Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors ofBordetella pertussisAdenylyl Cyclase Toxin

Abstract

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 Ն100-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.

Inhibitors of membranous adenylyl cyclases

“…MANT-nucleotides and the structurally related TNP-nucleotides inhibit ACs 1, 5 and 6 with similar potencies, whereas AC2 is inhibited only ∼10-fold less potently [22,26-29]. Hence, as for the classic P-site inhibitors, no compelling isoform-specificity was observed [15].…”

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

“…CaCl 2 , MnCl 2 tetrahydrate and MgCl 2 hexahydrate (highest quality) were purchased from Merck (Darmstadt, Germany). Mono-and bis-(M)ANT nucleotides were synthesised as described (Taha et al 2009;Geduhn et al 2011).…”

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

“…Despite these reservations regarding MANT-ITP, the long-term goal of obtaining isoform-specific AC inhibitors is not elusive. Specifically, we have shown that certain bis-MANT-substituted nucleotides are very potent inhibitors of the Bordetella pertussis AC toxin CyaA (Geduhn et al, 2011), with high selectivity relative to mammalian ACs. The identification of Bis-MANT nucleotides as potent and selective CyaA inhibitors resulted from a relatively small medicinal chemistry program and not from an extensive high-throughput screening effort.…”

“…Specifically, upon binding to the substrate-binding site, nonfluorescent inhibitors would quench the large basal or FS-stimulated direct fluorescence or FRET signals. This assay is even feasible to obtain information on relative inhibitor affinity through the analysis of cumulative concentration/quench curves in a single cuvette (Geduhn et al, 2011). Fluorescence assays with MANT-ITP could also be useful to identify allosteric AC inhibitors.…”