Molecular analysis of mammalian adenylyl cyclases and edema factor, a bacterial adenylyly cyclase toxin

Adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP, an important second messenger central to many signaling pathways. Nine different isoforms of mammalian ACs (mACs) are present, each with distinct localization, physiological function and regulatory mechanisms by activators and inhibitors. In addition to mACs, bacterial AC toxins such as edema factor (EF) from Bacillus anthracis and CyaA from Bordetella pertussis have also been identified. Following infection, the AC toxins cause a dramatic increase in cAMP levels, thereby disrupting several intracellular signaling pathways. This thesis is broadly divided into two parts and is aimed at validating the active-site nucleotide analogs of mACs and EF. The first part of the thesis focuses on understanding mAC regulation and the mechanism of interaction of mACs with fluorescent 2', 3'-O-(2, 4, 6-Trinitrophenyl) (TNP)- nucleotides. Using purified catalytic subunits of mAC (C1/C2) as model for mACs, we have observed the binding of TNP-nucleotides to C1/C2 and the resulting conformational changes monitored by fluorescence spectroscopy. The enzymatic assays have shown that TNP-nucleotides potently inhibit C1/C2 as well as holo-AC isoforms. An isoform-selective inhibition of AC1, AC2 and AC5 by TNP-nucleotides has been reported for the first time indicating that TNP-nucleotides can serve as models for rational design of potent isoform-specific AC inhibitors. Furthermore, biophysical and biochemical analysis of the effects of TNP- and 2' (3')-O-(N-methylanthraniloyl) (MANT) -nucleotides on the individual subunits C1 and C2 show that C1 and C2 can exist as homodimers. This homodimerization may play an important physiological role in cAMP signaling. The second part of the thesis addresses the structure-activity relationship in regulating the catalytic activity of EF and its interaction with calmodulin (CaM) using MANT derivatives of ATP and GTP as probes. Our enzymatic assays have shown that MANT-nucleotides are highly potent at inhibiting EF. MANT-nucleotides are also favorable for FRET studies indicating that our robust fluorescence assays can be used for High-Throughput Screening (HTS) of EF inhibitors. Additionally, EF-CaM interaction was probed by MANT-nucleotides. We have observed that binding and activation of EF by CaM are two independent processes in the presence and absence of calcium. Furthermore, our fluorescence assays to monitor binding of oxidized CaM to EF also indicate that methionine residues in CaM play an important role in binding to EF

Distinct Interactions of 2′- and 3′-O-(N-Methyl)anthraniloyl-Isomers of ATP and GTP with the Adenylyl Cyclase Toxin of Bacillus anthracis, Edema Factor

Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. Bacillus anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study we showed that various 2′(3′)-O-N-(methyl)anthraniloyl (MANT)-substituted nucleoside 5′-triphosphates are potent inhibitors (Ki values in the 0.1-5 μM range) of purified EF. Upon interaction with calmodulin we observed efficient fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine residues of EF and the MANT-group of MANT-ATP. Molecular modelling suggested that both the 2′- and 3′-MANT-isomers can bind to EF. The aim of the present study was to examine the effects of defined 2′- and 3′-MANT-isomers of ATP and GTP on EF. 3′-MANT-2′-deoxy-ATP inhibited EF more potently than 2′-MANT-3′-deoxy-ATP, whereas the opposite was the case for the corresponding GTP analogs. Calmodulin-dependent direct MANT-fluorescence and FRET was much larger with 2′-MANT-3′-deoxy-ATP and 2′-MANT-3′-deoxy-GTP compared to the corresponding 3′-MANT-2′-deoxy-isomers and the 2′(3′)-racemates. Ki values of MANT-nucleotides for inhibition of catalysis correlated with Kd values of MANT-nucleotides in FRET studies. Molecular modelling indicated different positioning of the MANT-group in 2′-MANT-3′-deoxy-ATP/GTP and 3′-MANT-2′-deoxy-ATP/GTP bound to EF. Collectively, EF interacts differentially with 2′-MANT- and 3′-MANT-isomers of ATP and GTP, indicative for conformational flexibility of the catalytic site and offering a novel approach for the development of potent and selective EF inhibitors. Moreover, our present study may serve as a general model of how to use MANT-nucleotide isomers for the analysis of the molecular mechanisms of nucleotide/protein interactions

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

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

Double-blind, placebo-controlled clinical evaluation of an Ayurvedic formulation (GlucoCare capsules) in non-insulin dependent diabetes mellitus

Diabetes mellitus describes a metabolic disorder of multiple etiologies characterized by insulin resistance, relative insulin deficiency and hyperglycemia with disturbances of carbohydrate, fat and protein metabolism. The goal for treatment of diabetes is to prevent its acute manifestations and long-term microvascular and macrovascular complications. The present study was conducted to evaluate the efficacy and safety of an Ayurvedic formulation (GlucoCare Capsules) in non-insulin dependent diabetes mellitus. Fifty NIDDM patients of pitta-kapha prakriti attending the outpatient department of the Government Ayurvedic Medical College, Guwahati, Assam, India were included in the study, and randomly divided into 2 groups, GlucoCare and placebo. All received either GlucoCare or placebo in a dose of 2 capsules twice daily, before meals for 3 months. All 50 patients completed the study - no drop outs, withdrawals or patients lost to follow up. The GlucoCare group showed significant improvement in symptoms from the 2nd month till the end of the study. GlucoCare was well tolerated by all patients throughout the treatment period with no evidence of adverse effects. The study indicates clinical efficacy of GlucoCare Capsules in the management of NIDDM in those belonging to pitta-kapha prakriti. The formulation is well tolerated and appears safe in the dosage used

Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2(3)-O-(N-methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides.

Abbreviations AC, adenylyl cyclase; ANT, anthraniloyl-; CaM, calmodulin; CyaA, Bordetella pertussis adenylyl cyclase toxin; ESI, electrospray ionization; FRET, fluorescence resonance energy transfer; HPLC, high pressure liquid chromatography; k, capacity factor; mAC, mammalian membranous adenylyl cyclase; MANT, methylanthraniloyl-; MS, mass spectroscopy; MW, molecular weight; NDP, nucleoside 5´-diphosphate; NTP, nucleoside 5´-triphosphate; PMEApp, {9-[2-(phosphonomethoxy)ethyl]adenine diphosphate}; EF, full-length edema factor adenylyl cyclase toxin; EF3, catalytic domain of edema factor adenylyl cyclase toxin; R f , retention factor; R t , retention time; TLC, thin layer chromatography. MOL #52340 3 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). However, conventional antibiotic treatment is ineffective against either toxemia or antibioticresistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous A