Structure-activity relationships for the interactions of 2\u27- and 3\u27-(O)-(N-methyl)anthraniloyl-substituted purine and pyrimidine nucleotides with mammalian adenylyl cyclases

Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that 2\u27,3\u27-(O)-(N-methylanthraniloyl) (MANT)-substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic structure-activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5. (M)ANT-nucleotides inhibited fully activated VC1:IIC2 in the order of affinity for bases hypoxanthine\u3euracil\u3ecytosine\u3eadenine∼guanine≫xanthine. Omission of a hydroxyl group at the 2\u27 or 3\u27-position reduced inhibitor potency as did introduction of a γ-thiophosphate group or omission of the γ-phosphate group. Substitution of the MANT-group by an ANT-group had little effect on affinity. Although all nucleotides bound to VC1:IIC2 similarly according to the tripartite pharmacophore model with a site for the base, the ribose, and the phosphate chain, nucleotides exhibited subtle differences in their binding modes as revealed by fluorescence spectroscopy and molecular modelling. MANT-nucleotides also differentially interacted with the VC1:VC1 homodimer as assessed by fluorescence spectroscopy and modelling. Similar structure-activity relationships as for VC1:IIC2 were obtained for recombinant ACs 1, 2 and 5, with AC2 being the least sensitive AC isoform in terms of inhibition. Overall, ACs possess a broad base-specificity with no preference for the cognate base adenine as verified by enzyme inhibition, fluorescence spectroscopy and molecular modelling. These properties of ACs are indicative for ligand-specific conformational landscapes that extend to the VC1:VC1 homodimer and should facilitate development of non-nucleotide inhibitors

Structural basis for the high-affinity inhibition of mammalian membranous adenylyl cyclase by 2\u27,3\u27-o-(N-methylanthraniloyl)-inosine 5\u27-triphosphate

2\u27,3\u27-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K(i), 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the G(s)α- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC

Anthraniloyl-derived nucleotides as potent and selective adenylyl cyclase inhibitors

Mammals express nine membranous adenylyl cyclases (AC 1-9) for the catalysis of the important second messenger cAMP in the intracellular signaling pathway. Inhibitors of AC1 and AC5 may be useful drugs for neuroprotection, heart failure and longevity. 2�,3�-O-(N-Methylanthraniloyl) (MANT)-substituted nucleotides are competitive AC inhibitors and were synthesized with different nucleobases in high purity after reversed phase HPLC separation. MANT-nucleotides were tested for their inhibitory potency at recombinant AC1, 2, and 5 expressed by a Sf9 insect cell system. In our primary investigations MANT-ITP was identified as the most potent AC1 and 5 inhibitor known so far with Ki values of 1 - 3 nM. Interestingly, bis-substituted MANT-nucleotides were discovered as side products in the regular synthesis of MANT-nucleotides. The synthesis of new mono- and bis-substituted anthraniloyl-group derived purine nucleotides was expanded. The anthraniloyl moiety differ by halogens of chlorine and bromine and acetylated amino group in 5 position of the phenyl ring system. Moreover, substitution at the amino function of the ANT-group lead to propyl derivatives. The immobile and aerobic bacterium Bordetella pertussis secretes the key virulence factor, the adenylyl cyclase toxin CyaA. Overall, 32 compounds were prepared to compare 16 pairs of mono- and bis-substituted (M)ANT-nucleotides for their potencies at inhibiting CyaA and membranous ACs (mAC). For the first time we found inhibitors combining high inhibition potency for CyaA toxin with selectivity towards mammalian ACs. Bis-substituted halogen anthraniloyl-derived purine nucleotides inhibited CyaA in the nanomolar range in a competitive manner (Ki = 13 - 20 nM). Moreover, bis-substituted halogen anthraniloyl-derived nucleotides of adenine displayed not only high affinity to the bacterial AC, but also revealed high selectivity by 50- to 150-fold depending on the chosen mAC 1, 2 or 5. The modeling study demonstrate an impression for the obvious alignment of the (M)ANT-nucleotide binding mode to CyaA. The docking of bis-substituted halogen anthraniloyl derivatives exposed additional hydrophobic interactions between enzyme and inhibitor causing higher potency for this class of compounds. Bis-substituted (M)ANT-nucleotides offered the advantage of excellent signal to noise ratio in fluorescence spectroscopy, compared to mono-substituted (M)ANT-nucleotides, with applications in HTS for assessment of non-fluorescent inhibitor potencies

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

Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors of Bordetella pertussis Adenylyl Cyclase Toxin

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

Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 monoand bis-(M)ANT-substituted nucleoside 5′-triphosphates

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 nucleotides 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)ANT-substituted 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 (K (i), 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

Relation between Intensity of Biocide Practice and Residues of Anticoagulant Rodenticides in Red Foxes (<i>Vulpes vulpes</i>)

<div><p>Anticoagulant rodenticides (ARs) are commonly used to control rodent infestations for biocidal and plant protection purposes. This can lead to AR exposure of non-target small mammals and their predators, which is known from several regions of the world. However, drivers of exposure variation are usually not known. To identify environmental drivers of AR exposure in non-targets we analyzed 331 liver samples of red foxes (<i>Vulpes vulpes</i>) for residues of eight ARs and used local parameters (percentage of urban area and livestock density) to test for associations to residue occurrence. 59.8% of samples collected across Germany contained at least one rodenticide, in 20.2% of cases at levels at which biological effects are suspected. Second generation anticoagulants (mainly brodifacoum and bromadiolone) occurred more often than first generation anticoagulants. Local livestock density and the percentage of urban area were good indicators for AR residue occurrence. There was a positive association between pooled ARs and brodifacoum occurrence with livestock density as well as of pooled ARs, brodifacoum and difenacoum occurrence with the percentage of urban area on administrative district level. Pig holding drove associations of livestock density to AR residue occurrence in foxes. Therefore, risk mitigation strategies should focus on areas of high pig density and on highly urbanized areas to minimize non-target risk.</p></div

Residue distribution of anticoagulant rodenticides (ARs) in German districts.

<p>Number (N) of red foxes (<i>Vulpes vulpes</i>) containing anticoagulant rodenticide (AR) residues in their livers and concentrations in μg/g of individuals where ARs were present: all anticoagulants (AR), brodifacoum (BR) and bromadiolone (BM). German districts are located in four federal states (Brandenburg: BB, Baden-Wuerttemberg: BW, Lower Saxony: LS, North Rhine-Westphalia: NW).</p

Residues of anticoagulant rodenticides (ARs) in red fox liver samples.

<p>Number (N) of red foxes (<i>Vulpes vulpes</i>) containing residues and concentrations in μg/g of residues from AR positive individuals are stated for all eight analyzed ARs. % refers to the total number of 331 samples.</p