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Biochem Pharmacol. Author manuscript; available in PMC 2012 August 15.
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Published in final edited form as:
Biochem Pharmacol. 2011 August 15; 82(4): 358–370. doi:10.1016/j.bcp.2011.05.010.
Structure-Activity Relationships for the Interactions of 2’- and 3’(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine
Nucleotides with Mammalian Adenylyl Cyclases
Cibele Pinto,
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS, USA
Gerald H. Lushington,
Molecular Graphics & Modeling Laboratory, University of Kansas, Lawrence, KS, USA
Mark Richter,
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
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Andreas Gille,
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS, USA
Jens Geduhn,
Institute of Organic Chemistry, University of Regensburg, Germany
Burkhard König,
Institute of Organic Chemistry, University of Regensburg, Germany
Tung-Chung Mou,
Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT, USA
Stephen R. Sprang, and
Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT, USA
Roland Seifert
Institute of Pharmacology, Medical School of Hannover, Hannover, Germany
Abstract
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Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising
drug targets. In previous studies we showed that 2’,3’-(O)-(N-methylanthraniloyl) (MANT)substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic
structure-activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC
subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5.
(M)ANT-nucleotides inhibited fully activated VC1:IIC2 in the order of affinity for bases
hypoxanthine > uracil > cytosine > adenine ~ guanine ≫ xanthine. Omission of a hydroxyl group
at the 2’ or 3’-position reduced inhibitor potency as did introduction of a -thiophosphate group or
omission of the -phosphate group. Substitution of the MANT-group by an ANT-group had little
effect on affinity. Although all nucleotides bound to VC1:IIC2 similarly according to the tripartite
pharmacophore model with a site for the base, the ribose, and the phosphate chain, nucleotides
© 2011 Elsevier Inc. All rights reserved.
Corresponding author Dr. Roland Seifert, Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Str. 1, D-30625
Hannover, Germany, Telephone: +49-511-532-2805. Fax: +49-511-532-4081. seifert.roland@mh-hannover.de.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
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Pinto et al.
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exhibited subtle differences in their binding modes as revealed by fluorescence spectroscopy and
molecular modelling. MANT-nucleotides also differentially interacted with the VC1:VC1
homodimer as assessed by fluorescence spectroscopy and modelling. Similar structure-activity
relationships as for VC1:IIC2 were obtained for recombinant ACs 1, 2 and 5, with AC2 being the
least sensitive AC isoform in terms of inhibition. Overall, ACs possess a broad base-specificity
with no preference for the “cognate” base adenine as verified by enzyme inhibition, fluorescence
spectroscopy and molecular modelling. These properties of ACs are indicative for ligand-specific
conformational landscapes that extend to the VC1:VC1 homodimer and should facilitate
development of non-nucleotide inhibitors.
Keywords
Adenylyl Cyclase; MANT-nucleotides; fluorescence spectroscopy; molecular modeling;
conformational landscape
1. Introduction
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Mammals express nine membranous AC isoforms that play an important role in signal
transduction [1-3]. ACs are activated by the G-protein Gs via receptors for hormones and
neurotransmitters and catalyze the production of the second messenger cAMP. ACs 1-8 are
also directly activated by the diterpene, FS [1-4]. The analysis of AC knock-out mice
provided important insights into the function of specific AC isoforms and potential
therapeutic applications of AC inhibitors [2, 5]. Currently, there is much interest in ACs 1
and 5. Specifically, AC1 knock-out mice are protected against neuronal toxicity mediated by
ionotropic glutamate receptors [6, 7]. AC5 knock-out mice are protected against heart failure
and stress and show reduced chronic pain responses as well as increased longevity [8-11].
Thus, dual AC1/5 inhibitors may be useful drugs for the treatment of various age-related
ailments including heart failure, neurodegenerative diseases, stroke and chronic pain [7, 8,
12].
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2’,3’-O-(N-Methylanthraniloyl) (MANT)-substituted nucleotides are competitive AC
inhibitors [13, 14]. ACs 1 and 5 are more sensitive to inhibition by MANT-nucleotides than
AC2 [14]. MANT-GTP S inhibits recombinant AC5 expressed in Sf9 insect cells with a Ki
values of ~35 nM [14] and blocks activation of voltage-dependent calcium channels in
cardiomyocytes via AC5 [12]. Moreover, MANT-nucleotides are fluorescence probes to
monitor ligand-protein interactions [15]. Upon binding of MANT-nucleotides to purified
catalytic subunits of mammalian AC (C1 subunit of AC5 (VC1) and C2 subunit of AC2
(IIC2), an increase in direct fluorescence is observed, reflecting binding of the MANT-group
into a hydrophobic pocket in the catalytic site [16, 17]. Moreover, FRET between Trp1020
in IIC2 and the MANT-group is observed, FS increasing the signals as a reflection of
optimization of the MANT binding pocket [16, 17]. However, it should be emphasized that
VC1:IIC2 is only a general model for membranous ACs and not a specific model for a given
AC isoform. The problem is that the VC2- and IIC1 subunits are difficult to express. We
tried this already in previous studies [16, 17], but we failed.
Enzymatic, fluorescence spectroscopy, crystallographic and molecular modelling studies
showed that ACs exhibit a high degree of conformational flexibility, allowing the catalytic
site to accommodate structurally diverse bases [13, 14, 18-21]. Even the VC1:VC1
homodimer, although exhibiting only exceedingly low catalytic activity, is capable of
binding MANT-GTP with high affinity [22]. It is possible that in vivo, C1:C1 dimers form
through interaction of the C1-subunits of neighboring AC molecules, ensuring low basal AC
activity and hence, providing a novel site for pharmacological intervention [22].
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The aim of our present study was to provide a systematic structure-activity relationship of a
series of 21 (M)ANT-nucleotides for VC1:IIC2 in terms of inhibition of catalysis,
fluorescence spectroscopy and molecular modelling. As basis for molecular modelling, the
crystal structures of VC1:IIC2 in complex with MANT-GTP, MANT-ATP and MANT-ITP
are now available [16, 17, 23]. Moreover, we studied the properties of (M)ANT-nucleotides
in terms of inhibition of recombinant ACs 1, 2 and 5 expressed in Sf9 insect cells. Finally,
we analyzed the nucleotide-binding properties of the VC1:VC1 homodimer in terms of
fluorescence spectroscopy and modelling. A detailed understanding of the structure-activity
relationship of nucleotides for the interaction with various ACs is essential for the future
development of AC isoform-selective non-nucleotide inhibitors that could be used as
potential drugs.
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Fig. 1 shows the structures of the nucleotides examined herein. In 2’,3’-MANT-nucleotides,
the MANT-group spontaneously isomerizes between the 2’- and 3’-group [15], but in the
VC1:IIC2 crystal structures, the 3’-MANT isomers are favored [16, 17, 23]. Therefore, we
also studied the defined 2’-d-3’-MANT- and 3’-d-2’-MANT-isomers of MANT-GTP (2 and
3) and MANT-ATP (6 and 7). These isomer pairs for MANT-ATP and MANT-GTP have
provided valuable insights into ligand-protein interaction for a bacterial AC toxin, edema
factor, from Bacillus anthracis [24]. Note, that in comparison to 2’,3’-MANT-nucleotides,
the defined 2’- and 3’-MANT-isomers lack a hydroxyl group that may be important for
hydrogen bonding with AC. Moreover, we studied MANT-ITP (8), the most potent AC
inhibitor known so far [21, 23], differing from MANT-GTP (1) only by the lack of an NH2group at C2 of the purine ring. For comparison, we also studied MANT-XTP (10), bearing a
keto group at C2 and inhibiting VC1:IIC2 much less potently than MANT-GTP [17, 23].
Considering the relatively high potency of 2’,3’-O-(2,4,6-trinitrophenyl)-UTP and 2’,3’-O(2,4,6-trinitrophenyl)-CTP for VC1:IIC2 (Ki, ~100-300 nM range) [17] and ACs 1, 2 and 5
(Ki ~10-100 nM) [20], we examined the interaction of VC1:IIC2 with MANT-UTP (11) and
MANT-CTP (12) as well. ANT-nucleotides differ from MANT-nucleotides by the lack of
the methyl group at the anthraniloyl ring (Fig. 1) and were used for the fluorescence analysis
of various proteins [15]. Therefore, we included various ANT-nucleotides (13-15, 21) into
our studies as well. Finally, the length of the polyphosphate tail critically determines the
affinity of AC for 2’,3’-substituted nucleotides [14]. Hence, we examined several (M)ANTNDPs (15-19) and (M)ANT-NMPs (20, 21), too.
2. Materials and Methods
2.1. Materials
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MANT- and ANT-substituted nucleotides 8, 11, 12, 14, 15 and 17-21 were synthesized
according to Hiratsuka [25] with the previously described modifications [26, 27]. MANTGTP (1), 2’-d-3’-MANT-GTP (2), 3’-d-2’-MANT-GTP (3), MANT-GTP S (4), MANTATP (5), 2’-d-3’-MANT-ATP (6), 3’-d-2’-MANT-ATP (7), MANT-ITP S (9), MANT-XTP
(10), ANT-GTP (14) and MANT-ADP (16) were obtained from Jena Bioscience (Jena,
Germany). Catalytic AC subunits VC1 and IIC2 and GTP S-activated Gsα (Gsα-GTP S) were
expressed and purified as described [28]. [α-32P]ATP (800 Ci/mmol) was purchased from
PerkinElmer (Wellesley, MA, USA). Aluminum oxide 90 active, neutral (activity 1, particle
size 0.06-0.2 mm) was purchased from Merck (Darmstadt, Germany). Bovine serum
albumin, fraction V, highest quality, was from Sigma-Aldrich (St. Louis, MO, USA). MnCl2
tetrahydrate (highest quality) was from Merck. FS was from LC Laboratories (Woburn, MA,
USA).
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2.2. Cell culture and membrane preparation
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Cell culture and membrane preparation were performed as previously described [29].
Briefly, Sf9 cells were cultured in SF 900 II medium supplemented with 5% (vol/vol) fetal
bovine serum and 0.1 mg/ml gentamicin. High-titer baculoviruses for ACs 1, 2 and 5 were
generated through two sequential amplification steps as previously described [14, 29]. In
each amplification step the supernatant fluid was harvested and stored under light protection
at 4°C. For membrane preparation Sf9 cells (3.0 × 106 cells/ml) were infected with
corresponding baculovirus encoding different mammalian ACs (1:100 dilutions of high-titer
virus) and cultured for 48 hours. Membranes expressing each construct and membranes from
uninfected Sf9 cells were prepared as described [29]. Briefly, cells were harvested and cell
suspensions were centrifuged for 10 min at 1,000 × g at 4°C. Pellets were resuspended in 10
ml of lysis buffer (1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 g/ml
leupeptine and 10 g/ml benzamide, pH 7.4). Thereafter, cells were lyzed with 20-25 strokes
using a Dounce homogenizer. The resultant cell fragment suspension was centrifuged for 5
min at 500 × g and 4°C to sediment nuclei. The cell membrane-containing supernatant
suspension was transferred into 30 ml tubes and centrifuged for 20 min at 30,000 × g and
4°C. The supernatant fluid was discarded and cell pellets were resuspended in buffer
consisting of 75 mM Tris/HCl, 12.5 mM MgCl2, and 1 mM EDTA, pH 7.4. Membrane
aliquots of 1 ml were prepared, stored at -80°C and protein concentration for each
membrane preparation was determined using the Bio-Rad DC protein assay kit (Bio-Rad,
Hercules, CA, USA).
2.3. AC activity assay
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AC activity in Sf9 membranes expressing ACs 1, 2 or 5 was determined essentially as
described [14]. Before experiments, membranes were sedimented by a 15 min centrifugation
at 4°C and 15,000 × g and resuspended in 75 mM Tris/HCl, pH 7.4. Reaction mixtures (50
l final volume) contained 30 g of membrane protein, 40 M ATP/Mn2+ plus 5 mM
MnCl2, 100 M FS, 10 M GTP S and (M)ANT-nucleotides at concentrations from 0.1 nM
to 1 mM as appropriate to obtain saturated inhibition curves. Following a 2 min preincubation at 37°C, reactions were initiated by adding 20 l of reaction mixture containing
(final) 1.0-1.5 Ci/tube [α-32P]ATP and 0.1 mM cAMP. AC assays were conducted in the
absence of an NTP-regenerating system to allow for the analysis of 2’,3’-substituted
(M)ANT-NDPs that could otherwise be phosphorylated to the corresponding (M)ANTNTPs [14]. For determination of Km values, reactions mixtures contained 20 M - 1 mM
ATP/Mn2+ as substrate [14]. Reactions were conducted for 20 min at 37°C and were
terminated by adding 20 l of 2.2 N HCl. Denatured protein was precipitated by a 1 min
centrifugation at 25°C and 15,000 × g. Sixty-five l of the supernatant fluid were applied
onto disposable columns filled with 1.3 g neutral alumina. [32P]cAMP was separated from
[α-32P]ATP by elution of [32P]cAMP with 4 ml of 0.1 M ammonium acetate, pH 7.0.
Recovery of [32P]cAMP was ~80% as assessed with [3H]cAMP as standard. [32P]cAMP
was determined by Čerenkov radiation.
For experiments with purified catalytic AC subunits, reaction mixtures contained 100 M
ATP/Mn2+, 10 mM MnCl2 and (M)ANT-nucleotides at concentrations from 0.1 nM to 1
mM as appropriate to obtain saturated inhibition curves. Additionally, assay tubes contained
VC1 (8 nM) and IIC2 (40 nM). For experiments with Gsα-GTP S, tubes contained VCI (3
nM), IIC2 (15 nM) and Gsα-GTP S (51 nM). We have used the 1:5 ratio of VC1:IIC2 in all of
our previous studies to ensure that all available VC1 molecules find a IIC2 partner [see e.g.
14, 16 and 17]. The formation of IIC2 dimers is not problematic because these dimers
neither bind nucleotides nor FS and do not exhibit catalytic activity [22]. Moreover, the VC1
homodimer exhibits only exceedingly low catalytic activity [22]. Thus, under the conditions
chosen, we almost exclusively analyze the VC1:IIC2 heterodimer. Reactions were
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conducted in the presence of 100 M FS. Following a 2 min pre-incubation at 30°C,
reactions were initiated by adding 20 l of reaction mixture containing (final) 1.0 Ci/tube
[α-32P]ATP, 0.1 mM cAMP and 100 mM KCl in 25 mM HEPES/NaOH, pH 7.4. AC assays
were conducted in the absence of an NTP-regenerating system to allow for the analysis of
2’,3’-substituted (M)ANT-NDPs that could otherwise be phosphorylated to the
corresponding (M)ANT-NTPs [14]. Reactions were conducted for 10-20 min at 30°C.
Competition isotherms were analyzed by non-linear regression using the Prism 4.0 software
(GraphPad, San Diego, CA, USA).
2.4. Fluorescence spectroscopy
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All experiments were conducted using a Cary Eclipse fluorescence spectrophotometer
equipped with a Peltier-thermostated multicell holder at 25°C (Varian, Palo Alto, CA,
USA). Measurements were performed in a quartz fluorescence microcuvette (Hellma,
Plainview, NY, USA). The final assay volume was 150 l. Reaction mixtures contained a
buffer consisting of 100 mM KCl, 10 mM MnCl2 and 25 mM HEPES/NaOH, pH 7.4.
Steady-state emission spectra were recorded at low speed with ex = 350 nm ( em = 370-500
nm) and ex = 280 nm ( em = 300-500 nm) with various (M)ANT-nucleotides (1 M each)
in the absence and presence of 5 M VC1 plus 25 M IIC2 without and with 100 M FS.
We have used a 1:5 ratio of VC1:IIC2 in all of our previous studies to ensure that all
available VC1 molecules find a IIC2 partner [see e.g. 14, 16 and 17]. The formation of IIC2
dimers is not problematic because these dimers neither bind nucleotides nor FS and do not
give rise to fluorescence signals [22]. Moreover, the VC1 homodimer gives rise to very
different fluorescence signals than the VC1:IIC2 heterodimer [22]. Thus, under the
conditions chosen, we almost exclusively analyze the VC1:IIC2 heterodimer. In experiments
with VC1 alone, the protein concentration was 5 M, and the MANT-nucleotide
concentration was 1 M. Fluorescence recordings were analyzed with the spectrum package
of the Cary Eclipse software (Varian). Baseline fluorescence (buffer alone) was subtracted
from all recordings. Figs. 2, 4 and 6 show superimposed original fluorescence recordings
representative for three independent experiments with different batches of VC1 and IIC2.
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Molecular modelling studies—Predicted structures of MANT-GTP, MANT-ITP,
MANT-ATP, MANT-UTP, MANT-CTP, MANT-XTP, ANT-GTP and ANT-ATP bound to
VC1:IIC2 were generated via molecular simulations. The complexes were constructed from
the crystal structure of VC1:IIC2 bound to MANT-GTP (PDB ID 1TL7) [16]. In each case,
the new complex was constructed by editing the structure of co-crystallized ligand in
SYBYL 8.0 [30] to represent the desired ligand while retaining the original atomic
coordinates for all structurally conserved portions of the ligand. The full complex was then
protonated using SYBYL. All ligands were assumed to have a fully anionic triphosphate tail,
while the receptor valences were assumed to correspond to physiological pH, with cationic
lysine and arginine residues, and anionic aspartates and glutamates. Based on these assumed
valences, Gasteiger-Marsili partial charges [31] were assigned to all atoms and the system
was permitted some modest relaxation via an 11 ps molecular dynamics simulation in
SYBYL (1 ps warming from 0 to 300 K, followed by 10 ps thermal equilibration) in which
the ligand and all receptor residues within 5.0 A of the ligand were left conformationally
mobile. The resulting relaxed complex structures were then optimized to within SYBYL
default convergence thresholds via molecular mechanics.
The bound conformation of the 3’-MANT analogs was taken to correspond to the structures
reported in the 2GVZ (MANT-ATP) [17] and 1TL7 (MANT-GTP) [16] crystal structures.
Bound conformations for the corresponding 2’-MANT analogs were determined by
sketching the structures in SYBYL 8.0 [30] and docking them into the VC1:IIC2 receptor
via Autodock [32]. Ligand charges were assigned via the Gasteiger-Marsili formalism [33],
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assuming a net charge state of -4, with oxygens on the α- and -phosphate units sharing a
charge of -1 per unit, and oxygens on the -phosphate sharing a -2 charge. For all docking
calculations, the receptor was prepared by removing the co-crystallized ligand and
crystallographic waters from the 2GVZ crystal structure, protonating the structure in
SYBYL (assuming physiological pH: cationic arginine and lysine residues; anionic
aspartates and glutamates), and adding Gasteiger-Marsili charges. The docked poses for
each ligand were determined to be the lowest energy conformer of the most populous cluster
as observed among 100 Lamarckian genetic algorithms searches in Autodock. The resulting
poses were refined in SYBYL via a 10 ps molecular dynamics simulation at 300K followed
by molecular mechanics optimization to full convergence according to default thresholds. In
both of the latter refinement processes, all receptor atoms were held rigidly fixed, while
ligand atoms were permitted to relax according to the Tripos molecular force field [34] and
Gasteiger-Marsili electrostatics.
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To generate the VC1:VC1 homodimer model, the first VC1 subunit was retained as reported
in the crystal structure previously reported [35], and the second VC1 subunit was mapped to
the framework via the Modeller program [36]. Sequence alignment between the VC1 target
and the IIC2 template was performed with the Custal-W program [37] using the
BLOSUM-30 substitution matrix [38], and standard gap penalties of 10 for opening and 0.1
for extension. Ligand interactions with residue 1029 were quantified in SYBYL 8.0 [30] via
the Tripos Molecular Force Field [34] and Gasteiger-Marsili electrostatics [33].
3. Results
3.1. Inhibition of the catalytic activity of VC1:IIC2 heterodimer by (M)ANT-nucleotides
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Table 1 summarizes the inhibitory effects of (M)ANT-nucleotides on the catalytic activity of
VC1:IIC2. All nucleotides reduced catalysis according to monophasic sigmoidal inhibition
isotherms. Defined 3’-O and 2’-O-isomers of both MANT-GTP (2, 3) and MANT-ATP (6,
7) exhibited 5-20-fold lower inhibitory potencies for the maximally stimulated VC1:IIC2
(Mn2+ + FS + Gsα-GTP S) than MANT-GTP (1) and MANT-ATP (5), respectively. 3’-d-2’MANT-GTP (3) was two-fold less potent than the 2’-d-3’-MANT derivative (2). Although
previous studies showed that MANT-ATP also binds to VC1:IIC2 preferably as 3’-isomer
[17], contrary to MANT-guanine nucleotides 1-3, 2’-d-3’-MANT-ATP (6) was less potent
than the 2’-MANT-derivative (7) at inhibiting VC1:IIC2 catalytic activity. MANT-UTP (11)
and MANT-CTP (12) exhibited two- to three-fold higher inhibitory potencies at VC1:IIC2
than MANT-GTP (1) and MANT-ATP (5) under maximally stimulatory conditions. In
contrast to the MANT-GTP/MANT-GTP S pair (1→4), exchange of the -thiophosphate by
a phosphate in hypoxanthine nucleotides increased the inhibitory potency by almost 30-fold
(9→8). Introduction of a keto group at the C2 carbon atom of the purine ring (8→10)
decreased inhibitor potency several hundred-fold. Deletion of the methyl group from the
fluorophore at the 2’,3’-O-ribosyl substituent in NTPs (compare 1 and 13, 5 and 14) did not
largely change their affinity for VC1:IIC2 in the presence of Gsα-GTP S (Table 1). Deletion
of the -phosphate reduced the inhibitory potency of (M)ANT-NDPs ~10-80-fold compared
to the corresponding (M)ANT-NTPs (compare 14 and 15, 5 and 16, 11 and 18, 12 and 19)
(Table 1).
Under submaximally stimulatory conditions, i.e. in the absence of Gsα-GTP S, the potencies
of nucleotides were generally lower than under maximally stimulatory conditions. However,
since fluorescence studies with VC1:IIC2 were performed in the absence of Gsα-GTP S (Figs.
2 and 4), it was important to determine inhibitor potencies under these experimental
conditions as well. Under submaximally stimulatory conditions, potencies of nucleotides
were 3.4- to 84-fold lower compared to maximally stimulatory conditions. The affinitydifference was most pronounced for ANT-ADP (15, 84-fold) and least pronounced for 3’Biochem Pharmacol. Author manuscript; available in PMC 2012 August 15.
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d-2’-MANT-GTP (3, 3.4-fold). In the absence of Gsα-GTP S, the order of potencies of
MANT-NTPs was MANT-ITP > MANT-UTP > MANT-ATP ~ MANT-GTP > MANTCTP ≫ MANT-XTP.
3.2. Analysis of the interaction of VC1:IIC2 heterodimer with (M)ANT-nucleotides by
fluorescence spectroscopy
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We determined the emission spectra of nucleotides at ex = 350 nm for direct excitation of
the (M)ANT group [15], and at ex = 280 nm for analysis of FRET between Trp1020 in IIC2
and the (M)ANT group [16, 17]. We performed fluorescence studies with a large molar
excess of C1 and C2 relative to (M)ANT-nucleotides to allow for quantitative nucleotide
binding to VC1:IIC2 [16, 17]. At ex= 280 nm, (M)ANT-nucleotides were only minimally
excited, whereas at ex= 350 nm they showed substantial intrinsic fluorescence signals with
an emission peak at ~ 450 nm (Fig. 2, blue tracings). The dashed black line indicates the
endogenous tryptophan (and tyrosine) fluorescence of VC1:IIC2 at ex= 280 nm, i.e. the
fluorescence in the absence of (M)ANT-nucleotide. Following the addition of VC1:IIC2, at
ex= 280 nm, MANT-GTP exhibited a higher basal FRET signal, as revealed by a second
emission peak at em= 420 nm, than MANT-ATP (green tracings, Figs. 2M and O). At ex=
350 nm, the interaction of MANT-GTP with VC1:IIC2 resulted in a two-fold higher
increase in fluorescence and a “blue-shift” of the emission peak compared with MANT-ATP
(green tracings, Figs. 2N and P). FS (100 M) increased basal FRET and direct fluorescence
with MANT-GTP and MANT-ATP (red tracings), with the differences between MANTGTP and MANT-ATP still being present. We also observed differences in both basal FRET
between purine and pyrimidine nucleotides (MANT-ITP > MANT-GTP > MANT-ATP >
MANT-CTP > MANT-UTP > MANT-XTP) (Figs. 2A, C I, L, M and O). The stimulatory
effect of FS on FRET followed the order MANT-CTP > MANT-UTP > MANT-GTP >
MANT-ATP > MANT-ITP ~ MANT-XTP. At ex= 350 nm, the increase in basal
fluorescence signal following the addition of VC1:IIC2 varied among the different MANTsubstituted nucleotides (MANT-ITP > MANT-GTP > MANT-ATP > MANT-UTP >
MANT-CTP > MANT-XTP) (Figs. 2B, D, J, L, N and P). For direct fluorescence increase
by FS, the order was MANT-UTP > MANT-CTP ~ MANT-GTP > MANT-ATP ~ MANTITP > MANT-XTP.
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As previously reported [25], ANT-GTP (Fig. 2F) and ANT-ATP (Fig. 2H) were also excited
at ex = 350 nm, but exhibited a shorter wavelength of the emission maximum (420 nm).
This difference in peak of emission can be also seen at ex= 280 nm, as the peak of basal and
FS-stimulated FRET is shifted to the left compared with the respective MANT-nucleotides
(compare Figs. 2E and M, and G and O). As with the MANT-GTP/MANT-ATP pair, ANTGTP and ANT-ATP showed significant differences in their emission spectra following the
addition of VC1:IIC2 and FS. At ex= 280 nm, ANT-ATP, contrary to ANT-GTP, showed
essentially no basal or FS-stimulated FRET (Figs. 2E and G). Furthermore, at ex = 350 nm,
the addition of VC1:IIC2 and FS (100 M) resulted in a two-fold higher increase in ANTGTP fluorescence compared with ANT-ATP (Figs. 2F and H). Moreover, the relative
stimulatory effect of FS in the direct fluorescence assay at ex= 350 nm with ANTnucleotides was about two-fold larger than with MANT-nucleotides. The stimulatory effect
of FS on direct fluorescence of ANT-GTP bound to VC1:IIC2 was much greater than with
any other (M)ANT-nucleotide studied and amounted to about 2.5-fold.
3.3. Analysis of the interaction of VC1:IIC2 heterodimer with (M)ANT-nucleotides by
molecular modelling
Fig. 3 shows a model of the interactions of MANT-GTP, MANT-ATP, MANT-ITP,
MANT-XTP, MANT-UTP, MANT-CTP, ANT-GTP and ANT-ATP with the catalytic site
of VC1:IIC2 based on the crystal structure of VCI:IIC2 in complex with MANT-GTP [16].
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We predict that MANT-XTP and MANT-ITP share very similar binding modes with the
originally co-crystallized MANT-GTP, characterized by strong H-bonding between the base
and the side chains of Lys 839 and Asp 1018 within the base-binding subpocket (Fig. 3A).
In comparing ANT-GTP and MANT-GTP (Fig. 3B), essentially no significant difference is
predicting in the binding conformation. However ANT- ATP is predicted to adhere to a
substantially different binding mode than MANT-ATP, courtesy of a reorientation of the
F400 ring that enables the ANT-group to occupy space that was not available to the other
ligands. In accordance with this model, we did not observe significant basal or stimulated
FRET with MANT-ATP (Fig. 2G), a finding that is in clear contrast to the data obtained for
ANT-ATP (Fig. 2O), MANT-GTP (Fig. 2M) and ANT-GTP (Fig. 2E).
NIH-PA Author Manuscript
MANT-ATP and MANT-UTP engage in some H-bonding with this subpocket, but are
predicted to be appreciably weaker in their interactions (Figs. 3A and 3C). For MANT-UTP,
this weaker H-bonding permits the ligand to achieve stronger lipophilic interactions between
the non-polar (-CH=CH-) portion of the base and the receptor hydrophobic pocket around
Leu 438. MANT-CTP is predicted to completely forgo base H-bonding interactions in favor
of a combination of ethylene – Leu 438 interactions similar to MANT-UTP plus stronger
lipophilic coupling between the MANT-group and the receptor hydrophobic pocket.
Specifically, for MANT-CTP, the MANT aryl group interacts closely with Leu412, Leu416
and Trp1020 (above the plane of thus not shown), the methyl on the MANT methyl amine
group has a favorable interaction with Ala404, and the methyl amine proton donates an Hbond to the side chain carbonyl-O of Asn 1025 (above plane), whereas for all other ligands
the MANT group has substantially less stabilization. The stronger hydrophobic interactions
of MANT-CTP with the hydrophobic pocket translated into a larger FRET signal compared
to MANT-UTP (Figs. 2A and C).
3.4. Analysis of the interaction of VC1:IIC2 heterodimer with 2’- and 3’-MANT-nucleotides
by fluorescence spectroscopy
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The overall basal fluorescence signals of VC1:IIC2 bound to 2’-d-3’-MANT-GTP and 3’d-2’-MANT-GTP were similar to the signals of VC1:IIC2 in complex with MANT-GTP
(compare Figs. 4A, E and I, as well as B, F and J), but the magnitude of FS-stimulated
FRET signals at ex= 280 nm was higher with the defined isomers (2’-d-3’-MANT-GTP and
3’-d-2’-MANT-GTP) than with MANT-GTP. With adenine nucleotides, the position of the
ribosyl-substituent had a very different impact on the conformations of VC1:IIC2 stabilized
by these nucleotides than with guanine nucleotides (Figs. 4 C, D, G, H, K and L). 3’-d-2’MANT-ATP showed a particularly large basal FRET and direct fluorescence (Figs. 4 G and
H). Unexpectedly, FS (100 M) decreased FRET and direct fluorescence with 3’-d-2’MANT-ATP. In contrast, the basal FRET and direct fluorescence increase by VC1:IIC2
with 2’-d-3’-MANT-ATP was small as was the stimulatory effect of FS (Figs. 4K and 4L).
3.5. Analysis of the interaction of VC1:IIC2 heterodimer with 2’- and 3’-MANT-nucleotides
by molecular modelling
There are three families of potentially justifiable conformers for the 2’-MANT analogs: i)
Close analogs to the 3’-MANT conformation, but with the MANT ring flipped (Fig. 5). ii)
Poses, where the binding site of the 2’-MANT group and the nucleotide are switched (not
shown). iii) Poses, where the nucleotide maintains its binding site, but the 2-MANT group
couples instead with lipophilic groups on FS (not shown). Although we here only show the
poses from family i), the poses from families ii) and iii) were computed as having fairly
comparable docking free energies. Thus, additional ligand conformations cannot be
excluded. This may be particularly the case for 3’-d-2’-MANT-ATP, since in this case, in
contrast to all other nucleotides, FS did not increase FRET and direct fluorescence signals,
but rather decreased fluorescence (Figs. 4G and H). It is clear that the positioning of the
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nuceotides in the receptor is similar. Yet, it is also evident that there are subtle differences in
the positioning of the MANT-group with the different isomers, fitting to the different
fluorescence signals and Ki values.
3.6. Analysis of the interaction of VC1:VC1 homodimer with 2’- and 3’-MANT-nucleotides
by fluorescence spectroscopy
The basal FRET and direct fluorescence signals determined for the interaction of VC1:VC1
with MANT-GTP were similar as for the interaction of VC1:IIC2 with MANT-GTP
(compare Fig. 2M with Fig. 6A and Fig. 2N with Fig. 6B, respectively). However, a major
difference between both experimental settings is the fact that only in the presence of IIC2
stimulatory effects of FS were observed. IIC2 alone did not show fluorescence changes upon
incubation with MANT-nucleotides [22], indicative for the absence of a nucleotide-binding
site. With VC1:VC1, we observed much larger basal FRET and direct fluorescence bound to
3’-d-2’-MANT-GTP or 2’-d-3’-MANT-GTP than with VC1:IIC2 (Figs. 6C and D versus
Figs. 4E and F as well as Figs. 6E and F versus Figs. 4I and J). Again, with the VC1
homodimer, FS failed to increase fluorescence.
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Compared to basal direct fluorescence and direct FRET with VC1:IIC2 bound to MANTATP, the corresponding signals obtained with VC1:VC1 were much larger (compare Figs.
4C and D versus Figs. 6G and H). As was true for MANT-GTP, FS did not enhance the
fluorescence signals with MANT-ATP. Similar to the observations made for VC1:IIC2,
basal direct fluorescence and FRET of VC1:VC1 bound to 3’-d-2’-MANT-ATP were much
larger than the signals obtained with 2’-d-3’-MANT-ATP (compare Figs. 4G and H versus
Figs. 6I and J as well as Figs. 4K and L versus Figs. 6K and L). For several nucleotides,
most notably 3’-d-2’-MANT-ATP, FS reduced the fluorescence signal with VC1:VC1.
FRET from tryptophan residues to the (M)ANT-group entails that the increase in
fluorescence at the (M)ANT emission maximum at 420-450 nm should be accompanied by a
corresponding decrease in fluorescence at the tryptophan fluorescence emission maximum
of 350 nm [16]. However, we noticed that the extent of fluorescence decrease at em = 350
nm did not correlate with the increase in fluorescence at em = 450 nm. This was more
evident in the experiments with VC1:IIC2 than in experiments with VC1:VC1 (campare
Figs. 4 and 6). An explanation for these discrepancies could be that nucleotide binding to
VC1:IIC2 changes the endogenous tryptophan fluorescence properties of the protein which
is superimposed with the FRET signal. Changes in endogenous tryptophan fluorescence
have also been observed upon nucleotide binding to signal-transducing GTP-binding
proteins [39, 40].
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3.7. Analysis of the interaction of VC1:VC1 homodimer with 2’- and 3’-MANT-nucleotides
by molecular modelling
Fig. 7 shows the interactions of 2’-MANT- and 3’-MANT-ATP and -GTP isomers with
VC1:VC1. In the dimer, nucleotides are stabilized by hydrophobic and hydrophilic
interactions. Within a distance of 3-20 Å, the MANT-group is predicted to interact with
several lipophilic residues such as Ala409 and Leu413, as well as polar residues Gln410,
Ser408 and Asn509. Within the same distance, the MANT-group is predicted to interact
with tryptophan residues at positions 502 and 507 as well as tyrosine residues at positions
383, 442, 443, 535, 540 and 557 of VC1. These are the most likely amino acid residues
contributing to the pronounced FRET and direct fluorescence signals observed for MANTnucleotides bound to VC1:VC1, most notably 3’-d-2’-MANT-GTP (Fig. 6C), 2’-d-3’MANT-GTP (Fig. 6E) and 3’-d-2’-MANT-ATP (Figs. 6I and J). It is evident that the
positioning of the nucleotides in the receptor is similar. Yet, it is also clear that there are
subtle differences in the positioning of the MANT-group with the different isomers.
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3.8. Inhibition of the catalytic activity of recombinant ACs 1, 2 and 5 by (M)ANTnucleotides
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MANT-GTP S (4) and MANT-ITP S (9) were similarly potent AC5 inhibitors (Table 2).
Whereas substitution of the -thiophosphate by a -phosphate had no effect on potency in
case of guanine nucleotide (4→1), this substitution increased potency in case of
hypoxanthine nucleotides (9→8) by more than 25-fold, yielding MANT-ITP. MANT-UTP
was similarly potent as MANT-GTP, whereas introduction of adenine (5) or cytosine (12)
decreased affinity for AC5 by 2-3-fold relative to guanine (1). Among all bases studied,
xanthine (10) conferred the lowest inhibitor potency to MANT-NTPs. In case of guanine,
both the 2’-d-3’-MANT-substitution (2) and the 3’-d-2’-MANT-substitution (3)
substantially reduced inhibitor potency, whereas in case of adenine, only the 2’-d-3’-MANT
substitution (6) decreased inhibitor potency. Exchange of the MANT-group for an ANTgroup had little effect on inhibitor potency (5→14, 15→16 and 20→21). Deletion of the phosphate reduced inhibitor 5-30-fold (5→16, 8→17, 11→18 and 12→19) and deletion of
the -phosphate reduced inhibitor affinity almost 150-fold (17→20). Overall, with the
exception of 3’-d-2’-MANT-ATP (7), inhibitor affinities at AC1 resembled those at AC5.
Inhibitor affinities at AC2 were all lower than at ACs 1 and 5.
4. Discussion
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4.1. Analysis of the VC1:IIC2 heterodimer
The long-term goal of our group is the development of potent and isoform-specific AC
inhibitors. Such inhibitors could be useful drugs for the treatment of various diseases
including heart failure, chronic pain and neurodegeneration [6-12]. In order to achieve this
long-term goal, a systematic analysis of the structure-activity relationships of nucleotide
inhibitors is required. Accordingly, we examined 21 (M)ANT-nucleotides (Fig. 1) in terms
of inhibition of catalysis (Tables1 and 2), fluorescence spectroscopy (Figs. 2, 4 and 6) and
molecular modelling (Figs. 3, 5 and 7).
Overall, our data corroborate the tripartite pharmacophore model with a site for the base, the
ribose and ribosyl substituent, and the polyphosphate chain [16]. In agreement with our
previous data on 2’,3’-O-(2,4,6-trinitrophenyl)-substituted nucleotides [16, 20], the basespecificity of VC1:IIC2 is also very broad for 2’,3’-OMANT-substituted nucleotides with
the base preference being hypoxanthine > uracil > cytosine > adenine ~ guanine ≫ xanthine
under maximally stimulatory conditions and the preference hypoxanthine > uracil > adenine
~ guanine > cytosine ≫ xanthine under submaximally stimulatory conditions. Regardless of
the experimental conditions, at least with respect to inhibitors, adenine can no longer be
considered as “cognate” base for ACs.
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Our studies corroborate the importance of the -phosphate for high-affinity AC inhibition
[14]. Crystallographic studies showed that the Mn2+ ion in the B-site coordinates with the phosphate of MANT-nucleotides and that deletion of the -phosphate destabilizes the
polyphosphate chain in its binding site [16, 17]. In case of MANT-ITP and MANT-ITP S,
the bulky -thiophosphate impedes with coordination to the polyphosphate-binding region.
In the available crystal structures, the 3’-MANT-conformation is clearly favored relative to
the 2’-MANT conformation [16, 17, 23], but in terms of enzyme inhibition, this is not the
case, reflecting the conformational flexibility of VC1:IIC2. The lower affinity of defined 2’and 3’-MANT isomers for VC1:IIC2 compared to 2’,3’-MANT isomer mixtures with
respect to inhibition of catalysis is explained by the absent hydrogen bond between Asn1025
of IIC2 and the missing hydroxyl group in the defined 2’- and 3’-MANT isomers [17].
Finally, deletion of the methyl group of the MANT-substituent resulting in ANT-nucleotides
(1→13 and 5→15) had little effect on inhibitor-affinity.
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The analysis of the fluorescence signals obtained with (M)ANT-nucleotides bound to
VC1:IIC2 yielded important information on ligand/enzyme interaction. Most strikingly, the
addition of FS to VC1:IIC2 bound to 3’-d-2’-MANT-ATP reduced FRET and direct
fluorescence signals (Figs. 4G and H). These data indicate that for 3’-d-2’-MANT-ATP, the
hydrophobic pocket is already optimally preformed without the AC activator FS.
Preparation of the corresponding crystal structure is needed to elucidate the structural basis
for this unique effect. In contrast, for all all nucleotides, binding of FS to its specific
interaction site optimizes interaction of the (M)ANT-group with the hydrophobic pocket
adjacent to the catalytic site. These activator-dependent interactions of (M)ANT-nucleotides
with VC1:IIC2 open the possibility to develop catalysis-dependent AC inhibitors. Such
compounds may be quite interesting since the therapeutic goal will probably not be to
completely abrogate catalysis but only to abrogate pathologically relevant excessive
catalytic activity. Another indication that this ambitious goal is achievable is the fact that
full activation of VC1:IIC2 by Gsα-GTP S plus FS compared to FS alone differentially
increased inhibitor potencies, with the differences in potency increase differing by up to 25fold among various compounds. In this respect, ANT-ADP (15), originally only synthesized
as a rather uninformative “control compound”, turmed out to be the most interesting
inhibitor.
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The FRET and direct fluorescence signals obtained with (M)ANT-nucleotides did not
correlate with inhibitor-affinity. This can be illustrated by several examples. First, MANTGTP and MANT-ATP possess similar affinity, but the fluorescence signals with MANTGTP were much larger than with MANT-ATP Figs. 2M-P). A similar situation applies to the
pair ANT-GTP and ANT-ATP (Figs. 2E-H). Secondly, MANT-GTP and ANT-GTP exhibit
similar affinity as well, but with MANT-GTP, larger FRET signals were obtained (Figs. 2E
and M). The opposite was true for direct fluorescence signals (Figs. 2F and N). Third, 3’d-2’-MANT-ATP shows lower affinity for VC1:IIC2 than 2’-d-3’-MANT-ATP, but larger
fluorescence signals under basal conditions (Figs. 4G, H, K and L). As a last example, 3’d-2’-MANT-GTP and 2’-d-3’-MANT-GTP possess similar affinity for VC1:IIC2, but upon
exposure to FS, larger FRET signals were obtained with 2’-d-3’-MANT-GTP than with 3’d-2’-MANT-GTP (Figs. 4E and I). These data indicate that in terms of fluorescence
spectroscopy, each nucleotide imprints its specific signature on VC1:IIC2, reflecting unique
positioning of any given ligand into the tripartite pharmacophore. Our molecular modelling
data support this conclusion (Figs. 3 and 5). In other words, each ligand stabilizes a unique
conformational landscape in VC1:IIC2 with different functional consequences. These
properties of ligands and VC1:IIC2 will substantially facilitate the development of nonnucleotide-based inhibitors.
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4.2. Analysis of the VC1:VC1 homodimer
While the IIC2:IIC2 homodimer does not possess a functional nucleotide-binding site, the
VC1:VC1 homodimer does [22, 42]. Evidence for the latter notion comes for the highaffinity (although exceedingly low-efficacy) catalytic activity of VC1:VC1 and substantial
fluorescence increases of MANT-GTP as well as 2’,3’-O-(2,4,6-trinitrophenyl)-ATP upon
binding to VC1:VC1 [22]. In this study we show that, most impressively, the basal FRET
and direct fluorescence signals obtained with MANT-ATP, 3’-d-2’-MANT-ATP and 3’d-2’-MANT-ATP largely exceed the signals obtained with the VC1:IIC2 heterodimer (Figs.
4 and 6), indicative for a formation of a binding site with better fit for the respective
fluorescent nucleotides in the former receptor than in the latter one. We generated a
homology model of the VC1:VC1 homodimer based on a VC1:IIC2 crystal structure [35],
and this model also reveals a tripartite pharmacophore with a site for the base, a prominent
hydrophobic site for the MANT-group, resulting in large FRET and fluorescence signals,
and a site for the polyphosphate chain (Fig. 7). The model also shows that the MANT-
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groups of the different nucleotides are differentially positioned in the VC1:VC1 homodimer,
again pointing to a specific conformational landscape for each nucleotide.
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Intriguingly, with 3’-d-2’-MANT-ATP as probe, FS reduced fluorescence signals in the
VC1:IIC2 heterodimer and the VC1:VC1 homodimer (Figs. 4G and H and Figs. 6I and J),
pointing to some similarity in the interaction of the nucleotides with both receptors. In
contrast to the data obtained for the heterodimer, FS reduced fluorescence signals, most
notably FRET signals, in the VC1:VC1 homodimer bound to most other nucleotides.
Collectively, our data show that the nucleotide-binding site formed by the VC1:VC1
homodimer possesses different regulatory properties and structure-activity relationships for
nucleotides. It is conceivable that in vivo, C1:C1 homodimers could form through
interaction of the corresponding subunits of neighboring AC molecules [22]. The function of
such homodimers may be to ensure a low basal AC activity and effective stimulation of
catalysis upon subsequent formation of the catalytically far more active C1:C2 heterodimer.
Accordingly, the nucleotide-binding site formed by AC C1:C1 homodimers may constitute a
novel regulatory site that should be considered in the future design of AC inhibitors.
Blockade of the nucleotide-binding site of the C1:C1 homodimer may result in elevated
basal AC activity. Whether such activity is desired or not cannot yet be answered since to
this end, very little attention has been paid to the possible (patho)physiological relevance of
basal AC activity. However, it is clear that AC2 possesses a particularly high basal AC
activity that can be inhibited by certain FS derivatives [4, 43]. Since the catalytic activity of
the VC1:VC1 homodimer is so exceedingly low, the analysis of the homodimer by
fluorescence spectroscopy constitutes a feasible approach to study the newly identified
nucleotide-binding site in this protein. However, it should be noted that such analysis by
fluorescence spectroscopy is also not trivial since high concentrations of VC1 are required
and since purification of this protein is much more difficult than purification of IIC2 both in
terms of protein yield and protein stability.
4.3. Analysis of recombinant holo-ACs
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In accordance with previous data [14, 21, 22], we corroborated the generally lower
sensitivity of AC2 to (M)ANT-nucleotide inhibitors compared to ACs 1 and 5. These
difference can be partially explained by the Ala409Pro- and Val1108Ile exchanges in ACs 1
and 5 versus AC2. [16]. However, we also noted nucleotide-dependent differences in
potency between the various AC isoforms. Specifically, the potency difference of MANTGTP, MANT-UTP and 2’-d-3’-MANT-ATP amounted to >10-fold for comparison of AC5
versus AC2, whereas the smallest difference was observed for MANT-IMP (20) and ANTIMP (21) (smaller than two-fold). Again, like in the case of ANT-ADP (15) and VC1:IIC2,
“control compounds” of supposedly low interest, turned out to be much more informative
than originally assumed. These data indicate that in principle, it should be possible to
develop AC2-selective inhibitors. Such compounds should not exploit the polyphosphatebinding site and, therefore, should be of non-nucleotide structure.
Overall, the structure-activity relationships of VC1:IIC2 and holo-ACs 1, 2 and 5 are similar
in terms of broad base-specificity, preference for hypoxanthine and uracil as bases and
importance of the polyphosphate chain for high inhibitor affinity. However, we also noted
some interesting differences between VC1:IIC2 and holo-ACs. Particularly, VC1:IIC2 did
not exhibit prominent preference for 3’-MANT isomers compared to 2’-MANT-isomers in
terms of potency of enzyme inhibition, although in the crystals, the 3’-MANT isomers are
clearly favored [16, 17, 23]. However, crystals are static structures, and the catalytically
active VC1:IIC2 is a conformationally flexible and “breathing” protein, largely
compensating for constraints observed in static structures. In contrast, holo-ACs exhibited
more marked preference for 2’-d-3’-MANT-GTP compared to 3’-d-2’-MANT-GTP. This
difference was also observed for the 3’-MANT- and 2’-MANT isomers of ATP, except for
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AC1. These data indicate that the presence of the transmembrane domains in holo-ACs,
missing in the fully soluble VC1:IIC2 system, imposes a rigidifying effect on the catalytic
sites in ACs 1 and 5, and also, to a somewhat smaller extent, on AC1, increasing the
expected preference of the AC isoforms for 3’-MANT isomers based on static crystal
structures.
Unfortunately, nucleotides per se are not useful as therapeutic compounds since they do not
penetrate the plasma membrane [12, 44]. The design of pronucleotides, converted to the
actually active compounds in intact cells, may constitute a solution to the problem [44].
Alternatively, non-nucleotide-based isoform-selective AC inhibitors may be designed.
Although this goal is certainly ambitious, it is not totally elusive. This is illustrated by the
case of MANT-IDP (17) and MANT-IMP (20), once again, originally thought to be “control
compounds” of little interest. Usually, the deletion of the - and -phosphate decreases AC
inhibitor-affinity dramatically. However, MANT-IDP possesses still the same inhibitory
potency as MANT-UTP (11), and the inhibitory potency of MANT-IMP is still in the low
M-range. Hence, provided that the base- and ribosyl-binding domains are optimally
exploited with feasible structural elements, the polyphosphate-binding domain may be
dispensible for inhibitor design, yielding the desired non-nucleotide compounds.
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The high-affinity interactions of MANT-UTP (11) and MANT-CTP (12) with ACs are quite
amazing. In fact, under certain experimental conditions, these nucleotides even surpass
MANT-ATP in terms of affinity although adenine is the “cognate” base for ACs. Previously,
we had reported high-affinity interactions of 2’,3’-O-(2’,4’,6’-trinitrophenyl)-substituted
uracil- and cytosine nucleotides with various ACs. Even UTP and CTP bind to
submaximally and maximally activated VC1:IIC2 with similar affinity as GTP [18]. Hence,
ACs do not possess a striking specificity for the “cognate” base adenine. Collectively, all
these data raise the most intriguing question whether ACs, in addition to cAMP, can also
catalyze the production of cCMP and cUMP. In other words; are ACs rather purinylyl- and
pyrimidinylyl cyclase with a broader substrate-specificity than generally assumed? In case
of the highly active “AC” toxins CyaA from Bordetella pertussis and edema factor from
Bacillus anthracis, it has already recently been shown that these proteins can also catalyze
the formation of cCMP and cUMP [45].
4.4. Conclusions and future studies
NIH-PA Author Manuscript
Our present study provided a comprehensive analysis of the structure-activity relationships
of (M)ANT-nucleotides for AC inhibition, analysis of the fluorescence properties of
nucleotides bound to AC and molecular modelling of the interactions of (M)ANTnucleotides with AC. Similar structure-activity relationships as for VC1:IIC2 were obtained
for recombinant ACs 1, 2 and 5, with AC2 being the least sensitive AC isoform in terms of
inhibition. Overall, ACs possess a broad base-specificity with no preference for the
“cognate” base adenine as verified by enzyme inhibition, fluorescence spectroscopy and
molecular modelling. These properties of ACs are indicative for ligand-specific
conformational landscapes.
Our results yield numerous directions for future studies. First, 2’-MANT- and 3’-MANTisomers of MANT-ITP, MANT-CTP and MANT-UTP as well as ANT-pyrimidine
nucleotides, all of which have not yet been synthesized, should be examined. Second,
various homologous combinations of the C1- and C2 subunits of ACs should be studied, but
the expression of such functionally active subunits is not trivial. Third, the crystal structure
of the VC1:VC1 homodimer bound to some of the nucleotides analyzed herein should be
analyzed to better understand the structural basis for the strong basal fluorescence signals
and the inhibitory effects of FS on fluorescence. Fourth, different AC isoforms than ACs 1,
2 and 5 should be examined. Again, functional expression of such isoforms in Sf9 cells is
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not easy. Fifth, the broad base-specificity of AC inhibitors urgently calls for a systematic
analysis of the substrate-specificity of ACs. Sixth, a systematic analysis of the activitydependence of AC inhibitors is required, aiming at the identification of compounds that
selectively inhibit pathologically high enzyme activity while leaving unaffected
physiologically relevant catalysis. Seventh, our studies will facilitate the analysis of the
interaction of diterpenes with AC. Diterpenes interact with AC according to a two-step
model [46]. In the first step, diterpenes bind to AC, and in the second step, they induce or
stabilize a conformational change that initiates catalysis. To this end, the analysis of
conformational changes in AC by diterpenes has only been conducted with MANT-GTP as
fluorescence sensor [46]. However, MANT-GTP is not the most sensitive sensor in this
regard, and is clearly surpassed by ANT-GTP for the analysis of direct fluorescence changes
and 2’-d-3’-MANT-GTP for FRET (compare Figs. 2F and N and Fig. 4I). Moreover, 3’d-2’-MANT-ATP provides a novel probe to monitor inhibitory effects of diterpenes on the
nucleotides bound to VC1:IIC2 (Figs. 4G and H). These studies will also help us understand
mechanisms of AC regulation in more general terms. Lastly, concerning future design of
both inhibitors and activators for ACs, much can be learned from the analysis of G-proteincoupled receptors. Specifically, much evidence has been accumulated in favour of the
existence of complex conformational landscapes that are being modulated in a ligandspecific manner by orthosteric and allosteric ligands, resulting highly complex signal
transduction outcomes [47-50].
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All in all, the fields of ligand design for G-protein-coupled receptors and ACs, having
developed as completely separate entities for a long time, could be merged, perhaps even
with receptors modulating the pharmacological properties of ACs and vice versa. Recently,
we have identified inhibitors with a 100-fold selectivity for the bacterial AC toxin CyaA
from Bordetella pertussis relative to mammalian ACs [41]. These data show that in
principle, it is possible to obtain isoform-selective AC inhibitors. Ultimately, this is the
long-term goal of our research program.
Acknowledgments
This work was supported by Deutsche Forschungsgemeinschaft research grant Se 529/5-2 to R.S. and NIH grant
2R56 DK46371-14 to S.R.S. Thanks are due to the reviewers for their helpful critique.
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Abbreviations
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AC
membranous adenylyl cyclase
ANT
anthraniloyl-
FRET
fluorescence resonance energy transfer
FS
forskolin
GTPγS
guanosine 5’-[ -thio]triphosphate
MANT
methylanthraniloyl-
NDP
nucleoside 5′-diphosphate
NTP
nucleoside 5′-triphosphate. Specific fluorescent nucleotides studied were: 2’,3’O-(N-methylanthraniloyl)-guanosine 5’-triphosphate (MANT-GTP), 2’deoxy-3’-O-(N-methylanthraniloyl)-guanosine 5’-triphosphate (2’-d-3’-MANTGTP), 3’-deoxy-2’-O-(N-methylanthraniloyl)-guanosine 5’-triphosphate (3’d-2’-MANT-GTP), 2’,3’-O-(N-methylanthraniloyl)-guanosine 5’-[ thio]triphosphate (MANT-GTP S), 2’,3’-O-(N-methylanthraniloyl)-adenosine
5’-triphosphate (MANT-ATP), 2’-deoxy-3’-O-(N-methylanthraniloyl)adenosine 5’-triphosphate (2’-d-3’-MANT-ATP), 3’-deoxy-2’-O-(Nmethylanthraniloyl)-adenosine 5’-triphosphate (3’-d-2’-MANT-ATP), 2’,3’-O(N-methylanthraniloyl)-uridine 5’-triphosphate (MANT-UTP), 2’(3’)-O-(Nmethylanthraniloyl)-cytidine 5’-triphosphate (MANT-CTP), 2’,3’-O-(Nmethylanthraniloyl)-inosine 5’-triphosphate (MANT-ITP), 2’,3’-O-(Nmethylanthraniloyl)-inosine 5’-[ -thio]triphosphate (MANT-ITP S), 2’,3’-O(N-methylanthraniloyl)-xanthosine 5’-triphosphate (MANT-XTP), 2’,3’-Oanthraniloyl-guanosine 5’-triphosphate (ANT-GTP), 2’,3’-O-anthraniloyladenosine 5’-triphosphate (ANT-ATP), 2’,3’-O-anthraniloyl-adenosine 5’diphosphate (ANT-ADP), 2’,3’-O-(N-methylanthraniloyl)-guanosine 5’diphosphate (MANT-GDP), 2’,3’-O-(N-methylanthraniloyl)-adenosine 5’diphosphate (MANT-ADP), 2’,3’-O-(N-methylanthraniloyl)-inosine 5’diphosphate (MANT-IDP), 2’,3’-O-(N-methylanthraniloyl)-uridine 5’diphosphate (MANT-UDP), 2’,3’-O-(N-methylanthraniloyl)-cytidine 5’diphosphate (MANT-CDP), 2’,3’-O-(N-methylanthraniloyl)-inosine 5’monophosphate (MANT-IMP)
Biochem Pharmacol. Author manuscript; available in PMC 2012 August 15.
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Fig. 1. Structures of 2’,3’-O-ribosyl-modified nucleotides
Represented are the three pharmacophores contributing to the inhibitor potencies of these
nucleotides, i.e. the base, the tri- or diphosphate chain and the (M)ANT group. Studied
nucleotides differed from each other in the base (guanine, hypoxanthine, xanthine, adenine,
uracil and cytidine), -phosphate chain substitution (phosphate or thiophosphate), phosphate
chain length (5’-triphosphate, 5’-diphosphate or 5’-monophosphate analogs), ribosyl
substituent (MANT or ANT), and in the position of the MANT-group (2’- and 3’-MANT).
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Fig. 2. Fluorescence emission spectra of (M)ANT-nucleotides bound to VC1:IIC2 heterodimer
NIH-PA Author Manuscript
Shown are representative fluorescence emission spectra of MANT-CTP, MANT-UTP,
ANT-GTP, ANT-ATP, MANT-ITP, MANT-XTP, MANT-GTP and MANT-ATP at ex =
280 nm ( em = 300-500 nm) (Panels A, C, E, G, I, K, M and O, respectively) and at ex =
350 nm ( em = 370-500 nm) (Panels B, D, F, H, J, L, N and P, respectively). Experiments
were conducted in the presence of different (M)ANT-nucleotides (1 M each), VC1 (5 M)
and IIC2 (25 M) without FS and with FS (100 M). Fluorescence measurements were
performed in a quartz fluorescence microcuvette using a Cary Eclipse fluorescence
spectrophotometer at 25°C as described under “Materials and Methods”. Reaction mixture
contained 100 mM KCl, 10 mM MnCl2, 25 mM HEPES/NaOH, pH 7.4. The final assay
volume was 150 l, and the final DMSO concentration was 3% (vol/vol). Fluorescence
intensities are shown in arbitrary units. In FRET experiments, 100% of fluorescence
intensity was defined as the maximum signal obtained with VC1:IIC2 alone. In direct
fluorescence experiments, 100% of fluorescence intensity was defined as the signal obtained
with nucleotide alone. Fluorescence tracings are representative for two to three independent
experiments with at least two different batches of VC1:IIC2. Order of addition: Blue
tracings, addition of nucleotide; green tracings, addition of VC1:IIC2; red tracings, addition
of forskolin.
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Fig. 3. Model of the interaction of VC1:IIC2 heterodimer with (M)ANT-nucleotides
Computationally predicted conformations for (MA)NT nucleotides bound to VC1:IIC2
comparing A, all MANT-nucleotides; B, (M)ANT-ATP vs. (M)ANT-GTP; and C, MANTCTP vs. MANT-UTP. Atoms of each ligand and two key receptor residues are represented
as sticks (MANT ligands are thick sticks; ANT are thin) according to standard CPK
coloring, except for carbon atoms which are colored as follows: (M)ANT-GTP = grey,
(M)ANT-ATP = green, MANT-CTP = slate blue, MANT-ITP = black, MANT-UTP =
brown, MANT-XTP = pink. The receptor surface is represented as a solvent-accessible
Connolly surface, colored as follows: lipophilic regions are yellow, polar oxygens are red,
polar nitrogens are blue, donatable protons are cyan, and polarized alkyl or aryl moieties are
white. Approximate locations of the alpha carbons of key residues are labeled for reference.
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Fig. 4. Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to
VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP
NIH-PA Author Manuscript
Shown are representative fluorescence emission spectra of MANT-GTP, MANT-ATP, 3’d-2’-MANT-GTP, 3’-d-2’-MANT-ATP, 2’-d-3’-MANT-GTP and 2’-d-3’-MANT-ATP at
ex = 280 nm ( em = 300-500 nm) (Panels A, C, E, G, I and K, respectively) and at ex = 350
nm ( em = 370-500 nm) (Panels B, D, F, H, J and L, respectively). Experiments were
conducted in the presence of different MANT-nucleotides (1 M each), VC1 (5 M) and
IIC2 (25 M) without FS and with FS (100 M). Fluorescence measurements were
performed in a quartz fluorescence microcuvette using a Cary Eclipse fluorescence
spectrophotometer at 25°C as described under “Materials and Methods”. Reaction mixture
contained 100 mM KCl, 10 mM MnCl2, 25 mM HEPES/NaOH, pH 7.4. The final assay
volume was 150 l, and the final DMSO concentration was 3% (vol/vol). Fluorescence
intensities are shown in arbitrary units. In FRET experiments, 100% of fluorescence
intensity was defined as the maximum signal obtained with VC1:IIC2 alone. In direct
fluorescence experiments, 100% of fluorescence intensity was defined as the signal obtained
with nucleotide alone. Fluorescence tracings are representative for two to three independent
experiments with at least two different batches of VC1:IIC2. Order of addition: Blue
tracings, addition of nucleotide; green tracings, addition of VC1:IIC2; red tracings, addition
of forskolin.
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Fig. 5. Model of the interation of VC1:IIC2 heterodimer with 2’-MANT- and 3’-MANTnucleotides
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Computationally predicted conformations for the 2’-MANT-and 3’-MANT isomers of ATP
and GTP bound to VC1:IIC2 are shown. The heavy sticks represent the 3’-MANT analogs
(CPK atomic colors, with the molecules color codes as follows: grey carbons = MANTGTP; green carbons = MANT-ATP) whereas the thinner sticks depict the 2’-MANT species.
The AC receptor is depicted in cartoon form via red helices, yellow sheets and green coils,
and the two metal cations are represented as magenta spheres. Approximate locations of the
alpha carbons of key residues are labeled for reference.
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Fig. 6. Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to
VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP
NIH-PA Author Manuscript
Shown are representative fluorescence emission spectra of MANT-GTP, MANT-ATP, 3’d-2’-MANT-GTP, 3’-d-2’-MANT-ATP, 2’-d-3’-MANT-GTP and 2’-d-3’-MANT-ATP at
ex = 280 nm ( em = 300-500 nm) (Panels A, C, E, G, I and K, respectively) and at ex = 350
nm ( em = 370-500 nm) (Panels B, D, F, H, J and L, respectively). Experiments were
conducted in the presence of different MANT-nucleotides (1 M each) and VC1 (5 M)
without FS and with FS (100 M). Fluorescence measurements were performed in a quartz
fluorescence microcuvette using a Cary Eclipse fluorescence spectrophotometer at 25°C as
described under “Materials and Methods”. Reaction mixture contained 100 mM KCl, 10
mM MnCl2, 25 mM HEPES/NaOH, pH 7.4. The final assay volume was 150 l, and the
final DMSO concentration was 3% (vol/vol). Fluorescence intensities are shown in arbitrary
units. In FRET experiments, 100% of fluorescence intensity was defined as the maximum
signal obtained with VC1:VC1 alone. In direct fluorescence experiments, 100% of
fluorescence intensity was defined as the signal obtained with nucleotide alone.
Fluorescence tracings are representative for two to three independent experiments with at
least two different batches of VC1:VC1. Order of addition: Blue tracings, addition of
nucleotide; green tracings, addition of VC1:VC1; red tracings, addition of forskolin.
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Fig. 7. Model of the interation of VC1:VC1 homodimer with 2’-MANT- and 3’-MANTnucleotides
NIH-PA Author Manuscript
Computationally predicted conformations for the 2’-MANT-and 3’-MANT isomers of ATP
and GTP bound to VC1:VC1 are shown. The heavy sticks represent the 3’-MANT analogs
(CPK atomic colors, with the molecules color codes as follows: grey carbons = MANTGTP; green carbons = MANT-ATP) whereas the thinner sticks depict the 2’-MANT species.
The AC receptor is depicted in cartoon form via red helices, yellow sheets and green coils,
and the two metal cations are represented as magenta spheres. Approximate locations of the
alpha carbons of key residues are labeled for reference.
Biochem Pharmacol. Author manuscript; available in PMC 2012 August 15.
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Table 1
Inhibition of the catalytic activity of VC1:IIC2 by (M)ANT-nucleotides
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VC1:IIC2 Mn2++ FS + Gsα-GTPγSKi (nM)
(M)ANT-nucleotide
VC1:IIC2 Mn2+ + FS Ki (nM)
1
MANT-GTP
18 ± 6.0
130 ± 20
2
2’-d-3’-MANT-GTP
180 ± 6.1
890 ± 180
3
3’-d-2’-MANT-GTP
350 ± 43
1,200 ± 74
4
MANT-GTP S
24 ± 4.1
N.D.
5
MANT-ATP
16 ± 6.4
100 ± 27
6
2’-d-3’-MANT-ATP
190 ± 3.4
2,100 ± 160
7
3’-d-2’-MANT-ATP
90 ± 2.2
1,200 ± 100
8
MANT-ITP
0.7 ± 0.1
7.0 ± 3.2
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9
MANT-ITP S
19 ± 3.3
N.D.
10
MANT-XTP
1,200 ± 370
4,600 ± 510
11
MANT-UTP
6.1 ± 1.3
58 ± 8.4
12
MANT-CTP
9.2 ± 1.5
260 ± 25
13
ANT-GTP
10 ± 1.7
160 ± 45
14
ANT-ATP
17 ± 2.4
180 ± 57
15
ANT-ADP
250 ± 12
21,000 ± 1,600
16
MANT-ADP
260 ± 40
2,400 ± 110
18
MANT-UDP
170 ± 27
4,700 ± 1,200
19
MANT-CDP
140 ± 22
1,500 ± 75
Catalytic activities of VC1:IIC2 were determined as described in “Materials and Methods”. Reactions were conducted in the presence of 10 mM
MnCl2 and 100 M FS in the absence or presence of Gsα-GTP S. Data were analyzed by non-linear regression to calculate Ki values. The
catalytic activity of VC1:IIC2 in the presence of Mn2++ FS+ Gsα-GTP S with 100 M ATP as substrate was 2,700 ± 350 nmol/mg/min and in the
presence of Mn2++ FS, the activity was 300 ± 110 nmol/mg/min. The Km values for VC1:IIC2 were previously reported for each experimental
condition (430 and 620 M, respectively) [16] and were used to calculate Ki values from IC50 values. Data are given in nM and are the mean
values ± SD of 2-4 independent experiments performed in duplicates with at least two different batches of protein. N.D., not determined. Data for
1, 8 and 10 were taken from Ref. 23. Data for the other nucleotides shown in the table were obtained in parallel with those for 1, 8 and 10 so that
direct comparison is appropriate.
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Table 2
Inhibition of the catalytic activity of recombinant ACs 1, 2 and 5 by (M)ANT-nucleotides
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(M)ANT-nucleotide
AC1 (nM)
90 ± 18
AC2 (nM)
AC5 (nM)
610 ± 70
53 ± 12
1
MANT-GTP
2
2’-d-3’-MANT-GTP
270 ± 30
1300 ± 210
410 ± 35
3
3’-d-2’-MANT-GTP
1,800 ± 70
8,700 ± 1,800
1,800 ± 90
4
MANT-GTP S
63 ± 17
370 ± 81
34 ± 8
5
MANT-ATP
150 ± 40
330 ± 80
100 ± 30
6
2’-d-3’-MANT-ATP
320 ± 19
4,800 ± 560
360 ± 54
7
3’-d-2’-MANT-ATP
470 ± 20
540 ± 20
65 ± 5
8
MANT-ITP
2.8 ± 0.9
14 ± 0.5
1.2 ± 0.1
9
MANT-ITP S
40 ± 11
120 ± 23
32 ± 8
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10.
MANT-XTP
1,100 ± 100
3,000 ± 200
1,300 ± 400
11
MANT-UTP
46 ± 4.0
460 ± 60
32 ± 2.0
12
MANT-CTP
150 ± 30
690 ± 20
150 ± 30
14
ANT-ATP
130 ± 20
640 ± 70
120 ± 20
15
ANT-ADP
860 ± 10
2,900 ± 320
640 ± 70
16
MANT-ADP
1,300 ± 200
2,900 ± 500
790 ± 180
17
MANT-IDP
39 ± 12
86 ± 9.1
31 ± 12
18
MANT-UDP
390 ± 50
2,700 ± 300
340 ± 10
19
MANT-CDP
580 ± 10
3,700 ± 420
740 ± 30
20
MANT-IMP
8,500 ± 500
6,800 ± 800
4,400 ± 200
21
ANT-IMP
7,400 ± 1,200
7,500 ± 1,400
4,300 ± 600
AC activity in Sf9 membranes were determined as described in “Materials and Methods”. Reactions were conducted in the presence of 5 mM
MnCl2 and 100 M FS. Data were analyzed by non-linear regression to calculate Ki values. The Km values were 120 M (AC1), 100 M (AC2)
and 70 M (AC5) and were used to calculate Ki values from IC50 values. Data are given in nM and are the mean values ± SD of 4-5 independent
experiments performed in duplicates with at least two different membrane preparations. Data for 5, 8 and 12, 17 and 21 were taken from Ref. 41.
Data for the other nucleotides shown in the table were obtained in parallel with those for 5, 8 and 12, 17 and 21 so that direct comparison is
appropriate.
NIH-PA Author Manuscript
Biochem Pharmacol. Author manuscript; available in PMC 2012 August 15.