Structure-based Inhibitor Discovery against Adenylyl Cyclase Toxins from Pathogenic Bacteria That Cause Anthrax and Whooping Cough*
Sandriyana Soelaiman
,
Binqing Q. Wei
¶,
Pamela Bergson
,
Young-Sam Lee ||,
Yuequan Shen
,
Milan Mrksich ||,
Brian K. Shoichet ** 
and
Wei-Jen Tang

From the
Ben-May Institute for Cancer Research,
The University of Chicago, Chicago, Illinois 60637, the
¶Department of Molecular Pharmacology and
Biological Chemistry, Northwestern University Medical School, Chicago,
Illinois 60611, the ||Department of Chemistry, The
University of Chicago, Chicago, Illinois 60637, and the
**Department of Pharmaceutical Chemistry, University
of California, San Francisco, California 94143
Received for publication, February 4, 2003
, and in revised form, March 25, 2003.
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ABSTRACT
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Edema factor (EF) and CyaA are adenylyl cyclase toxins secreted by
pathogenic bacteria that cause anthrax and whooping cough, respectively. Using
the structure of the catalytic site of EF, we screened a data base of
commercially available, small molecular weight chemicals for those that could
specifically inhibit adenylyl cyclase activity of EF. From 24 compounds
tested, we have identified one quinazoline compound, ethyl
5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, that specifically
inhibits adenylyl cyclase activity of EF and CyaA with
20
µM Ki. This compound neither
affects the activity of host resident adenylyl cyclases type I, II, and V nor
exhibits promiscuous inhibition. The compound is a competitive inhibitor,
consistent with the prediction that it binds to the adenine portion of the ATP
binding site on EF. EF is activated by the host calcium sensor, calmodulin.
Surface plasmon resonance spectroscopic analysis shows that this compound does
not affect the binding of calmodulin to EF. This compound is dissimilar from a
previously described, non-nucleoside inhibitor of host adenylyl cyclase. It
may serve as a lead to design antitoxins to address the role of adenylyl
cyclase toxins in bacterial pathogenesis and to fight against anthrax and
whooping cough.
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INTRODUCTION
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The 2001 anthrax attacks in the United States have spurred an intense
effort to discover new drugs to combat this dangerous biowarfare agent
(1). Anthrax is caused by the
pathogenic bacterium Bacillus anthracis. The anthrax bacterium
secretes three major exotoxins, protective antigen
(PA),1 lethal factor
(LF), and edema factor (EF)
(2). PA is a pH-dependent
transporter that delivers LF and EF into host cells. To do so, 83-kDa PA
(PA83) first associates with the cell surface protein tumor
endothelial marker 8 (TEM-8)
(3). The N-terminal 20-kDa
domain of PA83 is then cleaved by a surface furin-like protease to
form a PA63 heptamer. Two PA63 domains within the
PA63 heptamer form a surface to bind an EF or LF molecule so that
up to 3 mol of EF/LF mixtures can be delivered by a PA63 heptamer
(4). Upon endocytosis and
acidification, PA forms a pore to deliver EF or LF into the cytosol of host
cells (5). EF is a calmodulin
(CaM)-activated adenylyl cyclase that can elevate intracellular cAMP to
pathological levels (6). LF is
a metallo-protease that can cleave and inactivate a family of mitogenactivated
kinase kinases including mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase and p38 kinase
(7,
8). All three toxins work in
concert with a poly-D-glutamate capsule to make the anthrax
bacterium deadly (9).
The molecular structures of all three anthrax toxins have been determined
recently, providing an excellent starting point to develop specific inhibitors
against the action of these toxins
(6,
10,
11). Several peptide-based
reagents including the extracellular domain of TEM-8, dominant-negative PA
mutants, and oligomers of PA-binding peptides have been developed to block PA
from interacting with TEM-8, forming a functional pore, and associating with
EF/LF, respectively (3,
12,
13). In addition, sensitive
assays to search for LF inhibitors and low nano-molar affinity inhibitors of
LF have been developed recently
(14,
15). However, to date no
inhibitor against EF has been identified.
We have determined the molecular structure of EF with and without CaM
(6). Based on the structure of
EF, we have found that the catalytic site of this enzyme is different from
host adenylyl cyclases. This contrast suggests that it should be feasible to
identify small molecular weight compounds that can specifically inhibit the
activity of EF without affecting host adenylyl cyclases. The deletion of the
EF gene in B. anthracis not only impairs the germination of the
anthrax bacterium in mouse peritoneal macrophages but also raises the
LD50 value by 2 orders of magnitude in a rodent model
(16,
17). These results suggest
that the blockage of adenylyl cyclase activity of EF may significantly reduce
the lethality of anthrax bacterium, thereby providing a wider window to treat
patients with anthrax infection. The adenylyl cyclase domain of EF also shares
homology with other adenylyl cyclase toxins, CyaA and ExoY
(18,
19). CyaA is vital for the
colonization of Bordetella pertussis in the respiratory tract;
successful colonization results in whooping cough, a major health threat to
infants (20). ExoY is a toxin
delivered by the type III secretion system of Pseudomonas aeruginosa,
a bacterium that accounts for 20% of hospital-acquired infections
(19). In addition, a secreted
fraction having adenylyl cyclase activity and a gene homologous to known
adenylyl cyclase toxins were found in Yersinia pestis, a bacterium
that causes bubonic and pneumonic plagues
(2123).
Thus, molecules that can block the action of adenylyl cyclase toxins may have
a broad usage to combat illness caused by several deadly human pathogens.
Here we describe the identification of specific inhibitors of EF and CyaA
among commercially available chemicals. We first docked
200,000 molecules
from the Available Chemical Directory (MDL Information Systems Inc., San
Leandro, CA) in multiple orientations and conformations into the ATP binding
site of EF. Twenty-four high scoring molecules were selected for experimental
studies to identify those that specifically inhibit EF and CyaA compared with
corresponding host adenylyl cyclases and subsequently to block the
intoxication of adrenocortical Y1 cells caused by edema toxin (a combination
of EF and PA). This study identified a family of quinazoline compounds, the
best of which specifically inhibited EF and CyaA with a
Ki value of 20 µM without
inhibiting mammalian type I, II, and V adenylyl cyclases.
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EXPERIMENTAL PROCEDURES
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MaterialsCompounds 2-phenylaminoadenosine,
(3ar,4s,7r,7as)-7-(carbobenzyloxyamino)-3a,4,7,7a-tetrahydro-2,2-dimethyl-1,3-benzodioxol-4-ol,
and 2,7-diamino-3-cyano-4-phenyl-4n-benzopyrane were purchased from
Sigma;
n(2)-(4-nitrophenyl)[1,3]thiazolo[5,4-d]pyrimidine-2,7-diamine
and
2-amino-4-(2-furyl)-5-oxo-5,6,7,8-tetrahydro-4n-chromene-3-carbonitrile
were from Bionet (Camelford, UK); PU120 and PU574 were from Menai (Gwynedd,
UK); 16/06-35 was from Buttpark (Bath, UK); 6,8-dibromoquinazolin-4-ol,
5-amino-8-(trifluoromethyl)pyrido[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine-3-carbonitrile,
3-phenyl-8-(trifluoromethyl)pyrido[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5-amine,
(2-amino-4,5-dimethyl-3-thienyl)(4-chlorophenyl) methanone, ethyl
5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate,
(5-amino[1,2,3]triazolo[1,5-a]quinazolin-3-yl)(morpholino)methanone,
n3-(4-pyridylmethyl)-5-amino
[1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide,
9-fluoro-5n-chromeno[4,3-d]pyrimidin-2-amine,
2-[(3-amino-4-oxo-4n-[1,2,4]triazino[3,4-b][1,3,4]thiadiazol-7-yl)thio]acetic
acid,
7-methoxy-1,2-dihydrobenzo[e][1,2,4]triazolo[3,4-c][1,2,4]triazin-1-one,
8-(methylthio)-4,5-dihydrothieno[3',4':5,6]benzo[c]isoxazole-6-carboxamide,
6,8-difluoro-2,3-dihydro-1n-pyrazolo[4,3-c]quinolin-3-one,
4-amino-1-hydroxy-5,5-dimethyl-2-phenyl-3-imidazoline-3-oxide,
7-chloro-1,2-dihydrobenzo[e] [1,2,4]triazolo[3,4-c]
[1,2,4]triazin-1-one,
n3-ethyl-5-amino[1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide,
and
(5-amino-7-chloro[1,2,3]triazolo[1,5-a]quinazolin-3-yl)(2-thienyl)methanone
were from Maybridge (Cornwall, UK). Anthrax protective antigen was purchased
from List Biological Laboratory (Campbell, CA), restriction enzymes were from
New England Biolabs (Beverly, MA), and the QuikChange kit for site-directed
mutagenesis was from Stratagene (La Jolla, CA). [
-32P]ATP
and the Big-Dye kit for automatic DNA sequencing were from PerkinElmer Life
Sciences. Mouse adrenocortical Y1 cells were obtained from ATCC. Tissue
culture reagents were obtained from Invitrogen and Cambrex Bio Science
Walkersville, Inc. (Walkersville, MD).
DockingThe Northwestern University version
(2427)
of DOCK (28,
29) was used to screen the
Available Chemical Directory (version 2000.2, MDL) against the 3'-dATP
binding site of the EF·CaM structure (Protein Data Bank code 1K90
[PDB]
). To
prepare the site for docking, 3'-dATP and all water molecules were
removed. The observed ytterbium ion was treated as a magnesium ion, which is a
tightly bound, nondisplaceable group. Protonation of enzyme residues was done
with Sybyl (Tripos, St. Louis, MO). To generate docking "spheres,"
which are used to orient ligand, we used both the positions of the
3'-dATP atoms and sphere positions identified by SPHGEN
(28). Several selective
spheres were labeled based on the chemical functionality of the nearby
residues. The program DISTMAP was used to compute the excluded volume grid of
EF (30), which is used as an
initial steric filter in docking calculation. Electrostatic and van der Waals
energy potential grids were calculated by DelPhi
(31) and CHEMGRID
(29), respectively.
Ligand partial atomic charges and solvation energies were calculated with
AMSOL (27,
32,
33). Conformations of ligand
molecules were calculated with Omega (OpenEye, Santa Fe, NM) before docking,
and up to 2000 conformers for each molecule were generated. An ensemble of
conformations was stored and docked in a hierarchical manner that allows for
rapid pruning of conformations that clash with the binding pocket
(25). Alternative protonation
states of ligand ionizable groups were sampled. The distance tolerance
parameter (dislim) for orientation matching was set to 0.8 Å. The ligand
and receptor bin sizes were each 0.3 Å, and ligand and receptor overlap
were each 0.2 Å. Chemical matching
(34) and "critical
clusters" were used to guide the matching of ligand atoms to the
spheres. Docking energies were corrected for the cost of desolvating the
ligands (26,
27). Each ligand orientation
that passed the steric filter was refined with up to 20 iterations of rigid
body minimization (35). The
500 top scoring molecules of the 205,226 Available Chemical Directory
molecules docked were displayed with Midas-Plus
(36), and 19 molecules were
selected for experimental testing as inhibitors of EF. Following initial
enzyme inhibition assays, the ISIS program (MDL) was used to select five high
scoring analogs of compounds 1 and 2, two initial docking
"hits" that were found to inhibit EF, for testing.
Purification of EF, EF3, CyaA-N, and CaMEF3 and CyaA-N, the
catalytic domains of EF and CyaA, respectively, as well as calmodulin were
purified as described previously
(37,
38). To express edema factor
that has a hexahistidine tag substituted for its leader peptide (amino acids
133) and can be delivered by anthrax-protective antigen into host
cells, a plasmid, pProEx-H6-EF, was constructed as follows. The 3.2-kb
EcoRI-XhoI fragment was excised from pSE42 (kindly provided
by S. Leppla, National Institutes of Health) and inserted into pBluescript. A
NotI site was then introduced at the sequence encoding amino acids
3234 of EF by site-directed mutagenesis, and the mutation was confirmed
by DNA sequencing. The 3.2-kb NotI-XhoI fragment encoding
amino acids 35800 of EF was subsequently moved into pProEx-H6. To make
recombinant H6-EF, an N-terminal hexahistidine-tagged EF, pProEx-H6-EF was
transformed into BL21(DE3) that harbored pUBS520, a plasmid that encoded tRNA
for the AGA and AGG codons. The resulting cells were grown in a modified T7
medium with 50 µg/ml ampicillin and 25 µg/ml kanamycin at 30 °C to
A600 = 0.4, induced by adding
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 200 µM, and harvested 19 h postinduction. The
purification of EF was done by using a nickel-nitrilotriacetic acid column
followed by Q-Sepharose column to yield
20 mg from each liter of
Escherichia coli culture. The protein concentrations of all
recombinant proteins were determined by Bradford assay using bovine serum
albumin as the standard.
Enzymatic AssaysAdenylyl cyclase activities were measured
in the presence of 20 mM HEPES (pH 7.2), the indicated ATP
concentration with a trace amount of [32P]ATP, 1 mM
EDTA, 10 mM MgCl2, and 1 µM free calcium
for a 10-min incubation at 30 °C. ATP and cAMP were separated by Dowex and
alumina columns (39). Sf9 cell
membrane containing type I, II, or V adenylyl cyclase was prepared as
described previously (39). The
compounds were dissolved in Me2SO, and a minimal volume (1 µl)
was added to the assay to avoid the inhibitory effect by Me2SO.
TEM-1
-lactamase was expressed and purified to homogeneity as described
previously (40). Kinetic
measurements of TEM-1 were performed in 50 mM Tris buffer (pH 7.0)
using 200 µM nitrocefin as a substrate in methacrylate cuvettes;
reactions were monitored at 482 nm. Reactions were initiated either by the
addition of enzyme or, if inhibitor-enzyme preincubation was being tested, by
the addition of substrate.
Dynamic Light ScatteringCompounds were dissolved to 20
mM in Me2SO and diluted with filtered 50 mM
pH 7.0 potassium phosphate buffer (KPi). All compounds were
analyzed with a 3-watt argon-ion laser at 514.4 nm with optical systems from
Brookhaven Instrument Corp. The laser power and integration times were
comparable for all experiments. Calculation of mean particle diameter was
performed by the cumulate analysis tool of a 400-channel BI9000AT digital
autocorrelator with the last four channels used for base-line measurement. The
detector angle was 90°. Three to five independent measurements were
performed for each concentration of each compound at 22 °C.
Cell Round-up Assay of Adrenocortical Y1 CellsY1 cells were
maintained at 37 °C with 5% CO2 in Dulbecco's modified Eagle's
medium/F-12 supplemented with 2.5% fetal bovine serum and 12.5% horse serum.
Plates and flasks were coated with 1% gelatin before cells were plated to
facilitate cell attachment and spreading. Y1 cells were plated in 96-well
plates at 200 µl/well and used when they reached 5080% confluence
(about 5 x 104 cells/well). For the round-up assay of Y1
cells, compounds were dissolved in Me2SO at concentrations ranging
from 50 mM to 390 µM in 2-fold dilutions, and 2 µl
of each concentration were added to the appropriate wells. After a 1-h
incubation, EF and PA were added to 3 and 25 ng/ml final concentrations,
respectively. The morphology of Y1 cells was examined after 1 h, 4 h, and
overnight incubation.
Surface Plasmon Resonance SpectroscopyThe ability of EF to
bind cutinase-CaM was monitored by surface plasmon resonance spectroscopy as
described previously (38,
41). In brief, EF (0.24
nM2 µM) in the binding buffer (10
mM Tris-HCl, pH 7.0, 1.0 mM EGTA, 10 mM
MgCl2, 100 mM KCl, 0.96 mM CaCl2)
was mixed with compound 3 or 5. This mixture was then allowed to
interact with cutinase-CaM immobilized on 2% phosphonate surface with a flow
rate of 3 µl/min for 20 min, and the amount of bound EF was determined from
the change of surface plasmon spectroscopic response.
 |
RESULTS
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Full-length EF (H6-EF) and the Catalytic Domain of EF (EF3) Have
Similar Sensitivities to Calcium and CaMDue to the problem in
expressing the full-length EF, we have expressed and characterized the 60-kDa
adenylyl cyclase domain of EF, named EF3
(37). By optimizing the
expression, we now have effectively expressed and purified recombinant 90-kDa
H6-EF, which contains both the catalytic domain and PA-binding domain of EF.
After nickel-nitrilotriacetic acid and Q-Sepharose columns,
20 mg of 90%
pure H6-EF was obtained from each liter of E. coli culture
(Fig. 1A), a 5-fold
improvement over the previously reported expression and purification protocols
(42). H6-EF can be stimulated
by CaM with Vmax and EC50 values identical to
those of EF3 (Fig.
1B). We have recently shown that physiological calcium
concentrations can promote the association between CaM and EF3 as well as
directly inhibit the catalytic rate of EF3; such regulation is also found in
H6-EF (Fig. 1C)
(38). Thus, our data showed
that the catalytic properties of EF are identical to those of EF3. For the
subsequent studies, we used EF3 for the in vitro enzymatic assay to
avoid the potential complication of the PA-binding domain of EF and used H6-EF
for tissue culture cells where the PA-binding domain is required for EF to
enter into cells.

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FIG. 1. Effect of CaM and calcium ion on adenylyl cyclase activity of EF, a
full-size EF, and EF3, a catalytic domain of EF. A, purified EF
and EF3. 1 µg of purified EF and EF3 were run on an SDS-polyacrylamide gel
and stained by Coomassie Blue. B, adenylyl cyclase assays were
performed with 0.78 nM EF (open circles) and 0.85
nM EF3 (filled circles) in the presence of 1.0
µM free Ca2+. C, adenylyl cyclase
activities were measured with 0.52 nM EF (open circles)
and 0.56 nM EF3 (filled circles) in the presence of 10
µM CaM. Mean ± S.E. are representative of at least two
experiments.
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Identification of Compounds That Can Inhibit the Catalytic Domain of
EFOur goal was to identify low molecular weight molecules that can
specifically inhibit adenylyl cyclase toxins without affecting host adenylyl
cyclases and block the cellular intoxication by edema toxin (PA and EF). To do
so, we targeted our structure-based inhibitor discovery to the catalytic site
of EF. The 3'-dATP binding site of the EF·CaM complex was
screened against a data base of 205,226 small molecules. On average, each
compound was sampled in 447 orientations and 294 conformations, and overall
2.7x 1010 configurations were scored. Top scoring molecules
were visually examined in the context of the binding site, and 19 compounds
were initially chosen based on electrostatic or polar complementarity as well
as favorable nonpolar interactions. These compounds were purchased and tested
for their ability to inhibit adenylyl cyclase activity of EF3, a recombinant
protein containing only the catalytic portion of EF. Two
pyrido[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine-5-amines
(compounds 1 and 2) and one phenyl-methanone (compound 8)
were found to have IC50 values lower than 300 µM
(Fig. 2 and
Table I). Five high scoring
analogs (compounds 37) of compounds 1 and 2, which
have IC50 values of about 100 µM, were picked from
the Available Chemical Directory based on chemical similarity and were also
tested for inhibition of EF (Fig.
2 and Table I).
Three of these (compounds 3, 5, and 7) have
IC50 values lower than 100 µM.

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FIG. 2. Effects of eight compounds on adenylyl cyclase activity of EF3. The
adenylyl cyclase assay was done in the presence of 16 pM EF3, 1
µM CaM, 1 µM free Ca2+, and
the indicated concentrations of compounds dissolved in Me2SO. To
avoid the effect of Me2SO, only 1 µl of the compound solution
(or Me2SO as the solvent control) was added into the 100-µl
reaction. Means ± S.E. are representatives of at least two experiments,
and specific activities of EF3 without compounds in these experiments were in
the range of 0.61.6 ms1.
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Filtering the Active Compounds by Promiscuity Assays
Chemical compounds may form aggregates that promiscuously inhibit the activity
of EF instead of specifically occupying its active site; such phenomena have
been observed for many inhibitors from virtual and high throughput screening
as well as for protein kinases
(4345).
To eliminate the compounds with this unwanted effect, we first investigated
the effect of preincubation of compounds with EF
(Fig. 3, A and
B; not shown for compound 8). We found that
compounds 7 and 8 had reduced IC50 values, an
indication of promiscuous inhibition. In addition, the same set of compounds
also had increased IC50 values when 10-fold more EF was used, which
also suggested promiscuous inhibition. We then investigated the activities of
compounds 1, 2, 3, and 5 against a completely
unrelated enzyme,
-lactamase. Compounds 1, 3, and 5
at 200300 µM concentrations did not inhibit
-lactamase and showed no preincubation effect, while compound 2
at 70 µM almost completely inhibited
-lactamase activity.
The inhibition of
-lactamase by compound 2 increased after
preincubation and decreased when the enzyme concentration was raised by
10-fold (data not shown). We then used dynamic light scattering experiments to
test whether compounds 1, 3, and 5 can form aggregates,
which is a characteristic of some promiscuous inhibitors
(43,
45). Compound 1 at a
concentration comparable to its IC50 for EF showed high intensity
scatter that decayed on the 1,000100,000 µs time scale, suggesting
that particles larger than 1 µm in diameter were present
(Fig. 3C). Compounds
3 and 5, at up to 200 µM, gave low intensity,
poorly defined autocorrelation functions, consistent with the absence of
particles (Fig. 3D,
not shown for compound 5). These phenomena are consistent with the
notion that compounds 3 and 5 are classical, specific inhibitors
of EF, while part of the inhibition of EF by compounds 1, 2,
7, and 8 may be caused by the aggregation-based mechanism
(43,
45).

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FIG. 3. The promiscuity of compounds based on the in vitro adenylyl
cyclase assays (A and B) and on dynamic light scattering
(C and D). Adenylyl cyclase assays were performed in the
same way as in Fig. 2 except
that 160 pM EF3 was used in the 10x EF condition and a 10-min
incubation at 20 °C prior to the assay was done in the 20 °C
condition. Means ± S.E. are representative of at least two experiments,
and specific activities of EF3 without compounds in these experiments were in
the range of 1.42.4 ms1. Autocorrelation
functions from dynamic light scattering of 100 µM compound 1
(C) and 200 µM compound 3 (D) were performed in 50
mM KPi.
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Compounds 3 and 5
Block Round-up of Adrenocortical Y1 Cells Induced by EF and
PAThe increase of intracellular cAMP can cause actin-cytoskeleton
rearrangement and rounding up of mouse adrenocortical Y1 cells
(46); such changes are
commonly induced by bacterial toxins
(47). To monitor whether
compounds 17 can block the production of cAMP by EF, we took
advantage of the rapid morphological change (within 1 h) seen in mouse
adrenocortical Y1 cells in response to agents that increase cAMP. When H6-EF
and PA were added to cells together, we observed the expected round-up of Y1
cells (Fig. 4). However,
neither H6-EF nor PA alone also could induce round-up of Y1 cells (not shown).
We then tested compounds 17 in
Table I and found that only
compounds 3 and 5 could block the cAMP-induced round-up of Y1
cells at concentrations of 125 µM and above
(Fig. 4 and
Table I). The other compounds
had no effect on the round-up of Y1 cells even at a concentration of 1
mM (data not shown). These data together with the results above led
us to focus our subsequent analysis on compounds 3 and 5.

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FIG. 4. Effect of compounds 3 and 5 on the morphology of Y1 cells. Pictures
were taken 1 h after addition of EF and PA to final concentrations of 3 and 25
ng/ml, respectively. Y1 cells were incubated without EF and PA; with EF and
PA; with EF, PA, and 250 µM compound 3; or with EF, PA,
and 250 µM compound 5 as indicated.
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Specificity of Compounds 3 and
5We then tested the specificity of compounds
3 and 5 on CyaA-N, the adenylyl cyclase domain of CyaA, which is
an exotoxin secreted by B. pertussis
(Fig. 5, A and
B). We found that compounds 3 and 5
inhibited CaM-activated activity of CyaA-N with IC50 values of 40
and 80 µM, respectively. There are nine isoforms of
membrane-bound adenylyl cyclase found in mammals
(48). We expressed three of
them (type I, II, and V adenylyl cyclase) using Sf9 cells and tested whether
compounds 3 and 5 could modulate the activity of these enzymes
(Fig. 5, C and
D). All three enzymes are activated by forskolin and
recombinant Gs
, the
subunit of the stimulatory G
protein Gs. We found that, up to 500 µM, compound
3 reduced by 2045% the activity of all three mammalian adenylyl
cyclases, while compound 5 only marginally reduced the activities of
those three enzymes.

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FIG. 5. Effect of compounds 3 and 5 on bacterial (A and B) and
mammalian (C and D) adenylyl cyclases. The adenylyl
cyclase assays with EF or CyaA were done in the presence of 16 and 22
pM enzyme, 1 µM CaM, 1 µM free
Ca2+, 100 µM ATP, and the indicated
concentrations of compounds. The mammalian adenylyl cyclase assay was done
using 20 µg of Sf9 membranes containing mAC1, mAC2, or mAC5 in the presence
of 1 µM Gs , 50 µM forskolin, and
100 µM ATP. Mean ± S.E. are representative of at least
two experiments.
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Mechanism of Inhibition of EF by Compounds 3
and 5We also examined the mechanism
of how compounds 3 and 5 inhibit the catalytic activity of EF3
(Fig. 6). By varying substrate
and inhibitor concentrations, the kinetics of inhibition by compounds 3
and 5 were found to fit well for a competitive inhibition mechanism,
indicating that they compete directly with the binding of ATP
(Fig. 6A, data not
shown for compound 3). The estimated Ki
values were 50 and 20 µM for compounds 3 and 5,
respectively. Both EF and CyaA are activated by CaM. By loading a cutinase-CaM
fusion protein to a self-assembled monolayer using active site-directed
immobilization, we have used surface plasmon resonance spectroscopy to show
that EF can specifically bind to the immobilized CaM in a calcium-dependent
manner (38). Using this
method, we then examined whether compounds 3 and 5 affected the
interaction between EF and CaM (Fig.
6B). We found that the addition of compounds 3 and
5 did not change the affinity of EF to CaM at 10 µM free
Ca2+. This result indicated that these compounds did not
affect the interaction between EF and CaM.

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FIG. 6. Characterization of compounds 3 and 5 on the mechanism of
inhibition. A, kinetic properties in the inhibition of EF by
compound 5. The adenylyl cyclase assay was done in the presence of 16
pM EF3, 1 µM CaM, 1 µM free
Ca2+, and the indicated concentrations of compounds. The
Vmax, Km, and
Ki values were estimated to be 5
ms1, 50 µM, and 20
µM, respectively. B, the effect of compounds 3
and 5 on the binding of EF to CaM. Apparent
Kd was calculated using a simple
one-to-one-binding model: Req = RMax
x ([EF]/([EF] + )) where,
Req, RMax, and
are responses at equilibrium,
maximum response at excess EF, and apparent dissociation constant,
respectively. The SPR spectroscopy was performed in the presence of
Me2SO alone (open triangles), 500 µM
compound 3 (filled circles), and 500 µM compound
5 (open circles). Mean ± S.E. are representative of at
least two experiments.
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DISCUSSION
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Compound 5 is a novel, specific inhibitor of adenylyl cyclase toxins
from B. anthracis and B. pertussis. It blocks the
morphological change in Y1 cells induced by edema toxin without the inhibition
of mammalian type I, II, and V adenylyl cyclases. Despite its modest affinity
(20 µM), its specificity and activity in cell culture make it a
potentially good lead for an antitoxin against anthrax and whooping cough.
Thus, it is appropriate to consider how the affinity of the inhibitor might be
improved. In the absence of a crystal structure of an EF·inhibitor
complex, we turned to the docking-predicted geometries to understand the
binding of this compound. Based on our docking model, compound 5
overlaps primarily with the adenine group of the 3'-dATP structure,
consistent with our data that the mechanism of inhibition is competitive
(Fig. 7A). The
quinazolino ring fragment fits snugly into the pocket where the adenine group
binds where it would form the same three hydrogen bonds with the backbone
atoms of residues Thr-579 and Thr-548 as the adenine group does (distances are
between 3.0 and 3.4 Å). In addition, compound 5 appears to form a
hydrogen bond with the O
of Thr-548 through its ester oxygen atom
(distance is 2.8 Å). The ethoxyl group of compound 5 fits into a
shallow groove on the enzyme surface.

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FIG. 7. Data interpretation using the structural models. A, docked
structure of compound 5 compared with the observed structure of 3'-dATP
bound to the EF·CaM complex. Pictures are in stereoview. Protein
backbones are represented as green tubes. 3'-dATP and selective
active residues are shown in stick representation. Carbon atoms of
the proteins are colored in gray, and carbon of compound 5 is
in cyan. Oxygen atoms are in red, nitrogen is in
blue, phosphorus is in orange, and magnesium ion is in
magenta. Four hydrogen bonds between EF and compound 3 are
illustrated by dashed lines, and the bond distances are between 2.8
and 3.4 Å. B, the modeled conformation of compound 6.
The intramolecular hydrogen bond is illustrated by a dashed line, and
the bond distance between the two nitrogen atoms is 2.9 Å.
|
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Our model suggests that it will be possible to improve the affinity of this
compound to EF without compromising the specificity. EF binds the ribose
moiety of ATP in a manner that differs significantly from mAC. His-351 of EF
is believed to interact with the 3'-OH of the ribose, while a catalytic
metal is proposed to serve as the catalytic base. In addition, a hydrophobic
pocket centered around Phe-586 of EF has also been shown to play a vital role
in the binding of 3'-anthranyl group of the
2'-deoxy-3'-anthranyl-ATP of EF·CaM complex
(38). This pocket is proximal
to the putative binding site of compound 5 but is not currently used by
this compound; derivatives might be able to do so, thus improving affinity.
Finally the highly positively charged pocket formed by a catalytic metal and a
group of basic amino acids (Arg-329, Lys-346, Lys-353, and Lys-372), which
interacts with the phosphate groups of the nucleotide substrate, is not
exploited by compound 5
(6).
Compound 5 represents the first non-nucleoside-based inhibitor of
adenylyl cyclase toxins. It is dissimilar from nucleoside analogs and NKY80, a
previously described non-nucleosidebased inhibitor of host adenylyl cyclases
(49,
50). Several
triazolo[1,5-a]quinazoline compounds, which are structurally similar
to compound 5
(5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate) have been
synthesized and characterized recently
(51). A subset of
triazolo[1,5-a]quinazoline compounds is found to act as antagonists
of the adenosine receptor and the benzodiazepine receptor. Together with our
result, this suggests that azolo[1,5-a]quinazolines may be well
suited to mimic adenine.
Our data show that substituting the ester of compound 5 with a
secondary amide (compound 6) decreased the affinity by 10-fold. The
presence of an intramolecular hydrogen bond in compound 6
(Fig. 7B) may favor a
conformation that poorly fits the catalytic site of EF. As a tertiary amide,
compound 3 cannot form this intramolecular hydrogen bond and adopts a
conformation better suited to the binding site. The lack of activity of
compound 4, which also has a secondary amide, is consistent with this
view. Other possibilities such as the difference in solvation energy may also
explain our observation. Further structure-activity studies are required to
resolve this issue.
Our data also show that inhibitors against the catalytic site of EF from
B. anthracis can be identified by structure-based inhibitor
discovery. The hit rate in this computational approach, about 5%, is
consistent with a recent docking screen for novel inhibitors of
-lactamase as is the potency of the inhibitors discovered
(52). The hit rate is almost
10-fold lower than that in a large scale effort against a tyrosine phosphatase
(53) and is lower than that
found by several other docking programs (for a recent review, see Ref.
54). However, we have gone to
considerable effort to consider only non-promiscuous, biologically active
molecules as "true" hits, which diminished their numbers. We note
that promiscuous, aggregating small molecules appear to be relatively common
in hit lists from both virtual and high throughput screening
(44), and even widely used
biological reagents such as kinase inhibitors rottlerin (against protein
kinase C-
) and K-252c (against cAMP-dependent protein kinase and
protein kinase C) can act this way at micromolar concentrations
(45). Therefore, care must be
taken to exclude these promiscuous aggregators from hit lists in inhibitor
discovery projects.
 |
FOOTNOTES
|
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* This research was supported by National Institutes of Health Grants GM62548
and GM53459, and an American Heart Association Established Investigator Award
(to W.-J. T.), National Institutes of Health Grant 59957 (to B. K. S.), and
Defense Advanced Research Projects Agency Grant N00173-01-G010 and National
Science Foundation Grant DMR-9808595 (to M. M.). The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Both authors contributed equally to this work. 

To whom correspondence may be addressed: Dept. of Pharmaceutical Chemistry,
University of California, Genentech Hall, 600 16th St., San Francisco, CA
94143. E-mail:
shoichet{at}cgl.ucsf.edu.

To whom correspondence may be addressed: Ben-May Inst. for Cancer Research,
The University of Chicago, 924 East 57th St., Chicago, IL 60637. E-mail:
wtang{at}midway.uchicago.edu.
1 The abbreviations used are: PA, protective antigen; EF, edema factor; LF,
lethal factor; CaM, calmodulin; EF3, catalytic domain of EF (amino acids
291800); CyaA-N, catalytic domain of CyaA (amino acids 1393);
TEM, tumor endothelial marker; mAC, mammalian adenylyl cyclase. 
 |
ACKNOWLEDGMENTS
|
---|
We thank MDL for use of the Available Chemical Directory data base and the
program ISIS and OpenEye Software for the conformation generation program
Omega. We thank the Northwestern Keck Biophysics Facility for the dynamic
light scattering instrument.
 |
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