A conformational transition in the adenylyl cyclase catalytic site yields different binding modes for ribosyl-modified and unmodified nucleotide inhibitors

Wang, Jenna L.; Guo, Zhixiong; Zhang, Qiyuan; Wu, Jay J.-Q.; Seifert, Roland; Lushington, Gerald H.

doi:10.1016/j.bmc.2007.02.014

Cited by 10 publications

(5 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In previous studies, we developed a three-site pharmacophore model for mAC and CyaA toxin with binding regions for the base, the MANT group, and the polyphosphate chain Mou et al, 2006;Göttle et al, 2007;Wang et al, 2007). Those studies revealed that the MANT group and the polyphosphate chain are the major determinants of inhibitor potency, whereas the base plays a relatively small …”

Section: Discussionmentioning

confidence: 99%

“…MANT-nucleotides are fluorescent, and we exploited this property to suggest conformational changes associated with activation in purified catalytic subunits of mAC (Mou et al, , 2006 and the Bordetella pertussis AC toxin CyaA (Göttle et al, 2007). In addition, by combining crystallographic and molecular modeling approaches, we developed a three-site pharmacophore model for mAC and CyaA, with binding domains for the base, the MANT group, and the polyphosphate chain Mou et al, 2006;Göttle et al, 2007;Wang et al, 2007). Those studies revealed that the catalytic sites of mAC and CyaA exhibit substantial conformational flexibility, accommodating both purine and pyrimidine nucleotides.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Taha¹,

Schmidt²,

Göttle³

et al. 2009

Molecular Pharmacology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Taha¹,

Schmidt²,

Göttle³

et al. 2009

Molecular Pharmacology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Third, we analyzed the crystal structure of VC1:IIC2 in complex with MANT-ITP and compared this structure with the known VC1:IIC2 structure in complex with MANT-GTP . Last, we conducted molecular dynamics simulations to further evaluate the structural requirements for the highaffinity interaction of MANT-ITP with AC5 in comparison to MANT-GTP (Wang et al, 2007). Here, we show that even subtle differences in ligand structure have profound effects for interaction with mAC.…”

Section: Introductionmentioning

confidence: 90%

“…This is reflected by the fact that MANT nucleotides exhibit up to 17,000-fold higher affinity for AC than their nonsubstituted parent nucleotides and that purine and pyrimidine nucleotides exhibit similar affinity for AC . On the basis of those data, we proposed that ACs exhibit a high degree of conformational flexibility, allowing the catalytic site to accommodate structurally diverse bases (Mou et al, 2006;Wang et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2′,3′-O-(N-Methylanthraniloyl)-Inosine 5′-Triphosphate

Hübner¹,

Dixit²,

Mou³

et al. 2011

Molecular Pharmacology

Self Cite

View full text Add to dashboard Cite

2Ј,3Ј-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 ␣-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.

show abstract

“…We exploited this property to monitor conformational changes associated with activation in purified catalytic subunits of mAC (Mou et al , 2006Hübner 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). …”

Section: Introductionmentioning

confidence: 99%

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

Taha

Dove

Geduhn

et al. 2011

Naunyn-Schmiedeberg's Arch Pharmacol

Self Cite

View full text Add to dashboard Cite

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.

show abstract

A conformational transition in the adenylyl cyclase catalytic site yields different binding modes for ribosyl-modified and unmodified nucleotide inhibitors

Cited by 10 publications

References 31 publications

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

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

Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2′,3′-O-(N-Methylanthraniloyl)-Inosine 5′-Triphosphate

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

Contact Info

Product

Resources

About