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J Med Chem. Author manuscript; available in PMC 2011 April 26.
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Published in final edited form as:
J Med Chem. 2008 August 14; 51(15): 4456–4464. doi:10.1021/jm800481q.
Structure-Based Development of Novel Adenylyl Cyclase
Inhibitors
Christine Schlicker†,§, Annika Rauch†,§, Ken C. Hess‡,§, Barbara Kachholz†, Lonny R.
Levin‡, Jochen Buck‡, and Clemens Steegborn*,†
Department of Physiological Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801
Bochum, Germany, Department of Pharmacology, Weill Medical College of Cornell University,
1300 York Avenue, New York, New York 10065
Abstract
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In mammals, the second messenger cAMP is synthesized by a family of transmembrane isoforms
(tmACs) and one known cytoplasmic enzyme, “soluble” adenylyl cyclase (sAC). Understanding
the individual contributions of these families to cAMP signaling requires tools which can
distinguish them. Here, we describe the structure-based development of isoform discriminating
AC inhibitors. Docking calculations using a library of small molecules with the crystal structure of
a sAC homologue complexed with the noncompetitive inhibitor catechol estrogen identified two
novel inhibitors, 3,20-dioxopregn-4-en-21-yl 4-bromobenzenesulfonate (2) and
1,2,3,4,5,6,7,8,13,13,14,14-dodecachloro-1,4,4a,4b,5,8,8a,12b-octahydro-11-sulfo-1,4:5,8dimethanotriphenylene-10-carboxylic acid (3). In vitro testing revealed that 3 defines a novel AC
inhibitor scaffold with high affinity for human sAC and less inhibitory effect on mammalian
tmACs. 2 also discriminates between sAC and tmACs, and it appears to simultaneously block the
original binding pocket and a neighboring interaction site. Our results show that compounds
exploiting the catechol estrogen binding site can produce potent, isoform discriminating AC
inhibitors.
Introduction
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The ubiquitous second messenger cAMP regulates a diverse set of essential biological
processes in mammals,1 and its dysfunction contributes to a variety of human diseases. In
mammals, it is generated by two families of enzymes from the class III adenylyl cyclase
superfamily (AC; E.C. 4.6.1.1).2,3 A family of transmembrane ACsa is encoded by nine
distinct genes (tmACs AC1 to AC9), and a second family of cytoplasmic enzymes, referred
to as “soluble” ACs (sAC), is generated by alternative splicing of a single gene.2,4,5 The
tmACs play key roles in cellular responses to extracellular signals:1 they are regulated
through heterotrimeric G-proteins in response to the stimulation of G-protein coupled
receptors (GPCRs). sAC enzymes, in contrast, are directly activated by calcium and by the
cellular metabolites bicarbonate and ATP6,7 thus, sAC has been postulated to act as an
intracellular metabolic sensor.8
*
To whom correspondence should be addressed. Phone: (49)(234)3227041. Fax: (49)(234)3214193. Clemens.Steegborn@rub.de.
Address: Ruhr-University Bochum, Department of Physiological Chemistry MA 2/141, Universitätsstrasse 150, 44801 Bochum,
Germany.
†Department of Physiological Chemistry, Ruhr-University Bochum
‡Department of Pharmacology, Weill Medical College of Cornell University
§These authors contributed equally to this work.
aAbbreviations: α, -Me-ATP, α, -methylene-ATP; AC, adenylyl cyclase; C, catalytic domain; CE, catechol estrogen; GST,
glutathione S-alkyl transferase; MANT, methylanthranyloyl; sAC, soluble adenylyl cyclase; tmAC, transmembrane adenylyl cyclase.
Schlicker et al.
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All known mammalian class III ACs are comprised of two related catalytic domains, C1 and
C2, and the crystal structure of a tmAC enzyme revealed that these domains are structurally
very similar.9 The C1/C2 heterodimer therefore resembles a homodimer, and the shared
active site at the dimer interface has a pseudosymmetric site that is catalytically inactive.
Sequence conservations and the crystal structure of the cyano-bacterial sAC homologue
CyaC showed that sAC enzymes, despite their unique regulation, have the same overall
structure as tmACs and employ the same two-metal ion mechanism for catalysis.9–11 The
active site at the dimer interface contains two magnesium ions in the so-called ion A and ion
B sites. Ion A acidifies the ribose 3′ hydroxyl and stabilizes the transition state, while ion B
serves as an anchoring point for the ATP - and -phosphates.10,11
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Because of the biological importance of cAMP signal transduction, it is essential to
understand the processes that regulate the cellular levels of this second messenger. There has
been significant progress toward development of pharmacological tools to distinguish
among the various phosphodiesterases, the enyzme responsible for cAMP degradation.12 In
contrast, there has been much less progress toward the development of specific tools to
distinguish among the various means to synthesize cAMP. The limited recent progress
toward development of specific AC inhibitors has not yet yielded compounds with both high
potency and high selectivity.13,14 Also, pharmacological modulation of tmAC isoforms has
been proposed for treatment of acute heart failure,14 whereas sAC is a target for
contraception15 and possibly for the treatment of hypercalciuria.16 The most widely
characterized class of AC inhibitors are the so-called P-site inhibitors, nucleotide analogues
that occupy the binding site for the substrate ATP.17 P-sites show a moderate AC isoform
specificity and bear the potential to bind to many cellular nucleotide binding proteins.18,19 A
second inhibitor class contains an adenine linked to ion chelators. In general, this class
suffers from the same predicament;20 however, a new compound series from this class can
discriminate between some tmAC isoforms.21 Related AC inhibitors are derivatives of 9-(2phosphonylmethoxyethyl)adenine,22 but these were initially identified as potent inhibitors of
nucleotide polymerases.22 A distinct nucleotide modification, the methylan-thranyloyl
(MANT) group in MANT-GTP, MANT-inosine-5′-( -thio)triphosphate and other
nucleotides, showed promising discrimination between tmACs and sAC.19 The MANT
group makes MANT-GTP a potent tmAC inhibitor, whereas sAC and, surprisingly, even
heterotrimeric G-proteins show little sensitivity against this compound.19 The MANT
nucleotides show little selectivity among tmAC isoforms,19 but they may be useful
complements to the sAC-selective compound, KH7, which was identified in a chemical
screen.23 The nucleotide part of MANT-GTP occupies the ATP binding site, but the MANT
group binds to a less conserved pocket at the C1/C2 interface,24 suggesting that varying the
group in this pocket may prove to be an attractive approach for achieving greater isoform
specificity. The same holds true for the binding pocket for the noncompetitive inhibitors of
the catechol estrogen (CE) family. These compounds bind close to the AC catalytic site and
inhibit sAC and tmACs by chelating the catalytic ion A.25 The residues lining the binding
pocket show little conservation between mammalian AC isoforms; therefore, we postulated
that exploiting this binding site may yield more specific compounds.
Here, we describe the structure-based development of novel AC inhibitors that exploit the
CE binding site. Using the crystal structure of an AC/CE complex, we searched a compound
library through docking calculations. Potential inhibitors were characterized in vitro for their
inhibition potency and specificity. Two novel compounds were identified that inhibit sAC
but show little to no inhibitory effect on a variety of transmembrane ACs. These inhibitors
reveal a novel AC inhibitor scaffold and a substituent that enables a steroid-based compound
to exploit an additional binding site.
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Results
Identification of Novel AC Inhibitors through Docking Calculations
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For the identification of novel inhibitors of class III AC enzymes, we started with the crystal
structure of the catalytic core of CyaC, a cyanobacterial homologue of human sAC, bound to
the CE compound 2-hydroxy-17 -estradiol (1; Figure 1a), a nonselective inhibitor (PDB
entry 2BW7).25 In this structure of CyaC with the ATP analogue α, -Me-ATP (α, methylene-ATP) and the noncompetitive inhibitor 1, the catalytic magnesium ion A is
moved out of its proper location due to a chelating interaction with the inhibitor hydroxyl
groups (Figure 1b). This structure further displays a partial active site closure upon inhibitor
binding. To identify other compounds that can exploit the binding site of 1, we performed
our docking study with this partially closed protein structure; the inhibitor 1 was removed,
but the substrate analogue and the divalent active site ions (ion A: Mg2+; ion B: Ca2+) were
included in the target structure. The ligand target site was defined as a cube covering the
binding site of 1 as well as the complete ATP binding pocket. We tested the system by
redocking 1 into the protein. This control resulted in a complex close to the experimental
structure (rmsd 0.3–0.5 Å for 8 of the 9 top solutions).
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For identifying potential novel inhibitor leads, we screened the NCI diversity set
(http://www.dtp.nci.nih.gov) of 1990 structurally diverse organic compounds. The
compound structures were docked into the CyaC target site by using the genetic algorithm of
AutoDock 3.0.26 The 17 hits with highest predicted affinity (Table 1), except for hit number
10 (compound 11), which is an alkylating agent, were then tested in an in vitro activity assay
for inhibition of CyaC. Six compounds showed significant inhibition at or below 250 μM,
corresponding to a high hit rate of 38%, comparable to hit rates in other high-throughput
docking studies.27,28 Five of these six hits were among the nine top-ranked compounds,
indicating that the virtual screen indeed yielded a significant enrichment of inhibitors at the
top of the hit list. Three compounds inhibited CyaC with an IC50 ≤ 70 μM (Table 1), and the
two compounds with highest affinities (docking hits 9 and 4; Figure 1c), 3,20-dioxopregn-4en-21-yl 4-bromobenzenesulfonate (2) and 1,2,3,4, 5,6,7,8,13,13,14,14-dodecachloro-1,4,4a,
4b,5,8,8a,12b-octahy-dro-11-sulfo-1,4:5,8-dimethanotriphenylene-10-carboxylic acid (3),
were characterized further.
2 Shows a High Affinity to Mammalian sAC and Exploits a Second Binding Site
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2 resembles the AC inhibitor 1, having a steroid scaffold, but it lacks the catechol
arrangement in ring A thought to be essential for AC inhibition (see below). Of the 16
compounds tested in vitro, however, the compound showed the second highest potency at
inhibiting CyaC, with an IC50 of 15 μM (Figure 2a). CyaC is a cyanobacterial AC that we
have used for modeling mammalian sAC; the two enzymes are closely related at both the
amino acid sequence and regulatory levels.10 Therefore, we tested whether 2 also inhibits
mammalian sAC. On purified, recombinant mammalian sAC, 2 revealed an even greater
affinity, with an IC50 of 1.1 μM (Figure 2b) compared to the reported IC50 of ~3 μM
observed with 1.25,29 Thus, this compound is among the highest affinity inhibitors known
for human sAC with an IC50 comparable to the 0.7 μM reported for the nucleotide 2′,5′didesoxy-3′-ATP.19
To examine effects of 2 on tmACs, we measured its ability to inhibit the forskolinstimulated activities of the endogenous tmACs expressed in HEK293T cells; forskolin
exclusively stimulates tmACs.4 2 showed no significant inhibition of forskolin-stimulated
AC activity in whole cell extracts of HEK293T cells at a concentration of 100 μM (Figure
2c). To examine its potency on tmACs further, we used whole cell extracts of HEK293T
cells transfected with a representative of each of the tmAC subfamilies30 in the presence of
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forskolin. At 100 μM, 2 did not significantly inhibit the activities of AC1 (representing
tmACs 1, 3, and 8), AC2 (representing tmACs 2, 4, and 7), or AC5 (representing tmACs 5
and 6) (Figure 2d). To examine whether 2 inhibited AC9, which is the lone tmAC
insensitive to forskolin stimulation, we tested its effect on basal activity, and once again, 2
did not appreciably affect AC9 activity at a concentration of 100 μM (Figure 2d). Thus, 1
shows high potency versus sAC and discriminates between sAC and tmACs, which makes it
an attractive lead for the further development of potent, isoform-specific AC inhibitors.
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The crystal structure of 1 inhibiting CyaC revealed that the two ortho-hydroxyl groups
chelate the catalytic magnesium, ion A and remove it from its correct position.25 Other
compounds with a catechol group, including catechol steroids and norad-renalin derivatives,
were also able to inhibit soluble adenylyl cyclase activity.31 Therefore, it was thought that
an undisturbed catechol moiety would be essential for sAC inhibition. But 2, which has a
steroid scaffold but no catechol in ring A, inhibits sAC with even higher potency than 1.
Analysis of the docked CyaC/2 complexes hints at an explanation for this finding (Figure
3a,b,c). The most favorable predicted binding orientation (representing cluster comprising
54% of all poses, top binding energy −13.4 kcal/mol) is rotated ~180° compared to 1,
bringing the D ring of the steroid scaffold with its hydrophilic groups, the keto function and
the 4-bromobenzenesulfonate moiety, into position for interactions with the active site
magnesium (Figure 3a); slightly moving the ion down would then again result in a tight
chelator interaction with the inhibitor. This binding mode predicts, however, a localization
for the 4-bromobenzene group of 2 in a more polar, possibly unfavorable environment next
to the ATP phosphates and ribose and the loop between 2 and 3, as more favorable
binding pockets appear to be blocked by the substrate. Redocking of 2 into CyaC without
bound substrate analogue indicates that 2, in addition to exploiting the CE binding site and
inhibition mechanism, might also block the ATP binding site with its 4-bromobenzenesulfonate group (Figure 3b,c). The inhibitor could either shift toward the ATP site, placing
the 4-bromobenzene group into the ribose and part of the adenine binding pocket (Figure
3b), or the inhibitor might occupy the second 1 binding site of the homodimer and the
complete adenine binding cleft (Figure 3c). Both binding orientations would predict 2
should compete with ATP for binding. Activity assays with various substrate concentrations
in the presence of different fixed amounts of inhibitor had previously revealed that 1 is a
noncompetitive inhibitor with respect to ATP,25 and by doing these experiment with 2, we
found that this compound also acts as noncompetitive inhibitor (Figure 3d). Consistently,
most docking orientations of 2 (80% of 30 poses) in the absence of a ligand for the
nucleotide binding site did not occupy this site, and solutions exploiting the site were
predicted to have relatively low binding energies (top binding energy −9.7 kcal/mol).
Instead, the top cluster of poses (27% of poses; top binding energy −11.2 kcal/mol)
positions the 4-bromobenzene group again close to the phosphate/sugar binding site. Thus, 2
indicates that steroids can take advantage of an alternative binding orientation at the 1
binding site, and it suggests that steroid derivatization to simultaneously use the 1 site and a
neighboring pocket may facilitate development of potent, isoform discriminating inhibitors.
The docking results suggest that this additional site might be formed by side-chains around
the loop between 2 and 3, but we will have to await future structural studies for the
definite identification of this additional interaction site.
3 Reveals an Alternative to the Estrogen Scaffold
Of the 16 compounds tested in vitro, 3 was the most potent inhibitor of CyaC, with an IC50
of 5 μM (Figure 4a). The steep slope of the dose–response curve hints at cooperativity,
which might be expected due to the homodimeric architecture of the bacterial CyaC (see
Discussion). The modeled complex of CyaC with 3 predicts an inhibition mode comparable
to 1 (Figure 4b,c,d) despite its novel inhibitor scaffold. The more hydrophobic ring systems
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of 3 occupy the pocket that also binds the steroid ring systems B to D of 1; this pocket is
formed by atoms from the central helix α4 (Asn1146, Ala1149, Arg1150, Gln1152,
Glu1153), the active site strands 2 and 3 (Val1059, Ala1062, Val1059*; * indicates the
partner monomer of the dimeric catalytic core), and strand 1 (Phe1015, Asp1017). The
polar carboxyl group and sulfonic acid moiety point toward the active site region harboring
the two divalent ions A and B. All top clusters of docked conformations showed an
interaction between 3 and ion A, although various arrangements were observed for this
interaction (Figure 4b,c,d). Both the carboxyl and the sulfonic acid groups may interact with
ion A in a chelating interaction (Figure 4b), as previously observed for 1, or either group
may interact with ion A by itself (Figure 4c,d).
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3 strongly inhibits the cyanobacterial sAC enzyme CyaC, and we next tested whether it also
inhibits mammalian sAC. The compound inhibited purified, recombinant mammalian sAC
in a concentration-dependent manner with an IC50 of 11 μM (Figure 4e). We then analyzed
its potency on mammalian tmACs, first by testing forskolin-stimulated endogenous tmAC
activity in HEK293T whole cell extracts. 3 inhibited this activity only slightly at a
concentration of 100 μM (Figure 2c). We then tested 3 on lysates of HEK293T cells
expressing representatives of the mammalian tmAC subfamilies.30 3 at a concentration of
100 μM showed only moderate inhibition of basal AC2 and AC9 activity and of forskolinstimulated AC1 and AC5 activity (Figure 4f). Interestingly, 3 displayed a different effect on
AC2 (stimulation) in the presence of forskolin (data not shown), which will be the focus of a
separate study. We can conclude, however, that 3 shows a high selectivity for inhibition of
mammalian sAC versus tmAC isoforms.
Discussion
The 10 mammalian AC subclasses control diverse sets of crucial physiological processes,
rendering them interesting targets for therapeutic intervention.1,14 Successful AC-targeted
drug development has been limited so far, as most known AC inhibitors bind to the
enzyme’s ATP binding site and thus tend to bind to a wide range of nucleotide binding
proteins.1,14,18 In contrast, inhibitors binding outside the active site, such as the nonnucleoside inhibitor nevirapine for reverse transcriptase,32 exploit more specific features of
a target protein. CEs such as 1 are AC inhibitors binding to a conserved pocket different
from the substrate binding site, which displays features varying between individual AC
enzymes.25 1 shows little isoform specificity,25 however, and thus does not exploit the full
potential of this binding site. Therefore, we explored the possibility of targeting the CE
binding site with novel compounds in order to achieve greater specificity and higher
potency.
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Because of its steroid scaffold, the novel inhibitor 2 is structurally related to CEs such as 1.
However, 2 displays some significant differences compared to CEs. It has no catechol
moiety in the A ring and therefore appears to bind in a 180° rotated orientation, with its Dring pointing toward the active site. This variation would be consistent with previous studies
that indicated that the hydrophobic part of the ligand attached to the chelating group can
vary widely. Compounds such as tyrphostatins33 and epinephrine derivatives,31,34 which
contain a catechol completely different from steroids, inhibit sAC activity and are likely to
exploit the CE binding site and inhibition mechanism. Our docking system reflected the
imperfect steric fit of the steroid scaffold to the CE site indicated by these two possible
binding orientations. Although nine of the ten top solutions for redocking 1 into the CyaC
part of the CyaC/1 structure reproduced the experimentally determined orientation, one
docking orientation corresponded to the orientation predicted for 2. It appears that the
steroid scaffold offers a hydrophobic, nonspecific binding partner for the CE interaction site,
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but as seen for 2, variations and substitutions on this scaffold can increase the specificity of
these compounds.
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A second interesting feature discriminating 2 from 1 is 2’s ability to simultaneously exploit
a neighboring binding sites in addition to the CE site. While the steroid part of 2 and the
chelating groups at the D-ring appear to mimic CEs, the bulky extension of 2 likely adds
interactions to a neighboring pocket of the enzyme. This extension of the binding area
provides an opportunity to find compounds with both increased affinity and specificity.
Consistently, 2 indeed shows a higher specificity and a slightly increased potency against
sAC as compared to CEs.29 A similar principle is seen for the inhibition of ACs by MANTGTP, where the nucleotide binds to the ATP binding site and the MANT group occupies a
hydrophobic patch at the C1/C2 dimer interface.24 The interactions of this GTP substituent
have not yet been refined, and MANT-GTP, in fact, is well-known as a ligand for
heterotrimeric G-proteins35 and many other GTP-binding proteins (see, e.g., refs 36, 37).
Thus, MANT-GTP is likely to inhibit many nucleotide binding proteins. However, although
they are not yet fully developed, 2 and MANT-GTP indicate a huge potential for potent and
specific AC inhibition by compounds exploiting the substrate or CE binding site and an
additional, apparently AC isoform specific pocket.
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The steroid scaffold of CEs and 2 renders these compounds potential ligands for the cellular
steroid binding proteins. 3 reveals an alternative to the steroid scaffold for binding to the CE
site. The geometry of 3 indeed would be a good starting point for further improvements due
to the large space that can be probed by modifications at various positions, although its
chemical nature makes it hard to introduce modifications at specific positions. In particular,
the potential cooperativity observed for 3 on CyaC hints at an attractive idea for inhibitor
improvement. The Hill coefficient of 3.2 indicates several binding sites for 3, most likely the
two symmetry-related CE sites of the homodimer and possibly even the substrate binding
site. The Hill coefficient of 2.0 for sAC inhibition also hints at a second binding site for 3;
the effect of 3 on AC2 in presence of forskolin indicates that the pseudosymmetric substrate
site which can accommodate forskolin and related diterpenes9,38 is still available for
forskolin. Assuming that AC2, like sAC, has two sites for 3, we speculate that this
compound can additionally occupy the second, pseudosymmetric CE site in C2 of the
mammalian C1C2 heterodimer, although definitive answers will have to await further
studies. In any case, in the homodimeric CyaC/1 crystal structure,25 the flat steroid binds
with one side to the protein and faces with its opposite side the second 1 molecule, which is
bound to the CE site of the partner monomer. Thus, bulkier compounds should be able to
exploit the CE binding site and the second site—the symmetry-related CE site in
homodimers and the pseudosymmetric site in heterodimers—simultaneously for higher
affinity and specificity.
Taken together, our docking and inhibition studies have identified two novel AC inhibitors,
2 and 3. Both compounds inhibit mammalian sAC potently. In contrast, they show weak or
no inhibition of mammalian tmACs. 2 shows the potential of exploiting the CE binding site
and an additional pocket simultaneously, whereas 3 indicates that bulkier compound
scaffolds also can exploit the CE binding site with an increased specificity compared to 1.
Materials and Methods
Target Preparation and Virtual Ligand Screening
The crystal structure of CyaC in complex with α, -Me-ATP and 1 (PDB entry 2BW7)25
was used for this docking study. All water molecules as well as the two molecules of 1 were
removed from the catalytic dimer. Polar hydrogen atoms were added and Kollman charges
assigned to all atoms with AutoDockTools (http://autodock.scripps.edu/resources/adt). The
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divalent active site ions A and B were treated as found in the target structure, i.e., as Mg2+
and Ca2+, respectively. One of the two active sites of the homodimer was chosen as docking
site, and 60 Å × 50 Å × 66 Å affinity grids centered on this active site plus 1 binding pocket
were calculated with 0.375 Å spacing by using Autogrid326 for each of the following atom
types: C, A (aromatic C), N, O, S, H, F, Cl, Br, I, P, and e (electrostatic). The NCI diversity
set of 1990 compounds with unique scaffolds selected from the NCI-3D structural database
(http://www.dtp.nci.nih.gov) was then docked into the target site. The modified set with all
hydrogens added and assignments of Gasteiger charges and rotate able bonds was obtained
from http://autodock.scripps.edu/resources/databases. The docking calculations were done
by using the Lamarckian genetic algorithm (LGA) for ligand conformational searching of
AutoDock version 3.0.26 For each compound, the docking parameters were as follows: trials
of 100 dockings, population size of 150, starting position, orientation, and conformation
randomized using the default AutoDock randomization, translation step ranges of 1.5 Å,
rotation step ranges of 35°, elitism of 1, mutation rate of 0.02, crossover rate of 0.8, local
search rate of 0.06, and 10 million energy evaluations. The calculations were done on an
Opteron Linux workstation. Final docked conformations were clustered by use of a tolerance
of 2.5 Å root-mean-square deviation (rmsd). The top 17 compounds with the best simulated
binding energies were selected for the in vitro inhibition assay. Figures of structural models
were made with PyMol.39
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Protein Purification and Activity Assay
The compounds corresponding to the top docking hits were obtained from Division of
Cancer Treatment and Diagnosis at the National Cancer Institute. The immunogenic cAMP
assay kit used for cAMP measurements was from Assay Designs (Ann Arbor, MI), and all
other chemicals from Sigma (Saint Louis, MO). Forskolin and inhibitors were dissolved in
DMSO (10 mM stock solutions), yielding a maximum DMSO concentration of 2% v/v for
the tmAC experiments and 0.5% v/v for CyaC experiments; control reactions were done
with the same solvent without compound. A GST fusion of human sAC7 and his-tagged rat
sAC6 were expressed and purified as described previously. Dose–response curves were
measured with both sAC enzymes, and the IC50 values obtained did not differ significantly
between both enzymes. Data shown are representative results obtained with either human or
rat sAC. The catalytic domain of CyaC from S. platensis was expressed and purified with an
N-terminal his-tag as described10 and stored at −80 °C or supplemented with 50% (v/v)
glycerol and stored at −20 °C for activity assays.
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Mammalian sAC assays were done in 50 mM Tris-HCl (pH 7.5) with 2.5 mM ATP as
substrate, 5 mM MgCl2, 2.5 mM CaCl2, and 40 mM bicarbonate. Assays were started by
addition of purified mammalian sAC, incubated 30 min at room temperature, and stopped
through 400-fold dilution into 0.1 M HCl. The cAMP produced was quantitated by cAMP
ELISA. Activity assays with CyaC were done in 50 mM Tris-HCl (pH 7.5), 5 mM ATP, 10
mM MgCl2, and 5 mM CaCl2. Reactions were incubated 30 min at 37°C, diluted 500-fold
into 0.1 M HCl, and tested with the cAMP ELISA. Activity assays with the various tmACs
were performed on 50 μg of protein of whole cell lysates of HEK293T cells transfected with
the indicated mammalian tmAC. Assays were performed in 50 mM Tris-HCl (pH 7.5), 1
mM ATP, 5 mM MgCl2, 80 μM CaCl2, and creatine kinase ATP regenerating system in the
presence of 100 μM forskolin. Reactions were incubated 30 min at 37 °C, diluted 20-fold
into 0.1 M HCl, and cAMP was measured with the cAMP ELISA.
Acknowledgments
Supply of chemicals from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis at the National Cancer Institute, is greatly acknowledged. This work
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Schlicker et al.
Page 8
was supported by funds from National Institutes of Health (L.R.L. and J.B.), Hirschl Weill-Caulier Trust (L.R.L.),
the American Diabetes Association (L.R.L.), and grant STE1701/1 of Deutsche Forschungsgemeinschaft (C.S.).
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Figure 1.
Novel AC inhibitors developed based on the CE binding site. (a) Chemical structure of the
noncompetitive AC inhibitor 1. (b) Crystal structure of the active site of CyaC in complex
with a substrate analogue and the inhibitor 1. The catechol moiety of the inhibitor chelates
the active site magnesium ion A and removes it from its normal position. This figure was
reproduced from ref 25. (c) Chemical structures of the novel AC inhibitors 2 and 3 identified
through docking calculations with the 1 binding pocket of CyaC, followed by in vitro
activity assays.
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Figure 2.
Inhibition of CyaC and mammalian sAC and tmACs by compound 2. (a) Dose–response
curve for 2 inhibition of CyaC activity which shows an IC50 of 15 μM. (b) The dose–
response curve for the effect of 2 on mammalian sAC activity, which shows that this
compound inhibits with high potency (IC50 of 1.1 μM). (c) Forskolin-stimulated AC activity
of HEK293T cell lysates in presence of 2, 3, or DMSO (control). (d) Forskolin-stimulated
(AC1 and AC5) or basal (AC2 and AC9) AC activity of cell lysates from HEK293T cells
transfected with the indicated tmAC isoform in absence (control) and presence of 100 μM
compund 2.
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Figure 3.
Mode of inhibition of CyaC by compound 2. (a) Model of the inhibitor 2 docked into the
CyaC/α, -Me-ATP complex. (b) Crystal structure of CyaC in complex with the substrate
analogue α, -Me-ATP, overlaid with 2 docked into CyaC without ligand, showing a
potential steric clash predicted between ATP and 2. (c) Alternative conformation for 2
docked into unliganded CyaC, with the 4-bromobenzene in the adenine binding cleft. (d)
Saturation curves for CyaC activity at various substrate concentrations in the presence of
different amounts of 2 (●: 0 μM; Δ: 1 μM; ■: 2.5 μM; □: 15 μM). The depicted nonlinear
regression using the Michaelis–Menten equation resulted in Km values fluctuating between
0.5 and 1 mM, whereas the Vmax values decreased with increasing inhibitor concentration
(0.51, 0.38, 0.29, and 0.08 nmol/(mg · min)), indicating noncompetitive inhibition by 2.
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Figure 4.
Compound 3 reveals a novel scaffold and chelating group for AC inhibition. (a) Dose–
response curve for the inhibition of CyaC AC activity by 3, yielding an IC50 of 5 μM. (b)
Representative of a cluster of docked conformations of 3, which leads to a chelating
interaction of the 3 carboxyl group and sulfonyl moiety with ion A. (c) Representative of a
cluster of docked conformations of 3, which leads to an interaction of ion A with the 3
carboxyl group. (d) Representative of a cluster of docked 3 conformations, which leads to an
interaction of the inhibitor sulfonyl group with ion A. (e) A dose–response curve for the
inhibition of mammalian sAC activity by 3, yielding an IC50 of 11 μM. (f) Forskolinstimulated (AC1 and AC5) or basal (AC2 and AC9) AC activities of cell lysates from
HEK293T cells transfected with the indicated tmAC isoform in absence (control) and
presence of 100 μM 3.
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Table 1
J Med Chem. Author manuscript; available in PMC 2011 April 26.
Docking rank
NSC number
(Compound
no.)
1
2
Compound name
IC50 against CyaC in
vitro
28081 (4)
1-(bromo(1-naphthyl)methyl)naphthalene
~200 μMa
150289 (5)
2,3-dibromo-1-(4-(hydroxy(oxido)amino)phenyl)-3-(4-quinolinyl)-1-propanone
~ 100 μMa
Chemical structure
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Highest Ranked Docking Hits
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3
J Med Chem. Author manuscript; available in PMC 2011 April 26.
Compound name
IC50 against CyaC in
vitro
371884 (6)
6-(2-methyl-2H-indol-3-yl)-6′-(2-methyl-3aH-indol-3-yl)-4,4′
-bipyrimidine-2,2′-diamine
No significant inhibition
up to 250 μM
4
270718 (3)
1,2,3,4,5,6,7,8,13,13,14,14-dodecachloro-1,4,4a,4b,5,8,8a, 12b-octahydro-11-sulfo-1,4:5,8
-dimethanotriphenylene-10-carboxylic acid
5 μM
5
123994 (7)
4,5,6,7-tetrachloro-3,3-bis(6-hydroxy[1,1′-biphenyl]-3-yl)-2benzofuran-1 (3H)-one
No significant inhibition
up to 250 μM
Chemical structure
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Docking rank
NSC number
(Compound
no.)
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6
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Compound name
IC50 against CyaC in
vitro
88135 (8)
2,3,6,23-tetrahydroxyurs-12-en-28-oic acid
~100 μM a
7
7524 (9)
4, 9-Epoxycevane-3,4,12,14,16,17,20-heptol 3-(3,4-dimethoxybenzoate)
No significant inhibition
up to 250 μM
8
371878 (10)
6,6′-di(naphthalen-2-yl)-4,4′-bipyrimidine-2,2′-diamine
No significant inhibition
up to 250 μM
Chemical structure
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Docking rank
NSC number
(Compound
no.)
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9
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Compound name
IC50 against CyaC in
vitro
88915 (2)
3,20-dioxopregn-4-en-21-yl 4-bromobenzenesulfonate
15 μM
10
46529 (11)
N,N-bis(2-bromopropyl)-9H-fluoren-2-amine
Not tested (non-specific
alkylating agent)
11
50352 (12)
4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-N-(1-pyrrolidinylmethyl)
-1,4,4a,5,5a,6,11,12a-octahydro-2-naphthacenecarboxamide
No significant inhibition
up to 250 μM
12
159628 (13)
{Spiro[2-cyclohexene-1,} {2′(1′H)cyclopenta[de]naphthacene]-9′-carboxamide,} 7′,7′a,8′,11′, 11′a, 12′-hexahydro-5′,6′,7′a,10′,11′a, 12′-hexahydroxy-3′methoxy-2,6,6-trimethyl-7′,8′-dioxo-, (2′α,7′aβ,11′aβ,12′β)-(−)-
No significant inhibition
up to 250 μM
13
371876 (14)
6,6′-di(naphthalen-1-yl)-4,4′-bipyrimidine-2,2′-diamine
No significant inhibition
up to 250 μM
Chemical structure
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Docking rank
NSC number
(Compound
no.)
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14
44480 (15)
2-bromo-1-(9-bromo-3-phenanthryl)-1-propanone
No significant inhibition
up to 250 μM
15
259968 (16)
17,24-dihydroxy-10-(4-methoxybenzyl)-4,7,9,13,15,29-hexamethyl-22-oxa-O3,6,9,12,15,29hexaazatetracyclo[14.12.2.2~18,21~.1~2 3,27~]tritriaconta-18,20,23(31),24,26,32-hexaene-2,5,8,11,14,30-hexone
No significant inhibition
up to 250 μM
16
168656 (17)
2,3-dibromo-4-(4-methoxyphenyl)-4-oxobutanoic acid
No significant inhibition
up to 250 μM
Chemical structure
Compound name
IC50 against CyaC in
vitro
Schlicker et al.
J Med Chem. Author manuscript; available in PMC 2011 April 26.
Docking rank
NSC number
(Compound
no.)
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95090 (18)
Compound name
5′-benzyl-12′-hydroxy-2′-methyl-3′,6′,
18-trioxoergotaman
NIH-PA Author Manuscript
17
Chemical structure
IC50 against CyaC in
vitro
~70 μMa
Schlicker et al.
Docking rank
NSC number
(Compound
no.)
J Med Chem. Author manuscript; available in PMC 2011 April 26.
a
IC50 estimated from experiments without inhibitor and with two different inhibitor concentrations, respectively.
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