±0.029
24.49±0.32
27.68±0.47
7.96±0.15
0.50±0.001
100
17.65
123.53
8.17
0.86
0.76
2.64
42
wild type
F586A
H577A
N583A
N583Q
N583H
K353A
K353R
Inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP at EF3 and various EF3 mutants were determined as described under “Materials and
methods”. Ki values are the means±SD of three experiments performed in triplicates. The relative potencies (rel. pot.) of bis-MANT-ATP and bisMANT-CTP are given, too, EF3 being the reference. For determination of the inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP at EF3
and EF3(F586A), reaction mixtures contained 10 μM free Ca2+ , 5 mM free Mn2+ , 100 μM EGTA, 40 μM ATP, 0.2 μCi/tube [α−32 P]ATP,
100 μM cAMP, 100 nM CaM and 10 pM enzyme in 75 mM Tris/HCl, pH 7.4. For other EF3 mutants, reaction mixtures contained 0.4 μCi
[α−32 P]ATP per tube. The enzyme concentration was 2 nM. Nucleotides were added at different concentrations as appropriate to obtain saturated
concentration–response curves. Inhibition curves were analysed by non-linear regression
In a classic FRET experiment, the appearance of the new
emission peak should be accompanied by a corresponding
decrease in the endogenous tryptophan- and tyrosinefluorescence peak. However, for bis-MANT nucleotides,
the appearance of the fluorescence peak at a λem of 425–
430 nm was not accompanied by a decrease at a λem of
350 nm. These findings are explained by a model in which
part of the endogenous tryptophan and tyrosine fluorescence of EF3 is quenched by surrounding polar amino acids
such as aspartate, glutamate and histidine (Taha et al.
2009). Upon EF3 activation by CaM, a large conformational change in EF3 occurs (Drum et al. 2002; Taha et al.
2009), annihilating, to a large extent, the quenching effects
of polar amino acids and masking the predicted decrease in
fluorescence at a λem of 350 nm.
We also examined the direct bis-MANT nucleotide
fluorescence at an excitation wavelength λex of 350 nm
(Fig. 4c, d). At a λex of 350 nm, bis-MANT nucleotides
exhibited a very low endogenous fluorescence with a
maximum at a λem of 445–450 nm. The addition of EF3
to samples did not significantly change this basal fluorescence. However, upon addition of CaM, we observed large
increases in the intensity of the fluorescence signals, i.e.
about 6–7-fold with bis-MANT-ATP and 2–3-fold with bisMANT-CTP at λem 440 nm.
Direct fluorescence was also examined with EF3 mutants
(Figs. S1–S7, panels C and D). In EF3(F586A), the emission
maximum at a λem of 440 nm was reduced by 35% with bisMANT-ATP and 15% with bis-MANT-CTP. The fluorescence signals of bis-MANT-ATP and bis-MANT-CTP were
reduced by 50% and 22%, respectively, in EF3(N583A). In
EF3(N583Q) and EF3(K353A), the fluorescence signals
with bis-MANT-ATP were reduced by 57% and 43%, while
with bis-MANT-CTP, they were reduced by 44% and 28%,
Table 2 Inhibitory potencies of mono- and bis-ANT-nucleotides at EF3 in the presence of Mn2+
Nucleotides
Ki (μM)
Nucleotides
Ki (μM)
Propyl-ANT-ATP
Bis-Propyl-ANT-ATP
Br-ANT-ATP
Bis-Br-ANT-ATP
Cl-ANT-ATP
Bis-Cl-ANT-ATP
AC-NH-ANT-ATP
Bis-AC-NH-ANT-ATP
0.08±0.01
1.02±0.15
0.20±0.01
0.26±0.04
0.17±0.02
0.22±0.02
0.55±0.01
0.67±0.03
Propyl-ANT-ITP
Bis-Propyl-ANT-ITP
Br-ANT-ITP
Bis-Br-ANT-ITP
Cl-ANT-ITP
Bis-Cl-ANT-ITP
AC-NH-ANT-ITP
Bis-AC-NH-ANT-ITP
0.2±0.02
1.9±0.01
0.4±0.25
0.5±0.1
0.3±0.05
0.4±0.09
1.0±0.13
1.3±0.25
Inhibitory potencies of various newly synthesised mono- and bis-ANT-nucleotides at EF3 were determined as described under “Materials and
methods”. Ki values are the means±SD of three independent experiments performed in triplicates. The Km value of EF3 for ATP in the presence of
Mn2+ was 82.6±8.2 μM. Reaction mixtures contained 10 μM free Ca2+ , 5 mM free Mn2+ , 100 μM EGTA, 40 μM ATP, 0.2 μCi/tube [α−32 P]
ATP, 100 μM cAMP, 100 nM CaM and 10 pM enzyme in 75 mM Tris/HCl, pH 7.4. Nucleotides were added at different concentrations as
appropriate to obtain saturated concentration–response curves. Inhibition curves were analysed by non-linear regression
64
respectively. The analysis of the EF3 mutants H577A and
N583H with bis-MANT-ATP and bis-MANT-CTP at an
excitation wavelength λex of 350 nm revealed a strong
reduction in the intensity of fluorescence signal (60–90%) at
a λem of 440 nm. In the K353R mutant, the fluorescence
signal was nearly the same as in case of EF3.
Figure 4 shows the kinetics of FRET experiments with
bis-MANT-ATP and bis-MANT-CTP at a fixed emission
wavelength of 440 nm. Sequential addition of EF3 and
CaM resulted only in small fluorescence increases, reflecting the far end of the tryptophan/tyrosine emission
spectrum. Addition of bis-MANT nucleotides to cuvettes
instantaneously resulted in substantial fluorescence
increases, reflecting FRET. Addition of the high-affinity
EF inhibitor and non-fluorescent nucleotide analog
PMEApp (1 μM) (Taha et al. 2009) to cuvettes reduced
the fluorescence signals with both bis-MANT nucleotides
(300 nM each).
Overall, the FRET and direct fluorescence responses with
bis-MANT-CTP were considerably smaller than with bisMANT-ATP. Thus, the question arose whether bis-MANTCTP is less responsive to changes in hydrophobicity than bisMANT-ATP. However, the sequential increase in concentration
of the organic solvent DMSO from 0% (v/v) to 100% (v/v)
gradually increased fluorescence of both bis-MANT-ATP and
bis-MANT-CTP (Fig. S8). In fact, the fluorescence increases
with bis-MANT-CTP were even larger than with bis-MANTATP, ruling out inferior responsiveness to hydrophobicity as
reason for the small fluorescence responses of bis-MANTCTP upon binding to EF3 and EF3 mutants.
The binding modes of mono- and bis-(M)ANT nucleotides to EF have been investigated by docking studies based
on crystal structures of EF3 in complex with CaM and 2′deoxy-3′-ANT-ATP (Shen et al. 2002) and EF in complex
with CaM and 3′-deoxy-ATP (Shen et al. 2005). Figure 5
exemplarily shows docking poses of 3′-MANT-CTP and
bis-MANT-ATP. The nucleotide binding site of EF is a
spacious cavity located at the interface of two structural
domains, CA (D294-N349, A490-K622) and CB (V350T489). A special loop region within CA, called switch B
(G578-N591), essentially contributes to the accommodation
of the nucleobases and the MANT groups. A metal ion
(Mg2+ or Mn2+) is coordinated with D491, D493, H577 and
the α-phosphate of the nucleotides. In the most recent EF
structure (Shen et al. 2005), a second Mg2+ ion is resolved,
which coordinates the α-, β- and γ-phosphates. The
phosphate groups form additional salt bridges with R329,
K346, K353 and K372. The ribosyl moiety contacts the
side chain of L348 and the amide NH2 of N583 (hydrogen
bond with the ring oxygen).
The nucleobases of 3′-MANT-CTP (Fig. 5a) and bisMANT-ATP (Fig. 5b) fit to a pocket mainly consisting of
amino acids of switch B. In both cases, the NH2 groups
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
may form two hydrogen bonds with backbone carbonyls
(CTP: G578, T579; ATP: T548, T579). These hydrogen
bonds are impossible in the case of the hypoxanthine base,
explaining the lower potency of the ITP derivatives
(Table 2). However, the hypoxanthine 1-NH or 6-CO
fragments may weakly interact with other counterparts
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
Interactions of MANT- and bis-MANT nucleotides with EF. If
not otherwise indicated, atoms are coloured as follows: C and some
essential H (grey), O (red), N (blue), P (orange), Mg2+ (magenta). a
Binding of 3′-MANT-CTP to EF. b Binding of bis-MANT-ATP to EF.
In both panels, the side chains of amino acids within a sphere of 3.5 Å
around the ligand and Mg2+ ions are drawn as sticks, heteroatoms as
balls, Cα-traces as lines; colours of C atoms and Cα-traces of EF
correspond to the domain: green CA, green blue CB, yellow switch B,
blue switch C. c Binding of bis-MANT-ATP to EF, the EF binding site
is represented by the lipophilic potential mapped onto a Connolly
surface (MOLCAD, Darmstadt, Germany), hydrophobic areas
(brown), polar areas (green and blue); bis-MANT-ATP is shown as
stick model (heteroatoms as balls) with C and some H atoms coloured
yellow
Fig. 5
(e.g. the side chain of T548) by flexible fit. The ring planes
of the nucleobases are aligned with the side chain of N583.
The higher affinity of (M)ANT-CTP derivatives for EF
compared to their (M)ANT-ATP analog (Taha et al. 2009) is
in part due to the oxygen in 2-position of cytosine, which
may fit to the positive pole of the amide dipole.
Considering the farther environment of the cytosine
oxygen, additional reasons are possible. In the model, it is
3.3 Å distant from the guanidino group of R329. Moreover,
a water molecule can be placed in an ideal position where it
forms three hydrogen bonds, bridging the cytosine oxygen
with the side chains of R329 and E580. The affinity data of
bis-MANT-ATP and -CTP on N583 mutants (Table 1)
confirm the crucial role of this amino acid. The larger side
chains in N583Q and N583H seem to restrict the binding
cavity for the nucleobases so that the potency is strongly
reduced, whereas in the case of N583A, interactions are
only moderately weakened.
The EF binding mode of the (M)ANT-derivatives largely
corresponds to that of the nucleotides themselves (Shen et
al. 2002; Taha et al. 2009; Göttle et al. 2010). The 3′anthraniloyl group protrudes from the catalytic site due to a
3′-endo conformation of the ribosyl moiety in the case of
3′-(M)ANT isomers (Fig. 5a). Hydrophobic interactions
with F586 account for the generally higher affinity of the
(M)ANT-derivatives compared to the parent nucleotides
(Taha et al. 2009). The phenyl ring is π-stacked between
H351 and F386. Hydrophobic alkyl (methyl, propyl) and
halogen substituents may additionally contact F386 and,
thereby, increase affinity. However, the binding data result
from mixtures of 2′-(M)ANT and 3′-(M)ANT isomers.
Intriguingly, the binding mode of 2′-(M)ANT nucleotides
may be the same as in the case of the 3′-(M)ANT analog if
the ribosyl moiety adopts a 3′-exo conformation, allowing
an axial 2′-(M)ANT group also to protrude from the
catalytic site and to stack between H351 and F386 (Taha
et al. 2009).
Bis-MANT-ATP can be docked into EF (Fig. 5b) in a
similar pose like 3′-MANT-CTP (Fig. 5a). The ribosyl
moiety again adopts a 3′-endo conformation, leading to an
equatorial orientation of the 2′-MANT and an axial
65
orientation of the 3′-MANT group, which parallelly aligns
with F386. The 3′-MANT methyl substituent perpendicularly contacts the imidazole ring of H351. The 2′-MANT
group projects rather freely into the solvent, forming no
specific interactions with EF apart from weak hydrophobic
contacts of the methyl substituent with the phenyl ring of
F386. It is, therefore, not surprising that the affinities of all
tested bis-(M)ANT derivatives are similar. The lipophilic
potential of the binding site substantiates that hydrophobic
interactions in particular with F386 are the main cause for
the high affinity of mono- and bis-(M)ANT nucleotides
(Fig. 5c).
Discussion
In a recent study, we found that certain bis-(M)ANT
nucleotides, most notably bis-Cl-ANT-ATP, inhibit the
catalytic activity of CyaA with >100-fold potency than
mammalian mACs 1, 2 and 5 (Geduhn et al. 2011).
These data indicate that inhibitors of bacterial AC toxins
with high selectivity relative to mammalian ACs can be
obtained more easily than previously assumed (Johnson
and Shoshani 1990; Gille et al. 2004; Göttle et al. 2007).
Moreover, bis-MANT nucleotides exhibit very favourable
fluorescence properties, i.e. a high signal/noise ratio upon
binding to target proteins, facilitating development of nonradioactive high-throughput AC inhibitor screening assays
(Fig. S8) (Geduhn et al. 2011). These findings prompted
us to characterise the interaction of the catalytic site of EF
with a series of 18 mono- and bis-(M)ANT nucleotides
possessing various purine and pyrimidine bases to better
understand the molecular mechanisms of EF inhibition.
EF inhibitors could be useful compounds to treat EF
toxaemia and antibiotic-resistant B. anthracis strains
(Jedrzejas 2002).
The structure–activity relationships of nucleotides and their
(M)ANT derivatives at EF, CyaA and mAC are different,
indicating that in principle, the development of potent and
selective EF inhibitors is feasible. Previous studies from our
laboratory showed that CTP inhibited EF >400-fold more
potently than mAC (Gille et al. 2005; Taha et al. 2009).
Isomeric introduction of a MANT group at the 2′- or 3′-Oribosyl position of CTP decreased the Ki value from 5 μM to
100 nM, yielding an EF inhibitor that is even 5–10-fold more
potent than MANT-ATP in the presence of Mn2+; i.e. EF
exhibits high preference for the base cytosine (Taha et al.
2009). Among all compounds examined, propyl-ANT-ATP
was the most potent EF3 inhibitor (Table 2). Its inhibitory
potency is comparable to the potency of MANT-CTP
(100 nM) (Taha et al. 2009).
In contrast to the data obtained for CyaA (Geduhn et al.
2011), most bis-(M)ANT nucleotides exhibited a similar or
66
even lower potency than the corresponding mono- (M)ANT
nucleotides at EF (Tables 1 and 2). Only weak hydrophobic
interactions of the 2′-substituent with F586 could be
derived from docking studies (Fig. 5). However, mono(M)ANT nucleotides were up to 10-fold more potent at EF
than at CyaA. Therefore, the fit of a single (M)ANT group
to EF is more favourable than to CyaA, whereas the second
2′-(M)ANT moiety in bis-(M)ANT nucleotides more
strongly interacts with CyaA. The reasons for these differences are difficult to explain since the (M)ANT nucleotide
binding sites of both toxins are very similar. Fifteen out of
16 amino acids are identical, and the RMS distance of their
Cα atoms amounts to only 0.7 Å if the most recent
structures of EF (Shen et al. 2005) and CyaA (Guo et al.
2005) are compared. Nevertheless, docking studies indicate
that the 2′-substituent in the most potent bis-Cl and bis-BrANT nucleotides directly contributes to the high affinity for
CyaA via hydrophobic interactions with L60 (EF, L348),
F306 (EF, F586) and P305 (EF, D585) (Geduhn et al.
2011). It may be speculated whether the D585/P305
mutation at least in part accounts for the different potency
of bis-(M)ANT nucleotides at CyaA and EF.
Both bis-MANT-CTP and bis-MANT-ATP are competitive EF inhibitors (Fig. 2). These data rule out the existence
of a hitherto unidentified nucleotide binding site in the
structurally very complex EF protein (Drum et al.
2002; Shen et al. 2002). Kinetic FRET competition experiments with the non-fluorescent ATP analog PMEApp
revealed that both bis-MANT- ATP and bis-MANT-CTP
reversibly bind to the EF-catalytic site (Fig. 4), corroborating the competitive inhibition mode and the existence of a
single nucleotide binding site in EF.
Our study demonstrates a striking dissociation between
ligand affinity for EF as assessed by the inhibition of catalysis
and FRET on the one hand and both the maximum stimulation
of direct fluorescence and FRET upon activation of CaM on
the other hand. Most notably, both bis-MANT-ATP and bisMANT-CTP have the same Ki value at EF3 (Table 1).
However, in terms of maximum direct fluorescence and
FRET signals, bis-MANT-ATP clearly surpasses bis-MANTCTP (Fig. 3). Similar dissociations between affinity and
maximum fluorescence signals were observed in our
previous study for the comparison of MANT-ATP and
MANT-CTP at EF (Taha et al. 2009). An explanation for
these discrepancies could be differences in mobility of the
various fluorescence probes, with the more rigidly bound
ligands being more effective in terms of direct MANT
fluorescence and FRET (Göttle et al. 2007; Taha et al. 2009).
Specifically, the adenine base is larger than the cytosine base;
hence, bis-MANT-ATP may be bound to EF with fewer
degrees of rotational freedom than bis-MANT-CTP.
F586 mediates π-stacking interactions with 2′-deoxy-3′ANT-ATP, resulting in a fluorescence increase upon
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
excitation of the ANT-group (Shen et al. 2002). Our
previous study also showed that mutation of F586 resulted
in 5–6-fold reduction in potencies of both MANT-ATP and
MANT-CTP and largely reduced the CaM-dependent FRET
of MANT-ATP (Taha et al. 2009). F586 is important for the
interaction with bis-MANT-ATP and bis-MANT-CTP, too,
since the potencies at the F386A mutant are 5–6-fold
reduced as well. These similar results are not surprising if
the putative docking mode of bis-MANT nucleotides is
taken into account, which does not predict essential
additional interactions of the 2′-substituent (Fig. 5). Compared to EF3, the FRET signal with bis-MANT-ATP was
substantially lower in the F386A mutant (Figs. 3 and S2),
again highlighting the eminent importance of F586 for
ligand/protein interaction.
In analogy to the data obtained for MANT-CTP, FRET
signals for bis-MANT-CTP were small at EF3 (Fig. 3).
Molecular modelling suggested a similar binding mode of
MANT-CTP and its ATP analog with subtle differences in
mobility in the nucleobase region, accounting for the higher
potency and the small FRET signal of MANT-CTP. In
particular, the cytosine moiety may form water-mediated
hydrogen bonds with R329 and E580 and favourably fit to
the amide dipole of N583. Additionally, the flexibility of
the bound cytosine was greater than in the case of the
bulkier adenine ring (Taha et al. 2009). These factors may
lead to absorption and thus attenuation of the FRET energy,
which is mainly due to tyrosine and tryptophan residues in
switch C.
H577 plays a crucial role in catalysis as is reflected by
the very low catalytic activity of the H577A mutant (Drum
et al. 2002; Taha et al. 2009; Guo et al. 2004). In addition,
bis-MANT-ATP and bis-MANT-CTP exhibited no FRET
signal at all in H577A mutant and largely reduced signal
intensities in direct fluorescence studies. H577 participates
in the coordination of an Mg2+ or Mn2+ ion but is not
involved in strong interactions with (MANT) nucleotides
(see Fig. 5). Therefore, its replacement by alanine destabilises the catalytic site but does not exert detrimental effect
on substrate and inhibitor binding per se (Table 1) (Taha et
al. 2009).
N583 forms a crucial hydrogen bond with the ribosyl
moiety of nucleotides bound to the catalytic site of EF
(Drum et al. 2002; Taha et al. 2009). Accordingly,
replacement of N583 by a non-hydrogen bond-forming
amino acid (N583A) or a larger hydrogen bond-forming
amino acid with a different spatial arrangement of the
bonding partners (N583Q and N383H) substantially
decreases catalytic activity of the resulting EF mutants
(Drum et al. 2002; Taha et al. 2009). Moreover, N583
mutants substantially reduced the potencies of MANT-ATP
and MANT-CTP (Taha et al. 2009). Along the same line,
N583 mutants substantially reduced the potencies of bis-
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:57–68
MANT-ATP and bis-MANT-CTP. In addition, the bisMANT nucleotides potencies were more strongly reduced
by the N583Q and N583H than by N583A mutation. These
findings imply the substantial spatial constraint in this part
of the binding pocket and highlight the importance of the
spatial arrangement of N583 for the binding of bis-MANT
nucleotides.
K353 forms an ionic bond with the α-phosphate of
(MANT)-nucleotides. Disruption of this ionic bond by the
K353A mutation largely reduces catalytic activity and
lowers substrate affinity, but K353R displayed less severe
impairment of catalysis and no change in Km. The affinity
of bis-MANT-ATP and bis-MANT-CTP to the EF-catalytic
site, FRET and direct fluorescence are reduced in the
K353A mutant (Table 2 and Fig. S6). In contrast, the
positive charge-retaining K353R mutation had only a small
effect on inhibitor affinity as revealed by AC and
fluorescence assays (Table 2 and Fig. S7). Thus, binding
of bis-MANT nucleotides to the EF-catalytic site was
largely affected by disruption of the ionic bond by the
K353A mutation. The K353R mutation that alters the
spatial arrangement of the catalytic site but still allows ionic
bridge formation did not largely affect the inhibitor
potency.
Our data have conceptual and practical implications for
future drug development. Considering the fact that EF
exhibits a uniquely high affinity for the base cytosine, the
analysis of PMECpp will be particularly interesting. The
lower basal fluorescence of bis-MANT-analog and the
higher CaM-dependent fluorescence signals at the excitation wavelength of 350 nm as compared to mono-MANT
nucleotides could be used as non-radioactive highthroughput screening assay for EF inhibitors, including
compounds binding to the catalytic site as well as
compounds impeding with the interaction of EF and CaM
(Lee et al. 2004). Such FRET assay constitutes a valuable
complementation of the sensitive radiometric EF activity
assay used in this study and the recently described
fluorometric EF activity assay (Spangler et al. 2008).
In conclusion, using enzymatic, fluorescence spectroscopy and docking approaches, our present study shows that
bulky bis-MANT- and bis-propyl-ANT-substituted purine
and pyrimidine nucleotides are accommodated by the
nucleotide binding-site of EF. Thus, the results of this
study lend further support to the broad hypothesis that the
catalytic sites of various structurally unrelated ACs,
including mammalian membranous ACs and bacterial AC
toxins, exhibit substantial degrees of conformational flexibility, allowing for numerous structural modifications in
nucleotide inhibitors and the development of EF-potent and
selective inhibitors. We have also shown than bis-MANT
nucleotides are highly sensitive conformational probes for
EF and that these nucleotides could be used in high-
67
throughput screening studies for the identification of nonfluorescent and non-nucleotide EF inhibitors.
Acknowledgements This work was supported by Deutsche
Forschungsgemeinschaft grant Se 529/5-2 to R. S.
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