Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2\u27,3\u27-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5\u27-Triphosphates

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70: 878-886, 2006), we reported that 2\u27,3\u27-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5\u27-triphosphates (TNP-NTPs) are potent inhibitors (K(i) values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1: IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1: IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1: IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K(i), 3.7 nM) and sGC (K(i), 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K(i), 7.1 nM) and ACII (K(i), 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G(s)- and G(i)-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1: IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1: IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far

Bacillus anthracis edema factor substrate specificity: evidence for new modes of action

Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5'-triphosphate, uridine 5'-triphosphate and inosine 5'-triphosphate, in addition to adenosine 5'-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3':5'-monophosphate, cyclic uridine 3':5'-monophosphate and cyclic inosine 3':5'-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed

Molecular analysis of mammalian adenylyl cyclases and bacterial adenylyl cyclase toxins

The cAMP signaling pathway is crucial for many physiological processes and many disease states like neurodegenerative diseases, mood disorders, pain, drug dependency or heart failure. As mammalian AC isoforms are expressed in a tissue-specific manner, isoform-selective AC activation or inhibition may be a promising novel therapeutic strategy. Therefore, the aim of this doctoral thesis was to develop analytical methods for the investigation and characterization of AC enzyme activity in specific mammalian tissues. As the cAMP signaling pathway is essential for cardiac contractility, and as heart failure is one of the most important causes for morbidity and mortality in elderly patients, the heart - hence AC from cardiac tissue - was considered primarily. Thus, this work provides the basis for exploring AC as target for the treatment of heart failure. As one major achievement of this thesis, a biochemical method was developed to prepare membranous AC from cardiac tissue, yielding the components of the cAMP signaling cascade, i.e. GPCRs, G proteins and AC in intact form. Using forskolin analogs for stimulating AC activity and MANT-substituted nucleotides as inhibitors, and by investigating Michaelis-Menten enzyme kinetics, we found AC from heart tissue to correlate to some extent with recombinant AC5, compatible with the notion that AC5 is the major AC isoform in the heart. However, AC isoforms other than AC5 appear to contribute to total AC activity in mouse heart membranes, too. These findings were corroborated by results obtained by applying further techniques, i.e. real-time PCR and immunoblot analysis. In summary, by the use of several techniques, we provide a pharmacological profile of cardiac AC and an excellent starting point for the design of potent and selective inhibitors. When conventional treatment of heart failure is limited, e.g. due to ineffective beta-adrenoceptor antagonist therapy, as a promising novel therapeutic strategy, future clinical treatment strategies may be complemented by nucleotide prodrugs accomplishing beneficial effects on the heart and increased survival of the patient. The bacterial AC toxins CyaA and EF are key virulence factors impairing host immune responses and worsening the infections by Bordetella pertussis, the causative agent of whooping cough, and Bacillus anthracis, causing anthrax disease. The second aim of this doctoral thesis was to investigate the detailed modes of action of EF and CyaA and to provide the basis for the development of AC toxin inhibitors. Using radiochemical methods, we investigated the structure/activity relationships of substituted NTPs as CyaA inhibitors, revealing hypoxanthine nucleotides to be superior to other purine and pyrimidine nucleotides. One major achievement of this thesis is the development of fluorescence-based approaches allowing monitoring the binding of potential inhibitor molecules to CyaA. Selective CyaA inhibitors may be used to prevent dampening of the immune response upon Bordetella pertussis infection and to reduce mortality in severe courses of disease. The fluorescence-based approaches developed in this thesis are available for future high-throughput screening to support the development of highly potent and selective CyaA inhibitors. Cyclic cytidine 3´:5´-monophosphate (cCMP) was identified unambiguously in various mammalian tissues, protein kinase activity responsive to cCMP was observed and phosphodiesterase activity accounting for the selective degradation of cCMP was discovered. Therefore, cCMP may be a novel second messenger with potential importance in many physiological processes, including regulation of immune responses. However, so far, the precise identity of the cCMP-forming enzyme is unknown. For the first time, we provide evidence for cytidylyl cyclase (CC) activity of purified bacterial exotoxins, resulting in the conversion of CTP to cCMP. As a major achievement of this thesis, various nucleotidyl cyclase activities of bacterial AC toxins were investigated and monitored by the use of several techniques. First, the isotopic nucleotidyl cyclase assay based upon detection of radioactively labeled cNMPs showed that EF and CyaA possess CC activity, and additionally, we also observed the conversion of UTP to cUMP. We performed substrate saturation experiments to determine the kinetic properties of CC and UC activities. Second, the non-isotopic nucleotidyl cyclase assay basing upon an advanced sample preparation process and detection of cNMPs by HPLC was developed and used to monitor NTP consumption and cNMP formation by both AC toxins. Using this approach, in addition to the formation of cAMP, cCMP and cUMP, we also observed the formation of cIMP, cGMP and cTMP. The rank order of substrate preference was ATP > CTP > UTP > ITP > GTP > TTP, for both EF and CyaA. Third, mass spectrometry was used to unambiguously identify the corresponding cNMPs obtained from enzymatic reactions. Based on the fact that cCMP-analogs inhibit immune responses, we hypothesize that cCMP may be a novel endogenous second messenger and that the multiple nucleotidyl cyclase activities of bacterial AC toxins described in this thesis for the first time contribute to weakening of the immune defense by bacterial exotoxins, resulting in increased infection severity. Regarding future research, the endogenous CC enzyme remains to be discovered. The physiological role and the molecular targets of cCMP remain to be determined. What is the impact of multiple nucleotidyl cyclase activities of bacterial AC toxins on cyclic nucleotide metabolism in host cells? And finally, - regarding the occurrence of bacterial strains resistant to antibiotic treatment - can our findings lead to advancements in the prevention and therapy of bacterial infections

Molecular Analysis of the Interaction of Bordetella pertussis Adenylyl Cyclase with Fluorescent Nucleotides

The calmodulin (CaM)-dependent adenylyl cyclase (AC) toxin from Bordetella pertussis (CyaA) substantially contributes to the pathogenesis of whooping cough. Thus, potent and selective CyaA inhibitors may be valuable drugs for prophylaxis of this disease. We examined the interactions of fluorescent 2',3'-N-methylanthraniloyl (MANT)-, anthraniloyl- and trinitrophenyl (TNP)-substituted nucleotides with CyaA. Compared with mammalian AC isoforms and Bacillus anthracis AC toxin edema factor, nucleotides inhibited catalysis by CyaA less potently. Introduction of the MANT substituent resulted in 5- to 170-fold increased potency of nucleotides. Ki values of 3'MANT-2'd- ATP and 2'MANT-3'd-ATP in the AC activity assay using Mn2+ were 220 and 340 nM, respectively. Natural nucleoside 5'- triphosphates, guanine-, hypoxanthine- and pyrimidine-MANTand TNP nucleotides and d(i)-MANT nucleotides inhibited CyaA, too. MANT nucleotide binding to CyaA generated fluorescence resonance energy transfer (FRET) from tryptophans Trp69 and Trp242 and multiple tyrosine residues, yielding K(d) values of 300 nM for 3MANT-2d-ATP and 400 nM for 2'MANT-3'd-ATP. Fluorescence experiments and docking approaches indicate that the MANT- and TNP groups interact with Phe306. Increases of FRET and direct fluorescence with MANT nucleotides were strictly CaM-dependent, whereas TNP nucleotide fluorescence upon binding to CyaA increased in the absence of CaM and was actually reduced by CaM. In contrast to lowaffinity MANT nucleotides, even low-affinity TNP nucleotides generated strong fluorescence increases upon binding to CyaA. We conclude that the catalytic site of CyaA possesses substantial conformational freedom to accommodate structurally diverse ligands and that certain ligands bind to CyaA even in the absence of CaM, facilitating future inhibitor design

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70:878–886, 2006), we reported that 2′,3′-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5′-triphosphates (TNP-NTPs) are potent inhibitors (Ki values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (Ki, 3.7 nM) and sGC (Ki, 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (Ki, 7.1 nM) and ACII (Ki, 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by Gs- and Gi-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far

Molecular Analysis of the Interaction of Anthrax Adenylyl Cyclase Toxin, Edema Factor, with 2′(3′)-O-(N-(methyl)anthraniloyl)-Substituted Purine and Pyrimidine Nucleotides

Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous AC (mAC) exhibits broad specificity for purine and pyrimidine nucleotides (Mol Pharmacol 70 878-886, 200616766715). Here, we investigated structural requirements for EF inhibition by natural purine and pyrimidine nucleotides and nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl groups at the 2′(3′)-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (Ki, 100 nM) among 16 compounds studied. MANT-nucleotides inhibited EF competitively. 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 MANT-ATP, but FRET to MANT-CTP was only small. Mutagenesis studies revealed that Phe586 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. Docking approaches relying on crystal structures of EF indicate similar binding modes of the MANT nucleotides with subtle differences in the region of the nucleobases. In conclusion, like mAC, EF accommodates both purine and pyrimidine nucleotides. The unique preference of EF for the base cytosine offers an excellent starting point for the development of potent and selective EF inhibitors