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