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 calmodulindependent 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, 2006). 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 2Ј(3Ј)-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (K i , 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.

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 2Ј,3Ј-Nmethylanthraniloyl (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. K i values of 3ЈMANT-2Јd-ATP and 2ЈMANT-3Јd-ATP in the AC activity assay using Mn 2ϩ were 220 and 340 nM, respectively. Natural nucleoside 5Ј-triphosphates, guanine-, hypoxanthine-and pyrimidine-MANTand TNP nucleotides and di-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 3ЈMANT-2Јd-ATP and 400 nM for 2ЈMANT-3Јd-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.

Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces ␤-adrenoceptor-stimulated Ca 2ϩ inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg 2ϩ were similar. Moreover, the inhibitory profile of eight 2Ј(3Ј)-O-(N-methylanthraniloyl) (MANT)-nucleoside 5Ј-([␥-thio])triphosphates on mouse heart in the presence of Mg 2ϩ was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K i values in the 15 to 25 nM range in the presence of Mg 2ϩ and in the 1 to 5 nM range in the presence of Mn 2ϩ . However, in the presence of Mn 2ϩ , we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg 2ϩ , AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn 2ϩ masking AC5 effects were problems.

Cyclic adenosine 3′:5′-monophosphate (cAMP) and cyclic guanosine 3′:5′-monophosphate (cGMP) are second messengers for a numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3′:5′-monophosphate (cCMP) is discussed controversially, and almost nothing is known about cyclic uridine 3′:5′-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analog inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC- and UC activity was determined by a radiometric assay, using [α-32P]CTP and [α-32P]UTP as substrates, respectively, and by an HPLC method. The identity of cNMPs was confirmed by mass spectrometry. Based on available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate-specificity and exhibit cytidylyl- and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions.

Objective Lesch–Nyhan disease (LND) is caused by congenital deficiency of the purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Affected patients have a peculiar neurobehavioral syndrome linked with reductions of dopamine in the basal ganglia. The purpose of the current studies was to determine the anatomical basis for the reduced dopamine in human brain specimens collected at autopsy. Methods Histopathological studies were conducted using autopsy tissue from 5 LND cases and 6 controls. Specific findings were replicated in brain tissue from an HGprt-deficient knockout mouse using immunoblots, and in a cell model of HGprt deficiency by flow-activated cell sorting (FACS). Results Extensive histological studies of the LND brains revealed no signs suggestive of a degenerative process or other consistent abnormalities in any brain region. However, neurons of the substantia nigra from the LND cases showed reduced melanization and reduced immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. In the HGprt-deficient mouse model, immunohistochemical stains for TH revealed no obvious loss of midbrain dopamine neurons, but quantitative immunoblots revealed reduced TH expression in the striatum. Finally, 10 independent HGprt-deficient mouse MN9D neuroblastoma lines showed no signs of impaired viability, but FACS revealed significantly reduced TH immunoreactivity compared to the control parent line. Interpretation These results reveal an unusual phenomenon in which the neurochemical phenotype of dopaminergic neurons is not linked with a degenerative process. They suggest an important relationship between purine recycling pathways and the neurochemical integrity of the dopaminergic phenotype.