Bis-halogen-anthraniloyl-substituted nucleoside 5'-triphosphates as potent and selective inhibitors of Bordetella pertussis adenylyl cyclase toxin

Article abstract of DOI:10.1124/jpet.110.174219

Whooping cough is caused by Bordetella pertussis and still constitutes one of the top five causes of death in young children, particularly in developing countries. The calmodulin-activated adenylyl cyclase (AC) toxin CyaA substantially contributes to disease development. Thus, potent and selective CyaA inhibitors would be valuable drugs for the treatment of whooping cough. However, it has been difficult to obtain potent CyaA inhibitors with selectivity relative to mammalian ACs. Selectivity is important for reducing potential toxic effects. In a previous study we serendipitously found that bis-methylanthraniloyl (bis-MANT)-IMP is a more potent CyaA inhibitor than MANT-IMP (Mol Pharmacol 72:526-535, 2007). These data prompted us to study the effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)-substituted nucleotides on CyaA and mammalian ACs. The novel nucleotides differentially inhibited CyaA and ACs 1, 2, and 5. Bis-ANT nucleotides inhibited CyaA competitively. Most strikingly, bis-Cl-ANT-ATP inhibited CyaA with a potency ≥100-fold higher than ACs 1, 2, and 5. In contrast to MANT-ATP, bis-MANT-ATP exhibited low intrinsic fluorescence, thereby substantially enhancing the signal-to noise ratio for the analysis of nucleotide binding to CyaA. The high sensitivity of the fluorescence assay revealed that bis-MANT-ATP binds to CyaA already in the absence of calmodulin. Molecular modeling showed that the catalytic site of CyaA is sufficiently spacious to accommodate both MANT substituents. Collectively, we have identified the first potent CyaA inhibitor with high selectivity relative to mammalian ACs. The fluorescence properties of bis-ANT nucleotides facilitate development of a high-throughput screening assay. Copyright

Full text of DOI:10.1124/jpet.110.174219

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2010/10/20/jpet.110.174219.DC1.html
0022-3565/11/3361-104–115$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
JPET 336:104–115, 2011
Vol. 336, No. 1
174219/3654614
Printed in U.S.A.
Bis-Halogen-Anthraniloyl-Substituted Nucleoside
5Ј-Triphosphates as Potent and Selective Inhibitors of
□
S
Bordetella pertussis Adenylyl Cyclase Toxin
Jens Geduhn, Stefan Dove, Yuequan Shen, Wei-Jen Tang, Burkhard Ko¨ nig,
and Roland Seifert
Institute of Organic Chemistry (J.G., B.K.) and Department of Pharmaceutical and Medicinal Chemistry II (S.D.), University of
Regensburg, Regensburg, Germany; College of Life Sciences, Nankai University, Tianjin, People’s Republic of China (Y.S.);
Ben May Department of Cancer Research, University of Chicago, Chicago, Illinois (W.-J.T.); and Medical School of Hannover,
Hannover, Germany (R.S.)
Received August 19, 2010; accepted October 18, 2010
ABSTRACT
Whooping cough is caused by Bordetella pertussis and still nucleotides differentially inhibited CyaA and ACs 1, 2, and 5.
constitutes one of the top five causes of death in young chil- Bis-ANT nucleotides inhibited CyaA competitively. Most strikingly,
dren, particularly in developing countries. The calmodulin-acti- bis-Cl-ANT-ATP inhibited CyaA with a potency Ն100-fold higher
vated adenylyl cyclase (AC) toxin CyaA substantially contrib- than ACs 1, 2, and 5. In contrast to MANT-ATP, bis-MANT-ATP
utes to disease development. Thus, potent and selective CyaA exhibited low intrinsic fluorescence, thereby substantially enhanc-
inhibitors would be valuable drugs for the treatment of whooping ing the signal-to noise ratio for the analysis of nucleotide binding
cough. However, it has been difficult to obtain potent CyaA inhib- to CyaA. The high sensitivity of the fluorescence assay revealed
itors with selectivity relative to mammalian ACs. Selectivity is that bis-MANT-ATP binds to CyaA already in the absence of
important for reducing potential toxic effects. In a previous study calmodulin. Molecular modeling showed that the catalytic site of
we serendipitously found that bis-methylanthraniloyl (bis-MANT)- CyaA is sufficiently spacious to accommodate both MANT sub-
IMP is a more potent CyaA inhibitor than MANT-IMP (Mol Phar- stituents. Collectively, we have identified the first potent CyaA
macol 72:526–535, 2007). These data prompted us to study the inhibitor with high selectivity relative to mammalian ACs. The
effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)- fluorescence properties of bis-ANT nucleotides facilitate develop-
substituted nucleotides on CyaA and mammalian ACs. The novel ment of a high-throughput screening assay.
Pebody, 2006). Thus, novel strategies for the treatment of
whooping cough are urgently needed.
B. pertussis secretes two virulence factors that substan-
tially contribute to the pathogenesis of whooping cough. Per-
tussis toxin ADP-ribosylates Gi protein ␣-subunits and,
thereby, blocks the coupling of chemoattractant receptors to
Gi proteins and cellular effector systems in phagocytes that
kill invading bacteria (Carbonetti, 2010). This mechanism is
complemented by the AC toxin CyaA, a protein consisting of
1706 amino acids. After secretion from the bacteria, CyaA
inserts into the plasma membrane of host cells. CyaA then
binds calmodulin (CaM), stimulating its AC activity and re-
sulting in massive production of cAMP (Ladant and Ull-
mann, 1999; Vojtova et al., 2006). cAMP, like pertussis toxin,
blunts the host-defense function of phagocytes. Accordingly,
the synergistic actions of pertussis toxin and CyaA facilitate
Introduction
Whooping cough is caused by the Gram-negative bacte-
rium Bordetella pertussis (Guiso, 2009; Carbonetti, 2010).
Although vaccinations against whooping cough are available
and the disease can be treated with antibiotics, it is still one
of the five leading causes of death in young children, partic-
ularly in countries of the developing world (Crowcroft and
This work was supported by the Deutsche Forschungsgemeinschaft [Grant
529/5–2] (to R. S.) and the National Institutes of Health National Institute of
General Medical Sciences [Grant GM 062558] (to W.-J.T.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.174219.
□S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: AC, adenylyl cyclase; mAC, mammalian membranous AC; ANT, anthraniloyl; CaM, calmodulin; CyaA, Bordetella pertussis
adenylyl cyclase toxin; HPLC, high-pressure liquid chromatography; MANT, methylanthraniloyl; MS, mass spectroscopy; NDP, nucleoside
5Ј-diphosphate; NMP, nucleoside 5Ј-monophosphate; PMEApp, 9-[2-(phosphonomethoxy)ethyl]adenine diphosphate; Pr, propyl; Rt, retention
time; PDB, Protein Data Bank; DMSO, dimethyl sulfoxide; a.u., arbitrary units.
104

CyaA Inhibition by Bis-Halogen-ANT Nucleotides
105
colonization of the respiratory tract with bacteria (Ladant for this study was the serendipitous finding that bis-MANT-
and Ullmann, 1999; Vojtova et al., 2006; Carbonetti, 2010). IMP was an at least 5-fold more potent inhibitor of CyaA
As a result, the infection becomes more severe and lasts than mono-MANT-IMP (Go¨ttle et al., 2007). However, the Ki
longer.
value of bis-MANT-IMP is still very low, i.e., in the range of
Based on the pathophysiological function of CyaA, it is a 20 to 40 ␮M, depending on the experimental conditions.
logical approach to develop CyaA inhibitors. In fact, several Because the length of the phosphate chain is important for
noncompetitive so-called P-site inhibitors for mammalian high potency of AC inhibitors (Mou et al., 2005, 2006; Go¨ttle
ACs also inhibit the catalytic activity of CyaA (Johnson and et al., 2007; Taha et al., 2009), we focused our synthesis effort
Shoshani, 1990). However, the potency of these inhibitors is on NTPs. The ANT group was also modified by halogens and
rather low compared with mammalian ACs, rendering them an acetylated amino group at the 5-position of the phenyl
unsuitable as a starting point for drug development. High- ring system. Moreover, the amino function of the ANT group
throughput screening studies yielded a low-potency (Ki, 20 was modified with propyl groups. Figure 1 provides an over-
␮M) CyaA inhibitor (Soelaiman et al., 2003). To this end, 9-[2- view fn the structural properties of the newly synthesized
(phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the compounds. For comparison with CyaA, we studied ACs 1, 2,
active metabolite of the antiviral drug adefovir, is the most and 5 as representative membranous mammalian ACs
potent CyaA inhibitor known so far (Ki in the presence of Mn2ϩ
1 nM) (Guo et al., 2005). However, little is known about the and representative nucleotides and modeled the interaction
selectivity of PMEApp for CyaA relative to mammalian ACs. of CyaA with bis-MANT nucleotides. Here, we report that
,
(mACs). We also conducted fluorescence studies with CyaA
In other studies we have shown that 2Ј(3Ј)-O-MANT-sub- bis-Cl-ANT-ATP is a very potent CyaA inhibitor with sub-
stituted NTPs are moderately potent competitive CyaA in- stantial selectivity relative to mACs.
hibitors with Ki values in the range of 0.2 to 10 ␮M (Gille et
al., 2004; Go¨ttle et al., 2007). CyaA exhibits a broad base
specificity for purine and pyrimidine nucleotides (Go¨ttle et
Materials and Methods
al., 2007, 2010), indicating that the catalytic site possesses
substantial conformational flexibility and is spacious. Figure 1
provides an overview of structures of MANT nucleotides.
MANT nucleotides are fluorescent and can be used to moni-
tor the binding of nucleotides to CyaA and conformational
changes in CyaA upon binding of CaM. Interaction of the
MANT group with Phe306 results in a CaM-dependent in-
crease in fluorescence (Go¨ttle et al., 2007).
Materials. MANT-UTP, MANT-UDP, MANT-CTP, MANT-CDP,
MANT-ITP, MANT-IDP, MANT-IMP, ANT-IMP, ANT-ATP, and
ANT-ADP were synthesized according to Hiratsuka (1983) with mod-
ifications described in Taha et al. (2009). Methylisatoic anhydride,
isatoic anhydride, chloroisatoic anhydride, bromoisatoic anhydride,
aminoisatoic anhydride, ATP, ITP, CTP, IMP, and bovine serum albu-
min, fraction V, highest quality, were purchased from Sigma-Aldrich
(Seelze, Germany). MnCl2 tetrahydrate (highest quality) and alumi-
num oxide 90 active, (neutral, activity 1; particle size, 0.06–0.2 mm)
were from MP Biomedicals (Eschwege, Germany). PMEApp was sup-
plied by Gilead Sciences (Foster City, CA). The catalytic domain of B.
In an effort to obtain potent and selective CyaA inhibitors,
in the present study, we examined the effects of 32 substi-
tuted bis- and mono-(M)ANT nucleotides. The starting point pertussis AC protein (CyaA, amino acids 1–373) was purified as de-
Bases:
R2
Base
O
O
O
N
NH2
NH2
N
O
HO
P
O
P
O
2
O
N
N
N
N
N
NH
N
OH
OH
+
O
R1
0
N
O
OH
OH
N
O
hypoxanthine
cytosine
adenine
38 °C
pH = 8.6
Substituent
MANT
R1
H
R2
CH3
Base
Base
O
O
O
O
ANT
H
H
HO
P
O
P
O
HO
P
O
P
O
2
O
O
O
H
O
H
O
OH
OH
OH
OH
Cl-ANT
Cl
Br
H
H
0
2
0
H
H
OH
O
O
O
Br-ANT
Pr-ANT
Ac-NH-ANT
H
CH2CH2CH3
H
H
N
H
N
H
N
R2
R2
R2
R1 R1
R1
NH2COCH3
Fig. 1. Overview of the structures and synthesis of mono- and bis-(M)ANT-substituted nucleotides. Nucleotides differ from each other in terms of base,
phosphate chain length, and substitution of the 2Ј- and 3Ј-O-ribosyl group. In mono-(M)ANT-substituted nucleotides, the substituent can isomerize
spontaneously between the 2Ј and 3Ј positions (Suryanarayana et al., 2009). In bis-(M)ANT-substituted nucleotides, this isomerization cannot occur
anymore. In a single step, nucleotides and diversified isatoic anhydrides are reacted to the corresponding acylated nucleotides.

106
Geduhn et al.
scribed previously (Shen et al., 2002). [␣-32P]ATP (800 Ci/mmol) was more, the formation of bis-MANT nucleotides was also confirmed by
purchased from PerkinElmer Life and Analytical Sciences (Rodgau LC/MS online coupling. Under the basic reaction conditions, degra-
Ju¨gesheim, Germany). Lyophilized calmodulin from bovine brain was dation of the labile ␥-phosphate always occurred for mono- and
from Calbiochem (Darmstadt, Germany). Forskolin was supplied by LC bis-(M)ANT nucleotides. Thus, preparative HPLC was applied for
Laboratories (Woburn, MA). For all experiments double-distilled water the separation of (M)ANT-NTPs and (M)ANT-NDPs. In general, the
was used. Sources of all other biochemical reagents have been described purification by this method offered the possibility to obtain four
previously (Gille et al., 2004; Go¨ttle et al., 2007; Taha et al., 2009).
compounds simultaneously.
Mono- and Bis-MANT Nucleotide Synthesis General Proce-
Cell Culture and Membrane Preparation. Cell culture and
dure. Synthesis of new substituted mono- and bis-(M)ANT nucleo- membrane preparations (Gille et al., 2004) were performed as de-
tides followed the general reaction scheme shown in Fig. 1 to obtain scribed previously. In brief, Sf9 cells were cultured in SF 900 II
(bis-)Cl-ANT-ATP, (bis-)Cl-ANT-ITP, (bis-)Br-ANT-ATP, (bis-)Br- medium (Invitrogen, Carlsbad, CA) supplemented with 5% (v/v) fetal
ANT-ITP, (bis-)Br-ANT-ADP and (bis-)Pr-ANT-ATP, (bis-)Pr-ANT- bovine serum and 0.1 mg/ml gentamicin. High-titer baculoviruses for
ITP and (bis-)Ac-NH-ANT-ATP, and (bis-)Ac-NH-ANT-ITP. Furthermore, ACs 1, 2, and 5 were generated through two sequential amplification
we generated the bis-(M)ANT derivatives of known mono-(M)ANT steps as described previously (Gille et al., 2004). In each amplifica-
nucleotides, i.e., bis-MANT-ATP, bis-MANT-ITP, bis-MANT-CTP, tion step the supernatant fluid was harvested and stored under light
bis-MANT-ADP, bis-MANT-ADP, bis-MANT-IMP, and bis-ANT- protection at 4°C. For membrane preparation Sf9 cells (3.0 ϫ 106
IMP. Detailed synthesis procedures and chemical analysis of com- cells/ml) were infected with corresponding baculovirus encoding dif-
pounds, chemical structures, and their purity are documented in ferent ACs (1:100 dilutions of high-titer virus) and cultured for 48 h.
Supplementary Information 1. Under the basic reaction conditions Membranes expressing each construct and membranes from unin-
mono- and bis-(M)ANT-NTPs partially decomposed to the corre- fected Sf9 cells were prepared as described. Cells were harvested,
sponding NDPs. Those compounds were isolated as well.
and cell suspensions were centrifuged for 10 min at 1000g at 4°C.
During the synthesis of MANT-IMP, we observed a large new peak Pellets were resuspended in 10 ml of lysis buffer (1 mM EDTA, 0.2
at later retention times, when the crude reaction mixture was ana- mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptine, and 10
lyzed by reversed-phase HPLC. Because of the long retention time of ␮g/ml benzamide, pH 7.4). Thereafter, cells were lysed with 20 to 25
the unknown peak, a more lipophilic compound with additional non- strokes by using a Dounce homogenizer. The resultant cell fragment
polar groups was expected. Thus, substitution of a second MANT suspension was centrifuged for 5 min at 500g and 4°C to sediment
group was hypothesized. The analysis of LC/MS online coupling nuclei. The cell membrane-containing supernatant suspension was
corroborated the hypothesis. The esterfication of an additional transferred into 30-ml tubes and centrifuged for 20 min at 30,000g
MANT group was identified by the mass-per-charge ratio of 613.2 Da and 4°C. The supernatant fluid was discarded, and cell pellets were
for the negative electrospray ionization measurement. The chro- resuspended in buffer consisting of 75 mM Tris/HCl, 12.5 mM MgCl2,
matogram of the crude reaction mixture displayed the typical two- and 1 mM EDTA, pH 7.4. Membrane aliquots of 1 ml were prepared
peak system for the expected N-methyl-2Ј- and 3Ј-O-anthraniloyl and stored at Ϫ80°C, and protein concentration for each membrane
nucleotide isomers at a retention time of 21.4 and 22.0 min, respec- preparation was determined with the Bio-Rad DC protein assay kit
tively. However, despite separation by LC, purified isomers cannot (Bio-Rad Laboratories, Hercules, CA).
be used experimentally because of rapid reisomerization (Jameson and
Eccleston, 1997). Seven minutes later, the peak for bis-MANT-ITP ap- ACs 1, 2, or 5 was determined essentially as described previously
peared (Rt ϭ 29 min). The high polarity of nonreacted IMP resulted (Go¨ttle et al., 2009). Before starting experiments, membranes were
AC Activity Assay. AC activity in Sf9 membranes expressing
in a fast elution directly after the dead time (minor peak at Rt Ͻ 2 sedimented by a 15-min centrifugation at 4°C and 15,000g and
min). Further peaks were identified for the excess of methylisatoic resuspended in 75 mM Tris/HCl, pH 7.4. Reaction mixtures (50 ␮l,
anhydride (Rt ϳ17 min) and decomposition product of the nucleoside final volume) contained 20 to 40 ␮g of membrane protein, 40 ␮M
of hypoxanthine (Rt ϳ23 min). The assignment of all signals was ATP/Mn2ϩ plus 5 mM MnCl2, 100 ␮M forskolin, 10 ␮M GTP␥S, and
achieved by LC/MS online coupling. For the purification of IMP (M)ANT nucleotides at concentrations from 0.1 nM to 1 mM as
derivatives, only size-exclusion chromatography was required, yield- appropriate to obtain saturated inhibition curves. After a 2-min
ing MANT nucleotides of high purity of approximately 99%. Confir- preincubation at 37°C, reactions were initiated by adding 20 ␮l of
mation for production of bis-MANT nucleotides was provided by reaction mixture containing (final) 1.0 to 1.5 ␮Ci/tube [␣-32P]ATP
NMR spectroscopy. Structure determination of bis-MANT-IMP by and 0.1 mM cAMP. AC assays were conducted in the absence of an
one- and two-dimensional NMR measurements confirmed our as- NTP-regenerating system to allow for the analysis of (M)ANT-NDPs
sumption of the second acylated free hydroxyl group. The proton that could otherwise be phosphorylated to the corresponding
spectrum showed two sets of signals for the two MANT groups. (M)ANT-NTPs (Gille et al., 2004). Reactions were conducted for 20
Furthermore, the heteronuclear multiple bond coherence spec- min at 37°C and terminated by adding 20 ␮l of 2.2 N HCl.
trum definitely identified 3J correlation between protons and
quaternary carbons to ensure no substitution in the purine system 10 ␮l of inhibitor at final concentrations from 1 nM to 100 ␮M and 20
of the nucleobase. ␮l of CyaA protein (final concentration, 10 pM) in 75 mM HEPES/
For the determination of CyaA AC activity, assay tubes contained
At the beginning of our in-house MANT-NTP synthesis program, NaOH, pH 7.4, containing 0.1% (mass/v) bovine serum albumin.
we did not observe formation of bis-substituted MANT-NTPs. How- After a 2-min preincubation at 25°C reactions were initiated by the
ever, after the serendipitous discovery of bis-MANT-IMP (Go¨ttle et addition of 20 ␮l of reaction mixture consisting of (final) 100 mM
al., 2007), we addressed the question of whether bis-MANT-NTPs KCl, 10 ␮M free Ca2ϩ, 5 mM free Mn2ϩ, 100 ␮M EGTA, 100 ␮M
were produced as well. The standard purification procedure was
cAMP, 100 nM calmodulin, 40 ␮M ATP, and [␣-32P]ATP (0.2 ␮Ci/
performed by size-exclusion chromatography for separation of start- tube). For the determination of Km and Vmax values in kinetic stud-
ing materials. Nonreacted nucleotide and isatoic anhydride were ies, 10 ␮M to 2 mM ATP/Mn2ϩ plus 5 mM free Mn2ϩ were added. To
removed by this method as precleaning. Unfortunately, bis-MANT- ensure linear reaction progress, tubes were incubated for 10 min at
NTPs were lost by this separation as well. When we performed 25°C, and reactions were stopped by adding 20 ␮l of 2.2 N HCl.
HPLC analysis of the crude reaction mixture without the preclean- Denatured protein was precipitated by a 1-min centrifugation at
ing, similar peaks as for bis-MANT-IMP appeared in the chromato- 25°C and 15,000g. Sixty microliters of the supernatant fluid was
gram. Diode array detection and fluorescence detection for HPLC applied onto disposable columns filled with 1.3 g of neutral alumina.
analysis supported the assignment of bis-(M)ANT nucleotide signals
because of the similarity of spectroscopic properties to mono-substi- with 4 ml of 0.1 M ammonium acetate, pH 7.0. Recovery of
tuted (M)ANT nucleotides, especially of UV absorption. Further-
32P]cAMP was ϳ80% as assessed with [3H]cAMP as standard.
[
32P]cAMP was separated from [␣-32P]ATP by elution of [32P]cAMP
[

CyaA Inhibition by Bis-Halogen-ANT Nucleotides
107
Blank values were approximately 0.02% of the total added amount of were calculated and visualized by the program MOLCAD (MOLCAD,
[␣-32P]ATP; substrate turnover was Ͻ3% of the total added
Darmstadt, Germany) contained within SYBYL.
[␣-32P]ATP. Samples collected in scintillation vials were filled up
with 10 ml of double-distilled water and Cerenkov radiation was
Results
measured in
a Tri-Carb 2800TR liquid scintillation analyzer
(PerkinElmer Life and Analytical Sciences, Waltham, MA). Free
concentrations of divalent cations were calculated with Win-MaxC
(http://www.stanford.edu/ϳcpatton/downloads.htm). Competition iso-
therms were analyzed by nonlinear regression using Prism 4.0 software
(GraphPad Software Inc., San Diego, CA). Km values were 120 ␮M
(AC1), 100 ␮M (AC2), 70 ␮M (AC5) and were taken from Gille et al.
(2004). The Km value for CyaA was 45 ␮M and was determined in our
previous study (Go¨ttle et al., 2007).
Structure-Activity Relationships of Mono-(M)ANT
Nucleotides for mACs and CyaA. We examined the inhibi-
tory effects of 16 mono-MANT nucleotides on the catalytic ac-
tivity of mammalian ACs 1, 2, and 5 and bacterial CyaA (Table 1).
Nucleotides differed from each other in base (adenine, hypo-
xanthine, and cytosine) and phosphate chain length (mono-
phosphate, diphosphate, or triphosphate). Mono-substituted
compounds (1–16) undergo spontaneous isomerization under
physiological pH between the 2Ј- and 3Ј-O-ribosyl positions
(Suryanarayana et al., 2009). The ANT moiety differed by
hydrogen, chlorine, bromine, and an acetylamino group in the
5-position of the phenyl ring (Fig. 1; R1). Moreover, substitution
at the amino function of the ANT-group led to methyl (MANT)
and propyl (Pr-ANT) derivatives (Fig. 1; R2).
Recombinant ACs 1, 2, and 5 showed different sensitivity
to inhibition by (M)ANT nucleotides (Table 1). In accordance
with previous studies (Gille et al., 2004; Go¨ttle et al., 2009),
AC2 was the AC isoform with the lowest inhibitor sensitivity
because of the exchange of A409P and V1108I in AC1 and
AC5 versus AC2 (Mou et al., 2005). (M)ANT-NTPs with the
purine base hypoxanthine had the highest potency. Specifi-
cally, MANT-ITP is the most potent inhibitor known so far
for AC1 and AC5 with Ki values of 1 to 3 nM (Go¨ttle et al.,
2009). The exchange of adenine with cytosine had only mar-
ginal impact on potency (1 and 3). The halogenated ANT-ATP
derivatives (4 and 6), in comparison with MANT-ATP (1),
showed slightly more potent inhibition of ACs 1 and 5. The
corresponding inosine compounds (5 and 7) exhibited lower
potency than MANT-ITP on a still high level. Substitution of
bromine with chlorine in ANT-ITP yielded 2-fold more potent
inhibition of mACs (7 3 5). Elongation of the alkyl residue
from N-methylated to N-propylated ANT nucleotides lowered
Ki values 3- to 8-fold (1 3 8, 2 3 9). A further decrease in
potency was observed for the acetylated aminoanthraniloyl
nucleotides. The inhibition effect of Ac-NH-ANT-ATP (10)
dropped into the micromolar range, and the corresponding
inosine derivative (11) was 30- to 50-fold less potent than
MANT-ITP (2). It is noteworthy that Ac-NH-ANT-ITP (11)
displayed the highest 4-fold selectivity for AC5 compared
with AC1. Deletion of the ␥-phosphate reduced inhibitor af-
finity 3- to 26-fold (1 3 12, 2 3 13, 6 3 14) and is in
accordance with previous data. Crystallographic studies
showed that the Mn2ϩ ion in the B-site coordinates with the
␥-phosphate of MANT-NTPs (Mou et al., 2005, 2006). The lack
Fluorescence Spectroscopy. Experiments were conducted by
using a quartz UV ultra-microcuvette from Hellma (Mu¨llheim, Ger-
many) (light path length, 3 mm; center, 15 mm; total volume, 70 ␮l;
type 105.251-QS). Measurements were carried out in a Cary Eclipse
fluorescence spectrometer (Varian Inc., Palo Alto, CA) at a constant
temperature of 25°C (scan rate, 120 nm/min; averaging time, 0.5 s;
photomultiplier voltage, 700 V; data interval, 1 nm; slit width, 5 nm)
(Go¨ttle et al., 2007). Initially, the cuvette contained 64 ␮l of 75 mM
HEPES/NaOH buffer, pH 7.4, 100 ␮M CaCl2, 100 mM KCl, and 5
mM MnCl2. Next, nucleotide, CyaA, and CaM were added succes-
sively. The cuvette content was mixed after each addition to end up
with a total volume of 70 ␮l. (M)ANT nucleotides were excited at 350
nm, and steady-state emission spectra were recorded from 380 to 550
nm at low speed. The final concentrations of CyaA and CaM were 2.4
␮M each. Basal fluorescence in the presence of buffer alone was
subtracted. (M)ANT nucleotides were displaced from CyaA by PMEApp at
concentrations of 100 nM to 3 ␮M. For an estimation of the respon-
siveness of the MANT group to a hydrophobic environment, fluores-
cence of mono- and bis-(M)ANT nucleotides was determined with
dimethyl sulfoxide ranging from 0 to 100% (v/v). Fluorescence re-
cordings were analyzed with the spectrum package of Cary Eclipse
software (Varian).
Modeling of Nucleotide Binding Mode to mAC and CyaA.
Docking studies were performed with the molecular modeling pack-
age SYBYL 7.3 (Tripos, St. Louis, MO) on an Octane workstation
(SGI, Mountain View, CA). Initial computer models of bis-Br-ANT-
ITP in complex with mAC and bis-Br-ANT-ATP in complex with
CyaA were generated from the PDB crystal structures 3g82 (3Ј-
MANT-ITP bound to mAC) and 1zot (PMEApp bound to CyaA) (Guo
et al., 2005), respectively. In the case of CyaA, the starting confor-
mation of bis-Br-ANT-ATP was derived from complexes of 3Ј-MANT-
ATP and TNP-ATP with mAC (PDB structures 2gvz and 2gvd, re-
spectively) (Mou et al., 2005, 2006). An initial docking position
resulted from superposition of roughly optimized conformations with
PMEApp, allowing the modification of rotatable bonds, and from
consideration of the fluorescence data (interaction of the 3Ј-MANT
group with Phe306). In both cases, the ribosyl conformations were
adjusted to avoid clashes of the second Br-ANT group with the cores
of mAC and CyA, respectively. Hydrogens were added and charges
were assigned (proteins and water molecules, AMBER_FF99; li-
gands, Gasteiger-Hueckel). The ions (Mn2ϩ or Mg2ϩ) received formal of this phosphate group destabilizes the diphosphate chain in
charges of 2. Each complex was refined in a stepwise approach. First,
ϳ50 minimization cycles with fixed ligand using the AMBER_FF99
force field (Cornell et al., 1995) (steepest descent method); second,
ϳ100 minimization cycles of the ligand and the surrounding (dis-
tance up to 6 Å) protein residues (Tripos force field) (Clark et al.,
1989), and third, ϳ100 minimization cycles with fixed ligand
(AMBER_FF99 force field, Powell conjugate gradient) were performed.
The second and third steps were repeated with a larger number of
cycles until a root mean square force of 0.01 kcal/mol ϫ ÅϪ1 was
approached. To avoid overestimation of electrostatic interactions, a
distance-dependent dielectric constant of 4 was applied. Molecular
surfaces and lipophilic potentials (protein variant with the new
its binding site. The deletion of the ␤-phosphate group of the
MANT-NMP reduced inhibitor potency 120-fold (13 3 15). Ex-
change of the MANT group with an ANT group had only little
effect on inhibitor affinity (15 3 16).
Generally, the inhibitor potency of mono-(M)ANT-NTPs at
CyaA was lower than at mACs or maximally reached the
affinity range of AC2. MANT-ATP (1) was less potent than
MANT-ITP (2), but at all other combinations of adenosine
and inosine derivatives the corresponding ANT-ATPs exhib-
ited higher inhibition potency (4 3 5, 6 3 7, 8 3 9, 10 3 11).
MANT-CTP (3) is the most potent MANT nucleotide inhibitor
Crippen parameter table) (Heiden et al., 1993; Ghose et al., 1998) of edema factor AC toxin from Bacillus anthracis with a Ki

108
Geduhn et al.
TABLE 1
Inhibition of catalytic activity of recombinant mammalian ACs 1, 2, and 5 and bacterial AC toxin CyaA by (M)ANT nucleotides
Activity of ACs 1, 2, and 5 and CyaA was determined in the presence of (M)ANT nucleotides at increasing concentrations. Ki values were calculated by nonlinear regression.
Data are the mean values Ϯ S.D. of four to five independent experiments performed in triplicates with at least two different membrane preparations (for mACs).
(M)ANT Nucleotide
AC 1
AC 2
nM
AC 5
CyaA
1
2
MANT-ATP
150 Ϯ 40
2.8 Ϯ 0.9
150 Ϯ 30
80 Ϯ 4
3.3 Ϯ 0.3
120 Ϯ 20
7.0 Ϯ 0.1
440 Ϯ 20
22 Ϯ 1
6800 Ϯ 200
140 Ϯ 30
1300 Ϯ 200
39 Ϯ 12
560 Ϯ 10
4600 Ϯ 400
7400 Ϯ 1200
700 Ϯ 200
310 Ϯ 20
620 Ϯ 40
1700 Ϯ 100
66 Ϯ 1
330 Ϯ 100
13.5 Ϯ 0.5
690 Ϯ 20
100 Ϯ 30
1.2 Ϯ 0.1
150 Ϯ 30
45 Ϯ 1
2.0 Ϯ 0.2
70 Ϯ 20
4.6 Ϯ 0.4
360 Ϯ 60
10 Ϯ 2
3400 Ϯ 40
37 Ϯ 7
4300 Ϯ 400
600 Ϯ 100
1100 Ϯ 100
350 Ϯ 40
700 Ϯ 200
330 Ϯ 30
920 Ϯ 50
820 Ϯ 250
3800 Ϯ 500
710 Ϯ 40
4800 Ϯ 900
12,000 Ϯ 2000
11,000 Ϯ 3000
8700 Ϯ 1300
Ͼ100,000
Ͼ100,000
360 Ϯ 10
3000 Ϯ 400
2500 Ϯ 500
16 Ϯ 1
15 Ϯ 1
12.6 Ϯ 0.3
20 Ϯ 2
700 Ϯ 200
2100 Ϯ 100
280 Ϯ 20
7500 Ϯ 100
6500 Ϯ 800
6600 Ϯ 900
91 Ϯ 5
MANT-ITP
3
MANT-CTP
4
Cl-ANT-ATP
490 Ϯ 20
5
Cl-ANT-ITP
8 Ϯ 1
6
Br-ANT-ATP
450 Ϯ 40
7
Br-ANT-ITP
22 Ϯ 4
8
Pr-ANT-ATP
1100 Ϯ 100
68 Ϯ 12
9
Pr-ANT-ITP
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Ac-NH-ANT-ATP
Ac-NH-ANT-ITP
MANT-ADP
11,000 Ϯ 1000
390 Ϯ 80
2900 Ϯ 500
86 Ϯ 9
1700 Ϯ 100
8200 Ϯ 800
7500 Ϯ 1,400
2100 Ϯ 600
1100 Ϯ 200
7800 Ϯ 100
2400 Ϯ 100
200 Ϯ 10
800 Ϯ 200
31 Ϯ 12
MANT-IDP
Br-ANT-ADP
280 Ϯ 20
3400 Ϯ 200
4300 Ϯ 600
430 Ϯ 50
140 Ϯ 40
750 Ϯ 40
1600 Ϯ 100
65 Ϯ 3
MANT-IMP
ANT-IMP
Bis-MANT-ATP
Bis-MANT-ITP
Bis-MANT-CTP
Bis-Cl-ANT-ATP
Bis-Cl-ANT-ITP
Bis-Br-ANT-ATP
Bis-Br-ANT-ITP
Bis-Pr-ANT-ATP
Bis-Pr-ANT-ITP
Bis-Ac-NH-ANT-ATP
Bis-Ac-NH-ANT-ITP
Bis-MANT-ADP
Bis-MANT-IDP
Bis-Br-ANT-ADP
Bis-MANT-IMP
Bis-ANT-IMP
670 Ϯ 50
21 Ϯ 1
1100 Ϯ 100
71 Ϯ 2
900 Ϯ 90
15 Ϯ 2
18,000 Ϯ 3000
440 Ϯ 10
22,000 Ϯ 1000
1700 Ϯ 100
700 Ϯ 300
1000 Ϯ 100
1600 Ϯ 300
Ͼ100,000
Ͼ100,000
36,000 Ϯ 3000
1400 Ϯ 100
7000 Ϯ 1000
5600 Ϯ 300
1600 Ϯ 300
1400 Ϯ 100
3300 Ϯ 200
Ͼ100,000
Ͼ100,000
18,000 Ϯ 5000
250 Ϯ 20
6100 Ϯ 1300
480 Ϯ 30
510 Ϯ 70
700 Ϯ 200
1800 Ϯ 200
Ͼ100,000
Ͼ100,000
20,000 Ϯ 3000
12,000 Ϯ 3000
value of 100 nM (Taha et al., 2009). This preference for ATP (18) and bis-MANT-CTP (19) inhibited AC1 and AC5
cytosine was not observed for CyaA (Go¨ttle et al., 2007). with almost equal potency. Bis-halogen-ANT-ITPs (21 and
Insertion of halogens in ANT-ATPs reduced Ki values by 12- 23) showed Ki values between 15 and 66 nM for AC1 and
to 13-fold (1 3 4, 1 3 6). However, the Ki values of the AC5, but the corresponding adenine nucleotides (20 and 22)
corresponding inosine derivatives remained almost constant exhibited 25- to 60-fold lower potency. N-propylated instead
(2 3 5, 2 3 7). Pr-ANT-ATP (8) and Ac-NH-ANT-ATP (10) of N-methylated ANT nucleotides lost affinity dramatically
revealed still stable inhibitory potency, but Pr-ANT-ITP (9) only for bis-Pr-ANT-ATP (24) (17- to 42-fold) but not for
and Ac-NH-ANT-ITP (11) lost 6- to 8-fold inhibitor affinity bis-Pr-ANT-ITP (25). Bis-Ac-NH-ANT-ATP (26) exhibited
compared with MANT-ITP (1). Deletion of ␥-phosphate re- low inhibition potency, but possessed 3-fold higher sensitiv-
duced inhibitor potency 3- to 20-fold, and omission of ␥- and ity for AC2 and AC5 compared with AC1. Usually, the re-
␤-phosphates further decreased inhibition up to the high maining inhibitors showed higher potencies at ACs 1 and 5
micromolar range.
than AC2. It is noteworthy that bis-MANT-ADP (28) was as
Structure-Activity Relationships of Bis-Substituted potent as bis-MANT-ATP (17). Bis-MANT-IDP (29) and bis-Br-
(M)ANT Nucleotides for mACs and CyaA. We examined ANT-ADP (30) were only 1.3- to 5-fold less potent than the
the inhibitory effects of 16 bis-MANT nucleotides on the corresponding (M)ANT-NTPs. Bis-MANT-IMP (31) and bis-
catalytic activity of mACs 1, 2, and 5 and bacterial CyaA ANT-IMP (32) showed only exceedingly low affinity for mACs.
(Table 1). Overall, bis-(M)ANT nucleotides exhibited lower
At CyaA, bis-substituted (M)ANT-NTPs exhibited a broad
inhibition potencies for ACs 1, 2, and 5 than the correspond- variety in potency with Ki values from the nanomolar up to
ing mono-substituted derivatives. However, the reduction of the micromolar range. In general, inhibition potencies of
the potency does not consistently depend on the nucleobase. adenine nucleotides were the highest. Bis-MANT-CTP (19)
In the case of bis-MANT and bis-Ac-NH-ANT derivatives, and bis-MANT-ITP (18) exhibited 7- and 8-fold lower inhibi-
hypoxanthine led to the strongest decrease, whereas the af- tion potency than bis-MANT-ATP (17). It is noteworthy
finity of bis-halogen-ANT nucleotides was more reduced with that halogenated bis-ANT-nucleotide derivatives (20–23) in-
adenine. Comparing mono- and bis-(M)ANT nucleotides, the creased the inhibition potency 20- to 240-fold compared with
potency ratio of hypoxanthine versus adenine analogs was bis-MANT nucleotides (16–19). Bis-Br-ANT-ATP (22) showed
not markedly changed by the introduction of a second the lowest Ki value of 12.6 nM for CyaA, but the remaining
(M)ANT group. Exceptions were bis-MANT and bis-Ac-NH- halogen-ANT-NTPs were only slightly less potent (Ki values
ANT derivatives with a significantly lower ratio for the bis- 15–20 nM). The affinity of bis-Pr-ANT-ATP (24) decreased only
substituted compounds. For the highest inhibition affinities 2-fold compared with bis-MANT-ATP (17), whereas the corre-
of bis-(M)ANT-NTPs inosine was still required. Bis-MANT- sponding propylated inosine derivative (25) exhibited a slight

CyaA Inhibition by Bis-Halogen-ANT Nucleotides
109
increase in potency compared with bis-MANT-ITP (18). Again, inhibitors. Prototypical P-site inhibitors are adenine nucleo-
deletion of the ␥-phosphate reduced potency [bis-(M)ANT-NDPs tide analogs with a 3Ј-phosphate chain (Tesmer et al., 2000).
28–30]. For most (M)ANT-NMPs and (M)ANT-NTPs and all Most P-site inhibitors require pyrophosphate as a cofactor
(M)ANT-NDPs the inhibitor affinity at CyaA was increased or and bind to an AC-pyrophosphate conformation (Tesmer et
kept constant by introduction of a second (M)ANT group. In the al., 2000). MANT nucleotides are competitive mAC inhibitors
case of bis-halogen-ANT nucleotides, the increase was 18- to and possess a 5Ј- phosphate chain (Gille and Seifert, 2003).
73-fold (4–7 3 20–23). Bis-Ac-NH-ANT-ATP (26) was only Based on these data, for our newly synthesized mono- and
2.5-fold more and bis-Ac-NH-ITP (27) even slightly less potent bis-substituted (M)ANT nucleotides we expected identical
than the corresponding mono-substituted derivatives (10, 11). inhibition properties as for known mono-substituted MANT
A second Pr-ANT substitution did not significantly change af- nucleotides. To prove our hypothesis, enzyme kinetics of
finity (8, 9 3 24, 25). An exception to the rule was bis-MANT- CyaA were determined with two pairs of compounds, i.e.,
ITP (17), exhibiting 5-fold lower inhibitory potency compared MANT-ATP (1)/bis-MANT-ATP (17) and Br-ANT-ATP (6)/
with MANT-ITP (2). In most cases of (M)ANT-NTPs, the affin- bis-Br-ANT-ATP (22) (Fig. 2). In fact, Lineweaver-Burk dou-
ity order with respect to the nucleobase (adenine Ͼ hypoxan- ble-reciprocal plotting of CyaA inhibition kinetics displayed
thine) was not altered by the introduction of a second (M)ANT competitive inhibition pattern for mono- and bis-(M)ANT
group, but for halogen derivatives, the increase was greater nucleotides. The linear regression lines intersected at the
with ITP than with ATP. Again, the bis-MANT-NTPs were y-axis, i.e., Vmax remained constant, whereas Km increased
outlier, because the affinity order of the mono-MANT-NTPs with rising inhibitor concentrations.
(hypoxanthine Ͼ adenine) was reversed.
Fluorescence Experiments. Although the present nu-
Analysis of the Enzyme Kinetics of CyaA. Two classes cleotides differ in mono/bis substitution and in diversified
of AC inhibitors are known, i.e., so-called P-site inhibitors (M)ANT groups, emission spectra were similar. Nucleotides
that are noncompetitive or uncompetitive, and competitive were excited at 350 nm, and emission was scanned from 380
A
C
CyaA
CyaA
0.34
0.30
0.26
0.22
0.18
0.14
0.10
0.06
0.02
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
MANT-ATP [µM]
Br-ANT-ATP [µM]
0
2
10
20
0
1
2.5
5
-0.005
0.005
0.015
1/(ATP/Mn2+
0.025
0.035
-0.005
0.005
0.015
0.025
0.035
)
-1 [µM-1
]
1/(ATP/Mn2+) [µM-1
]
B
D
CyaA
0.11
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
CyaA
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Bis-MANT-ATP [µM]
Bis-Br-ANT-ATP [nM]
0
0
0.5
2.5
5
100
250
500
-0.005
0.005
0.015
0.025
0.035
-0.005
0.005
0.015
0.025
0.035
1/(ATP/Mn2+) [µM-1
]
1/(ATP/Mn2+
)
[µM-1
]
-1
Fig. 2. Enzyme kinetics of CyaA: inhibition by mono- and bis-substituted (M)ANT nucleotides. AC activity of CyaA was determined with the indicated
concentrations of MANT-ATP (0, 2, 10, and 20 ␮M) (A), bis-MANT-ATP (0, 0.5, 2.5, and 5.0 ␮M) (B), Br-ANT-ATP (0, 1.0, 2.5, and 5.0 ␮M) (C) or
bis-Br-ANT-ATP (0, 100, 250, and 500 nM) (D). Reaction mixtures contained 10 pM CyaA, 100 mM KCl, 10 ␮M free Ca2ϩ, 5 mM free Mn2ϩ, 100 ␮M
EGTA, 100 ␮M cAMP, 100 nM calmodulin, 0.2 ␮Ci/tube [␣-32P]ATP, and unlabeled ATP/Mn2ϩ concentrations as indicated. Data were plotted
double-reciprocally and analyzed by linear regression according to Lineweaver-Burk. The r2 values of the regression lines were 0.97 to 0.99. Shown
are the results of a representative experiment performed in triplicate. Similar results were obtained in at least two independent experiments.

110
Geduhn et al.
to 550 nm. Figure 3, A and B shows representative fluores- ide ranging from 0 to 100% (v/v). Fluorescence of MANT-ATP
cence emission spectra for the MANT-ATP/bis-MANT-ATP increased from water environment to pure dimethyl sulfoxide
pair. MANT-ATP was added first to the buffer, displaying just 5-fold (Fig. 3C). In marked contrast, bis-MANT-ATP
high autofluorescence at ␭ ϭ 449 nm (brown lines in Fig. exhibited a ϳ40-fold fluorescence increase upon exposure to
em
3). Addition of CyaA did not change the intrinsic fluorescence pure dimethyl sulfoxide (Fig. 3D). Thus, independently of the
significantly (blue lines in Fig. 3). However, upon addition of 12-fold higher potency for CyaA compared with MANT-ATP,
CaM, fluorescence increased by 36%. This result is in accor- bis-MANT-ATP displayed an improved signal-to-noise ratio
dance to our previous fluorescence resonance energy transfer for the fluorescence analysis of CyaA. Blue shifts were de-
studies (Go¨ttle et al., 2007). It is noteworthy that bis-MANT- tected for both compounds (MANT-ATP: ␭
ϭ 448 nm 3
max
ATP revealed a nearly 20-fold lower autofluorescence (brown 426 nm; bis-MANT-ATP: ␭
ϭ 445 nm 3 429 nm).
max
lines in Fig. 3) compared with MANT-ATP. The addition of
In kinetic experiments CyaA/CaM-evoked fluorescence was
CyaA into the cuvette containing bis-MANT-ATP increased inhibited by the nonfluorescent nucleotide analog PMEApp in a
fluorescence by 4-fold. Moreover, the emission maximum was concentration-dependent manner (Fig. 4). Because of the
shifted to shorter wavelengths (blue shift). Thus, CyaA higher potency of PMEApp, half-maximal displacement of 2
bound bis-MANT-ATP even without the activator CaM. Like- ␮M bis-MANT-ATP occurred at a PMEApp concentration of
wise, 2Ј,3Ј-O-(2,4,6-trinitrophenyl)-substituted NTPs bind to approximately 1 ␮M. Fluorescence changes in CyaA stimu-
CyaA in the absence of CaM (Go¨ttle et al., 2007). In contrast, lated by CaM and inhibition by PMEApp occurred within
for binding of mono-substituted MANT-NTPs to CyaA, inter- mixing time, i.e., a few seconds. These data show that the
action with CaM is required (Go¨ttle et al., 2007). Upon addi- fluorescence changes monitored were rapid, specific, and re-
tion of CaM, bis-MANT-ATP displayed an even larger versible. Because fluorescent nucleotides, e.g., bis-MANT-
fluorescence increase (6-fold higher compared with basal nu- ATP, were competitively displaced from CyaA by PMEApp,
cleotide fluorescence).
the affinity of nonlabeled inhibitors may also be estimated
We also studied the responsiveness of MANT-ATP and using this approach. Experiments with two additional com-
bis-MANT-ATP to a hydrophobic environment (Go¨ttle et al., pound pairs, i.e., MANT-ITP/bis-MANT-ITP and Br-ANT-
2007). Emission spectra were recorded with dimethyl sulfox- ATP/bis-Br-ANT-ATP, showed similar fluorescence proper-
MANT-ATP
Bis-MANT-ATP
A
B
50
40
30
20
10
0
12
10
8
+ CyaA/CaM
+ CyaA
Nucleotide alone
+ CyaA/CaM
+ CyaA
Nucleotide alone
6
4
2
0
380
420
460
500
540
380
420
460
500
540
Wavelength λ [nm]
Wavelength λ [nm]
C
MANT-ATP
D
Bis-MANT-ATP
200
160
120
80
80
70
60
50
40
30
20
10
0
100% DMSO
80% DMSO
60% DMSO
40% DMSO
20% DMSO
0% DMSO
100% DMSO
80% DMSO
60% DMSO
40% DMSO
20% DMSO
0% DMSO
40
0
380
420
460
500
540
380
420
460
500
540
Wavelength λ [nm]
Wavelength λ [nm]
Fig. 3. Fluorescence properties of MANT-ATP and bis-MANT-ATP: binding of nucleotides to CyaA and the effect of dimethyl sulfoxide. The assay
buffer contained 75 mM HEPES/NaOH, pH 7.4, 100 ␮M CaCl2, 100 mM KCl, and 5 mM MnCl2, pH 7.4. A and B, MANT-ATP (A) and bis-MANT-ATP
(B) were added to the buffer to yield a final concentration of 2 ␮M, and emission was scanned at an excitation wavelength of 350 nm. CyaA and CaM
were added successively to yield a final concentration of 2.4 ␮M each. Shown are superimposed recordings of a representative experiment. Similar data
were obtained in three independent experiments. C and D, MANT-ATP (C) and bis-MANT-ATP (D) were added to water-dimethyl sulfoxide (DMSO)
mixtures ranging from 0 to 100% (v/v) to yield a final concentration of 2 ␮M. Nucleotides were directly excited at ␭ ϭ 350 nm to mimic binding of
ex
the MANT group to a hydrophobic binding pocket. Shown are superimposed recordings of a representative experiment. Similar data were obtained
in two independent experiments. Fluorescence intensities are given in arbitrary units (a.u.).

CyaA Inhibition by Bis-Halogen-ANT Nucleotides
111
20.0
17.5
15.0
12.5
10.0
7.5
1
2
3
5
6
7
8
4
5.0
2.5
0.0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
Time (min)
Fig. 4. Time-resolved activation of CyaA by CaM and stepwise abolish-
ment of fluorescence by PMEApp. Excitation wavelength was 350 nm,
and emission was detected at 440 nm over time. Bis-MANT-ATP (2 ␮M)
(1), 2.4 ␮M CyaA (2), 2.4 ␮M CaM (3), and PMEApp in the given concen-
trations (addition steps: 4, 100 nM; 5, 500 nM; 6, 1 ␮M; 7, 2 ␮M; 8, 3 ␮M)
were added in sequence. A recording of a representative experiment is
shown. Similar data were obtained in three independent experiments.
Fluorescence intensities are given in a.u.
ties as with MANT-ATP/bis-MANT-ATP (data not shown).
Control experiments were conducted to rule out false-positive
fluorescence signals, i.e., combination of CaM and fluoro-
phore without CyaA caused no signal and denatured CyaA
(10 min at 95°C) reduced fluorescence to basal response (data
not shown).
Modeling of Nucleotide Binding to mAC and CyaA.
Models of binding modes of bis-(M)ANT nucleotides on mAC
were based mainly on the crystal structure of mAC in com-
plex with 3Ј-MANT-ITP. In this structure, the N- and C-
terminal halves of the catalytic core (VC1 and IIC2) originate
from AC isoforms 5 and 2, respectively. 3Ј-MANT-ITP occu-
pies the same binding pocket at the C1:C2 interface as 3Ј-
MANT-GTP and 3Ј-MANT-ATP in previous crystal struc-
tures (Mou et al., 2005, 2006). The ribosyl moiety adopts a
2Ј-endo envelope conformation, leading to an equatorial ori-
entation of the 2Ј-hydroxyl incompatible with binding of a 2Ј
(M)ANT group (strong sterical clashes with VC1). However,
the binding modes of mono- and bis-(M)ANT nucleotides
should be similar, because an exchange of hypoxanthine by
adenine leads to a related drop in potency in most cases of
mono- and bis-substituted derivatives (see Table 1). On the
base of the “mono-(M)ANT-NTP mode,” docking of bis-
(M)ANT-NTPs to mAC is possible in a 3Ј-endo envelope ribo-
syl conformation with an axial 2Ј- and an equatorial 3Ј-
substituent. This enables to align the 2Ј-(M)ANT group with
the 3Ј-(M)ANT moiety of mono-(M)ANT nucleotides and pro-
vides an open channel for binding of the 3Ј-(M)ANT group.
Figure 5A shows the suggested mAC binding mode of bis-
Br-ANT-ITP (23), the most potent bis-(M)ANT nucleotide
from the present series. The binding pocket is formed by
amino acids of VC1 (396–440) and IIC2 (938–1065). The first
Mn2ϩ coordinates Asp440 and the ␣- and ␤-phosphates,
whereas the second Mn2ϩ stabilizes the ligand by octahedral
coordination with Asp396, Ile397, Asp440, and the ␤- and
␥-phosphates, which additionally interact with the backbone
of Gly399 and the side chains of Thr401 and Lys1065. The
ribosyl oxygen is involved in a weak hydrogen bond with the
backbone NH of Asp440. The hypoxanthine moiety interacts
Fig. 5. Interactions of (M)ANT nucleotides with mAC. The models are
based on the crystal structures of mAC in complex with MANT-ITP, PDB
3g82, and with MANT-ATP, PDB 2gvz (Mou et al., 2006). Colors of atoms,
unless otherwise indicated, are: orange, phosphorus; red, oxygen; blue,
nitrogen; yellow, carbon, hydrogen; green, bromine; purple, Mn2ϩ. A,
suggested binding mode of bis-Br-ANT-ITP; amino acids within a sphere
of ϳ3 Å around the ligand are shown and labeled: carbon atoms of the
backbone, gray; carbon atoms of side chains, cyan; Mn2ϩ ions represented
by van der Waal radii. B, comparison of the binding modes of 3Ј-MANT-
ITP (ball and stick model, yellow C atoms) and 3Ј-MANT-ATP (stick
model, green C atoms); amino acids within a sphere of ϳ3 Å around the
nucleobases are shown and labeled (3g82, ball and stick model, cyan
labels and C atoms; 2gvz, stick model, light gray labels and C atoms; both
structures, dark gray labels). C, binding site of bis-Br-ANT-ITP, repre-
sented by the lipophilic potential mapped onto a MOLCAD Connolly
surface (brown, hydrophobic areas; green and blue, polar areas); bis-Br-
ANT-ITP in ball and stick model, the 2Ј-Br-ANT group is highlighted by
light yellow carbon atoms.

112
Geduhn et al.
with Lys938 and Asp1018 by two charge-assisted hydrogen 2Ј-MANT-3Ј-dATP for CyaA. 3Ј-MANT-2Ј-dATP adopted an
bonds of the 6-oxygen and the 1-NH functions, respectively. ideal position for ␲-stacking of the phenyl ring of the inhib-
All interactions described so far correspond to those of itor and Phe306. This stacking also accounts for the substan-
3Ј-MANT-ITP in the crystal structure (PDB 3g82). Obvious tial fluorescence increases. Docking of 2Ј-MANT-3Ј-dATP re-
differences to the complex of 3Ј-MANT-ATP with mAC (PDB produced the interaction pattern of 3Ј-MANT-2Ј-dATP. The
2gvz) may account for the higher inhibitory potency of ITP only difference is a 3Ј-endo conformation of the desoxyribosyl
derivatives (Fig. 5B). The hypoxanthine base adopts an anti moiety as is present, e.g., in A-DNA.
conformation with respect to the ribosyl moiety, which en-
Figure 6 shows bis-Br-ANT-ATP in complex with CyaA.
ables the hydrogen bonds with Lys936 and Asp1018. This Docking was based on 3Ј-MANT-2Ј-dATP, because the equa-
conformation is impossible in the case of 3Ј-MANT-ATP, torial position of the 2Ј-H atom to be replaced by the second
probably because of electrostatic repulsion with Asp1018. In Br-ANT group enables this substituent to be accommodated
the alternative syn conformation, the adenine base forms no outside of the active site and to form additional specific
hydrogen bonds, but only some weak van der Waals contacts interactions with CyaA. The ribosyl moiety adopts a half-
with mAC.
chair (2Ј-endo, 3Ј-exo) conformation. The initial hypothesis
The 2Ј-Br-ANT group binds in close analogy to the 3Ј- that the second ANT group may interact with Phe261 (Go¨ttle
MANT moiety of 3Ј-MANT-ITP in the same groove between et al., 2007) was not confirmed because of conformational
␤1-␣1-␣2 of VC1 and ␣4Ј-␤5Ј of IIC2 (compare Fig. 5, A and restrictions of the ester moiety (clash with the adenine base).
B). The model predicts a hydrogen bond of the ortho-amino Instead, an energetically favorable conformation of bis-Br-
substituent with the side-chain amide oxygen of Asn1025. In ANT-ATP is possible where the 2Ј-Br-ANT substituent can
the case of 3Ј-MANT-ITP, the methyl group prevents the easily expand to Phe306. The binding mode of the rest of the
formation of this hydrogen bond by sterical hindrance (clash molecule is largely the same as in the case of 3Ј-MANT-2Ј-
with Asn1022), but otherwise contributes to the high potency
of MANT-ITP by hydrophobic binding to Ala404. There are
significant hydrophobic interactions of the 2Ј-Br-ANT group
with the VC1 site: a face-to-edge approach of the phenyl ring
to Phe400 and contacts of the bromine with Leu412 and
Val413. With respect to the IIC2 site, the 2Ј-Br-ANT moiety is
aligned to the backbone of ␤5Ј (Trp1020, Gly1021, Asn1022),
forming only van der Waals contacts. This amphiphilic nature
of the 2Ј-Br-ANT binding site (hydrophobic VC1, hydrophilic
IIC2) becomes also obvious from the lipophilic potential map of
mAC (Fig. 5C).
Interactions of the 3Ј-Br-ANT group with mAC are rather
weak compared with the 2Ј-Br-ANT moiety. Van der Waals
contacts occur mainly with the side chains of Thr401,
Asn1025, Arg1029, Val1064, and Lys1065. Figure 5C shows
that the hydrophobic Br-phenyl fragment projects into a po-
lar environment, which may in part explain the drop in
potency of bis-(M)ANT nucleotides compared with their
mono-(M)ANT analogs. Other possible reasons are conforma-
tional strain caused by restricted space in the binding site
and electrostatic repulsion of the ester function in 3Ј-position
with the ␥-phosphate, leading to insufficient fit of the phos-
phate groups.
The crystal structure of CyaA in complex with CaM and
PMEApp (Guo et al., 2005) served as a base for predictions of
the binding mode of mono- and bis-substituted (M)ANT-ATP
derivatives. The AC domain of CyaA includes the catalytic
site at the interface of two structural domains, CA (Met1-
Gly61, Ala187-Ala364) and CB (Val62-Thr186). Compared
with PMEApp, the substrate ATP and the fluorescent nucleo-
tides are conformationally constrained because of the ribosyl
moiety. However, the spacious cavity between CA and CB
may accommodate the different scaffolds so that an align-
Fig. 6. Docking of bis-Br-ANT-ATP to CyaA. The models are based on the
crystal structure of CyaA in complex with PMEApp, PDB 1zot (Guo et al.,
ment of the adenine bases and the terminal phosphates is
possible. Hydrophobic interactions, especially of the 3Ј-
MANT-group with Phe306, significantly contribute to the
binding of MANT-ATP to CyaA (Go¨ttle et al., 2007). In sup-
port of this model, the CyaA-F306A mutant failed to increase
the fluorescence signal of 3Ј-ANT-2ЈdATP (Guo et al., 2005).
In our previous study (Go¨ttle et al., 2007), we observed
2005). Colors of atoms, unless otherwise indicated, are: orange, phospho-
rus; red, oxygen; blue, nitrogen; yellow, carbon, hydrogen; green, bro-
mine; purple, Mg2ϩ. A, overview of the binding site, represented by the
lipophilic potential mapped onto a MOLCAD Connolly surface (brown,
hydrophobic areas; green and blue, polar areas); bis-Br-ANT-ATP in ball
and stick model. The 2Ј-Br-ANT group is highlighted by light yellow
carbon atoms. B, detailed binding mode. Amino acids within a sphere of
ϳ3 Å around the ligand are shown and labeled: carbon atoms of the
backbone, gray; carbon atoms of side chains, cyan. For the two Mg2ϩ ions
relatively high inhibition potency of 3Ј-MANT-2Ј-dATP and interacting with the inhibitor, the van der Waal radii are shown.

CyaA Inhibition by Bis-Halogen-ANT Nucleotides
113
dATP, but the ideal ␲-stacking with Phe306 is not retained. that bis-MANT-IMP showed at least 5-fold higher potency at
The Br-ANT moieties rather enclose Phe306 from both sides CyaA than mono-MANT-IMP (Go¨ttle et al., 2007). Although
(Fig. 6A). Obviously, both Br (or Cl) substituents increase the the absolute potency of bis-MANT-IMP was still very low,
hydrophobic interactions with Phe306, thus accounting for i.e., in the 20- to 40-␮M range, depending on the experimen-
the high inhibitory potency of bis-halogen-ANT-ATPs com- tal conditions, we nonetheless decided to follow up on this
pared with their dehalogenated derivatives. Additional hy- observation and aim at optimizing inhibitor affinity. In fact,
drophobic contacts are formed between the 2Ј-Br-ANT group we achieved a 1000-fold increase in affinity with a relatively
and the side chains of Leu60 (phenyl ring) and Pro305 (Br). small number of novel compounds.
Figure 6B presents the putative binding mode of bis-Br-
Bis-halogen-ANT-adenine nucleotides possess the best se-
ANT-ATP in more detail. The adenine base, the ribosyl nu- lectivity ratio. Our prediction of facile accommodation of
cleus, and the phosphate groups form similar interactions as bulky bis-substituted (M)ANT nucleotides in the large cavity
described previously for 3Ј-MANT-2Ј-dATP (Go¨ttle et al., of the catalytic site of CyaA, causing an increase in affinity
2007). Two of the three Mg2ϩ ions are in positions like those (Go¨ttle et al., 2007), was confirmed experimentally. Bis-Cl-
in the CyaA-PMEApp complex. Both are coordinated with ANT-ATP (20) is the most interesting compound with a 100-
Asp188 and Asp190, one additionally with His298, and the to 150-fold selectivity for CyaA relative to mACs. The equi-
other with the ␣- and ␤-phosphate. The third Mg2ϩ ion in- potent CyaA inhibitor bis-Br-ANT-ATP (22) exhibited high
teracts with Glu308, the carbonyl oxygen of the 3Ј-ANT selectivity as well (50- to 90-fold). Generally, the CyaA selec-
group, and the ␥-phosphate, which also contacts Lys58 and tivity of the four bis-halogen-ANT derivatives is only caused
Lys65. These interactions account for the higher inhibitory by different affinities for mACs. Bis-halogen-ANT-ITPs (21
potencies of the NTPs compared with NDPs and NMPs (Ta- and 23) displayed only marginal selectivity because of higher
ble 1). The adenine moiety is sandwiched between the side mAC potency, probably depending on two specific hydrogen
chains of Leu60 and Asn304 whose amide NH2 group may bonds of the hypoxanthine base with Lys936 and Asp1018
form an additional hydrogen bond with the ribosyl ring oxy- (Fig. 5). Bis-Cl-ANT-ATP is 3- to 4-fold less potent at mACs
gen. Hydrogen bonds are also possible between the 6-amino than its bis-Br analog and therefore the most CyaA-selective
substituent of the nucleobase and the backbone oxygens of inhibitor. Docking of both structures on the mAC model con-
Val271 and Thr300. However, the loop between Gly299 and firms this difference. The calculated interaction energy
Asn304 may interact with different nucleobases. In the case (steric and electrostatic potentials) of bis-Br-ANT-ATP with
of ITP derivatives the backbone NH of Gly299 may serve as mAC is by ϳ3 kcal/mol greater than that of bis-Cl-ANT-ATP,
a hydrogen donor for the carbonyl oxygen in the 6-position. probably because of better fit of the 3Ј-Br-ANT group, be-
The diversity of possible interactions may account for incon- cause the 2Ј-substituents bind at the site of the 2Ј- or 3Ј-
sistent potency differences resulting from base substitution (M)ANT group of mono-substituted derivatives (Fig. 5). How-
(Table 1). In addition, the affinity of each nucleotide may be ever, in this case Cl leads to 2-fold higher potency than Br
affected by a specific arrangement of water molecules that (compounds 4 and 5 versus 6 and 7).
cannot be simply transferred from the PMEApp-bound CyaA
structure (Guo et al., 2005).
It is noteworthy that the more space-filling propyl and
acetylated amino group of ANT-ATPs implemented also se-
Hydrophobic interactions with the phenyl ring of Phe306 lectivity aspects. Bis-propyl-ANT-ATP (24) was 26- to 52-fold
are formed mainly by both Br substituents. Not only the side more selective for CyaA compared with mAC, and bis-Ac-NH-
chain, but also the backbone oxygen of Leu60, may interact ANT-ATP (26) was 20- to 80-fold more selective. For acylated
with the 2Ј-Br-ANT group by forming a hydrogen bond with amino ANT nucleotides even the mono-substituted deriva-
the amine. The amino group of the 3Ј-Br-ANT moiety con- tive (10) yielded a 5- to 15-fold selectivity. Docking of bis-
tacts the ␥-phosphate and is involved in a charge-assisted substituted halogen anthraniloyl derivatives to CyaA (Fig. 6)
hydrogen bond with the carboxylate of Glu308. All of these highlighted the critical importance of Phe306 for both high
interactions may be weaker or impossible in the case of inhibitor potency (Table 1) and large fluorescence increases
secondary amino groups caused by sterical hindrance. This upon binding of inhibitor (Fig. 3B). The second halogen-ANT
may explain subtle potency differences between ANT, group fits to CyaA via strong hydrophobic interactions with
MANT, and Pr-ANT derivatives.
Phe306, but aligns to a polar channel of mAC, forming only
van der Waals contacts and inducing conformational strain.
The resulting increase of CyaA and decrease of mAC potency
is the first contributing factor for CyaA selectivity, but not
Discussion
In previous studies we found MANT nucleotides to be effectual on its own. The second factor is caused mainly by
highly potent mAC inhibitors (Ki values in the 1- to 20-nM weak interactions of the adenine base with mAC (only van
range), but their affinity for CyaA was considerably lower (Ki der Waals contacts, no hydrogen bonds; see Fig. 5) (Mou et
values in the 0.2- to 10-␮M range) (Gille et al., 2004; Go¨ttle et al., 2006). Both factors together account for the high CyaA
al., 2007, 2009). In other previous studies, it was also difficult selectivity of bis-halogen-ANT-ATPs. In accordance with the
to identify highly potent and selective CyaA inhibitors (John- very low mAC potency of bis-Pr-ANT-ATP, nucleotides sub-
son and Shoshani, 1990; Soelaiman et al., 2003). In this study stituted with a bulky boron-dipyrromethene group are less
we found, for the first time, not only highly potent (M)ANT potent mAC inhibitors than mono-MANT nucleotides (Gille
nucleotides for CyaA inhibition with Ki values in the range of et al., 2004). Compared with the catalytic site of mACs, the
10 to 20 nM, but also inhibitors with substantial selectivity catalytic site of CyaA is more spacious and possibly more
for CyaA versus ACs 1, 2, and 5 (Table 1). The starting point flexible, offering novel and unexpected opportunities for in-
for our systematic analysis of 25 novel mono- and bis- hibitor design and selectivity.
(M)ANT-substituted nucleotides was the unexpected finding
Pro-drugs based on NMPs with protected phosphate could

114
Geduhn et al.
be feasible drug candidates for inhibition of CyaA and treat- assume that the enzymological analysis of edema factor with
ment of whooping cough (Laux et al., 2004). The lipophilic our new compound library will give different results than the
NMP pro drug has first to be activated by esterases and then analysis of CyaA. Beyond whooping cough and anthrax in-
phosphorylated by specific kinases to yield the active NTP fection, it will be very interesting to examine the effects of the
derivative. Moreover, it is conceivable that high-affinity bis- new compounds on the AC toxin ExoY from Pseudomonas
(M)ANT nucleotides injected intravenously could bind to ex- aeruginosa (Yahr et al., 1998) as well as mycobacterial ACs
tracellularly circulating CyaA and, thereby, block catalytic (Linder and Schultz, 2008).
and cytotoxic activity when the toxin inserts into the mem-
Since their initial description by Hiratsuka (2003), (M)ANT
brane of host cells. The compounds characterized in this nucleotides have been broadly used for the analysis of confor-
study can also serve as pharmacophore for the rational de- mational changes in various proteins involved in signal
velopment of non-nucleotide-based CyaA inhibitors that transduction, including GTP-binding proteins, protein ki-
could be used in the treatment of whooping cough (Soelaiman nases, phosphodiesterases, and ACs (Rensland et al., 1991;
et al., 2003).
Jameson and Eccleston, 1997; Ni et al., 2000; Bessay et al.,
Bis-substituted (M)ANT nucleotides offer the advantage of 2008). To this end, attention in the field was paid only to
excellent signal-to-noise ratio in fluorescence spectroscopy, mono-(M)ANT nucleotides, although mono- and bis-(M)ANT
compared with mono-substituted (M)ANT nucleotides (Fig. nucleotides are produced simultaneously during the syn-
3). Probably, intramolecular interactions of the two MANT- thetic procedure. Because purification procedures have fo-
groups with each other quench endogenous fluorescence, cused only on mono-(M)ANT nucleotides and bis-(M)ANT
which is then unmasked upon binding to a hydrophobic nucleotides have quite different physiochemical properties,
amino acid in the target protein (Phe306 in case of CyaA). most notably higher lipophilicity, formation of the latter nu-
Considering then the fact that CyaA can be purified in large cleotides has largely gone undetected. In this article, we
quantities (Guo et al., 2005), this fluorescence assay offers describe methods that allow for the convenient isolation of
interesting opportunities for high-throughput screening both mono- and bis-(M)ANT nucleotides. Based on the results
studies aimed at the identification of novel non-nucleotide- of this study, we anticipate that beyond the field of ACs,
based CyaA inhibitors (Fig. 4).
bis-(M)ANT nucleotides may have broader applications in
Noncompetitive/uncompetitive P-site inhibitors preferen- signal transduction research, specifically in cases where
tially bind to the catalytically active enzyme (Tesmer et al., there are no large spacious constraints in the nucleotide-
2000). Although an increase in catalytic activity also in- binding site. Small GTP-binding proteins are suitable candi-
creases the affinity of (M)ANT nucleotides for AC (Gille and dates (Rensland et al., 1991).
Seifert, 2003; Gille et al., 2004), it is also clear that certain
Collectively, in our present study, we have developed a
(M)ANT nucleotides can bind to mammalian and bacterial novel series of mono- and bis-(M)ANT-substituted nucleo-
ACs in the absence of an activator. For example, MANT-GTP tides. Some of these nucleotides possess surprisingly high
binds to the purified catalytic subunits of mAC in the absence selectivity for CyaA relative to mACs, showing that the de-
of the activator forskolin, with the diterpene further enhanc- velopment of selective CyaA inhibitors for the improved
ing the fluorescence increase (Mou et al., 2005, 2006). The treatment of whooping cough is not an elusive goal. Our data
effective binding of bis-MANT-ATP to CyaA in the absence of show that numerous chemical modifications such as haloge-
CaM reported in this study is a particularly impressive illus- nation and introduction of acetyl and propyl groups can be
tration for activator-independent interaction of inhibitors implemented in the (M)ANT group, resulting in substantial
with ACs. Crystallographic studies are required to precisely changes in pharmacological properties of compounds. Future
define differences in structure of CyaA bound to bis-ANT- studies will have to explore in detail the chemical opportu-
NTPs in the absence and presence of CaM. An approach to nities offered by the 2Ј- and 3Ј-O-ribosyl substituent of nu-
explore such potential conformational differences is the ex- cleotides, facing a spacious cavity in CyaA.
amination of the effects of CyaA inhibitors in the absence and
presence of CaM using the fluorescence assay with bis-
MANT-ATP. Thus, in a broader mechanistic perspective, our
data support the concept of multiple activation-dependent
AC conformations stabilized by nucleotide inhibitors.
Acknowledgments
We thank Dr. M. Go¨ttle for helpful discussions, Mrs. S. Bru¨gge-
mann for expert technical assistance, and the reviewers for their
constructive critique.
There are both similarities and dissimilarities between
CyaA and edema factor in terms of interaction with (M)ANT
nucleotides. Both toxins possess a phenylalanine residue in
the catalytic site (Phe306 in the case of CyaA and Phe586 in
the case of edema factor), being responsible for the fluores-
cence increases upon interaction with CaM (Go¨ttle et al.,
2007; Taha et al., 2009). Moreover, MANT nucleotides inhibit
the AC activities of both toxins competitively (Fig. 2) (Taha et
al., 2009). Nonetheless, the inhibitory profiles of edema fac-
tor and CyaA are different. Most notably, MANT-ATP, de-
fined 2Ј-MANT- and 3Ј-MANT-ATP isomers, and MANT-CTP
inhibit catalytic activity of CyaA with approximately 10-fold
lower potency than catalysis by edema factor, respectively
(Go¨ttle et al., 2007; Suryanarayana et al., 2009; Taha et al.,
2009). Based on these differences it is also reasonable to
Authorship Contributions
Participated in research design: Geduhn, Dove, Ko¨nig, and Seifert.
Conducted experiments: Geduhn.
Contributed new reagents or analytic tools: Geduhn, Shen, Tang,
and Ko¨nig.
Performed data analysis: Geduhn, Dove, and Seifert.
Wrote or contributed to the writing of the manuscript: Geduhn,
Dove, Shen, Tang, Ko¨nig, and Seifert.
Other: Ko¨nig provided funding and laboratory space. Dove con-
ducted molecular modeling. Seifert directed and coordinated study
among collaborators and provided funding and laboratory space.
References
Bessay EP, Blount MA, Zoraghi R, Beasley A, Grimes KA, Francis SH, and Corbin
JD (2008) Phosphorylation increases affinity of the phosphodiesterase-5 catalytic
site for tadalafil. J Pharmacol Exp Ther 325:62–68.

CyaA Inhibition by Bis-Halogen-ANT Nucleotides
115
Carbonetti NH (2010) Pertussis toxin and adenylate cyclase toxin: key virulence
factors of Bordetella pertussis and cell biology tools. Future Microbiol 5:455–469.
Clark M, Cramer RD III, and Van Opdenbosch N (1989) Validation of the general
purpose Tripos 5.2 force field. J Comput Chem 10:982–1012.
Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KMJ, Ferguson DM, Spellmeyer
DC, Fox T, Caldwell JW, and Kollman PA (1995) A second generation force field for
the simulation of proteins and nucleic acids. J Am Chem Soc 117:5179–5197.
Crowcroft NS and Pebody RG (2006) Recent developments in pertussis. Lancet
367:1926–1936.
Ghose AK, Viswanadhan VN, and Wendoloski J (1998) Prediction of hydrophobic
(lipophilic) properties of small organic molecules using fragmental methods: an
analysis of ALOGP and CLOGP. J Phys Chem A 102:3762–3772.
Gille A and Seifert R (2003) 2Ј(3Ј)-O-(N-methylanthraniloyl)-substituted GTP ana-
logs: a novel class of potent competitive adenylyl cyclase inhibitors. J Biol Chem
278:12672–12679.
Gille A, Lushington GH, Mou TC, Doughty MB, Johnson RA, and Seifert R (2004)
Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by
purine and pyrimidine nucleotides. J Biol Chem 279:19955–19969.
Go¨ttle M, Dove S, Kees F, Schlossmann J, Geduhn J, Ko¨nig B, Shen Y, Tang WJ,
Kaever V, and Seifert R (2010) Cytidylyl and uridylyl cyclase activity of Bacillus
anthracis edema factor and Bordetella pertussis CyaA. Biochemistry 49:5494–
5503.
Go¨ttle M, Dove S, Steindel P, Shen Y, Tang WJ, Geduhn J, Ko¨nig B, and Seifert R
(2007) Molecular analysis of the interaction of Bordetella pertussis adenylyl cy-
clase with fluorescent nucleotides. Mol Pharmacol 72:526–535.
Go¨ttle M, Geduhn J, Ko¨nig B, Gille A, Ho¨cherl K, and Seifert R (2009) Character-
ization of mouse heart adenylyl cyclase. J Pharmacol Exp Ther 329:1156–1165.
Guiso N (2009) Bordetella pertussis and pertussis vaccines. Clin Infect Dis 49:1565–
1569.
Guo Q, Shen Y, Lee YS, Gibbs CS, Mrksich M, and Tang WJ (2005) Structural basis
for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin.
EMBO J 24:3190–3201.
Heiden W, Moeckel G, and Brickmann J (1993) A new approach to analysis and
display of local lipophilicity/hydrophilicity mapped on molecular surfaces. J Com-
put Aided Mol Des 7:503–514.
Hiratsuka T (1983) New ribose-modified fluorescent analogs of adenine and guanine
nucleotides available as substrates for various enzymes. Biochim Biophys Acta
742:496–508.
(2004) Pro-nucleotide inhibitors of adenylyl cyclases in intact cells. J Biol Chem
279:13317–13332.
Linder JU and Schultz JE (2008) Versatility of signal transduction encoded in
dimeric adenylyl cyclases. Curr Opin Struct Biol 18:667–672.
Mou TC, Gille A, Fancy DA, Seifert R, and Sprang SR (2005) Structural basis for the
inhibition of mammalian membrane adenylyl cyclase by 2Ј(3Ј)-O-(N-methylan-
thraniloyl)-guanosine 5Ј-triphosphate. J Biol Chem 280:7253–7261.
Mou TC, Gille A, Suryanarayana S, Richter M, Seifert R, and Sprang SR (2006)
Broad specificity of mammalian adenylyl cyclase for interaction with 2Ј,3Ј-
substituted purine- and pyrimidine nucleotide inhibitors. Mol Pharmacol 70:878–
886.
Ni Q, Shaffer J, and Adams JA (2000) Insights into nucleotide binding in protein
kinase A using fluorescent adenosine derivatives. Protein Sci 9:1818–1827.
Rensland H, Lautwein A, Wittinghofer A, and Goody RS (1991) Is there a rate-
limiting step before GTP cleavage by H-ras p21? Biochemistry 30:11181–11185.
Shen Y, Lee YS, Soelaiman S, Bergson P, Lu D, Chen A, Beckingham K, Grabarek
Z, Mrksich M, and Tang WJ (2002) Physiological calcium concentrations regulate
calmodulin binding and catalysis of adenylyl cyclase exotoxins. EMBO J 21:6721–
6732.
Soelaiman S, Wei BQ, Bergson P, Lee YS, Shen Y, Mrksich M, Shoichet BK, and
Tang WJ (2003) Structure-based inhibitor discovery against adenylyl cyclase tox-
ins from pathogenic bacteria that cause anthrax and whooping cough. J Biol Chem
278:25990–25997.
Suryanarayana S, Wang JL, Richter M, Shen Y, Tang WJ, Lushington GH, and
Seifert R (2009) Distinct interactions of 2Ј- and 3Ј-O-(N-methyl)anthraniloyl-
isomers of ATP and GTP with the adenylyl cyclase toxin from Bacillus anthracis,
edema factor. Biochem Pharmacol 78:224–230.
Taha HM, Schmidt J, Go¨ttle M, Suryanarayana S, Shen Y, Tang WJ, Gille A,
Geduhn J, Ko¨nig B, Dove S, et al. (2009) Molecular analysis of the interaction of
anthrax adenylyl cyclase toxin, edema factor, with 2Ј(3Ј)-O-(N-methyl)anthra-
niloyl)-substituted purine and pyrimidine nucleotides. Mol Pharmacol 75:693–
703.
Tesmer JJ, Dessauer CW, Sunahara RK, Murray LD, Johnson RA, Gilman AG, and
Sprang SR (2000) Molecular basis for P-site inhibition of adenylyl cyclase. Bio-
chemistry 39:14464–14471.
Vojtova J, Kamanova J, and Sebo P (2006) Bordetella adenylate cyclase toxin: a swift
saboteur of host defense. Curr Opin Microbiol 9:69–75.
Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, and Frank DW (1998) ExoY, an
adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc
Natl Acad Sci USA 95:13899–14904.
Jameson DM and Eccleston JF (1997) Fluorescent nucleotide analogs: synthesis and
applications. Methods Enzymol 278:363–390.
Johnson RA and Shoshani I (1990) Inhibition of Bordetella pertussis and Bacillus
anthracis adenylyl cyclases by polyadenylate and “P”-site agonists. J Biol Chem
265:19035–19039.
Ladant D and Ullmann A (1999) Bordetella pertussis adenylate cyclase: a toxin with
multiple talents. Trends Microbiol 7:172–176.
Laux WH, Pande P, Shoshani I, Gao J, Boudou-Vivet V, Gosselin G, and Johnson RA
Address correspondence to: Dr. Roland Seifert, Institute for Pharmacology,
Medical School of Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Ger-
many. E-mail: seifert.roland@mh-hannover.de