Fluorescent analogs of cyclic and linear dinucleotides as phosphodiesterase and oligoribonuclease activity probes

Abstract

Cyclic dinucleotide-based second messengers, including c-di-GMP, c-di-AMP and cGAMP, are universal signaling molecules widely used by prokaryotes and eukaryotes. C-di-GMP and c-di-AMP play key roles in bacterial survival. Consequently, metabolism proteins that modulate cyclic dinucleotide concentrations, such as cyclic dinucleotide phosphodiesterases (PDE), are potential drug targets; thus, probes that report PDE activity could have great utility in high throughput screening for PDE inhibitors. However, there is a paucity of luminescent-based probes for monitoring cyclic dinucleotide PDE activity. In this study, we synthesized various fluorescent nucleobase (ethenoA and 2-AP) analogs of cyclic and linear dinucleotides. These analogs were readily cleaved by the cyclic dinucleotide PDE and oligoribonucleases (Orn). Cleavage of the fluorescent probes led to changes in fluorescence intensities. Our results suggest that these fluorescent analogs can be used to monitor the activity of cyclic dinucleotide PDEs in real time and that these probes could facilitate the identification of inhibitors of cyclic dinucleotide PDEs. Additionally these probes could be used to profile the activity of expressed PDEs and Orns.