Phospholipase Fluorogenic Probes and Molecular Determinants of Cyclic Guanosine Monophosphate–Adenosine Monophosphate Binding to Cyclic Oligonucleotide-Based Antiphage Signaling System CapV

Cyclic oligonucleotide-based antiphage signaling systems (CBASS) protect bacteria from bacteriophage infection by producing cyclic nucleotide second messengers that activate downstream effector proteins. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP)-activated phospholipase in Vibrio (CapV) is a prevalent CBASS effector whose activity is commonly monitored using the commercially available fluorogenic substrate resorufin butyrate; however, this probe is hydrolyzed by both basal and activated CapV, limiting its usefulness in CapV-related studies. Here, we report the rational design and evaluation of a series of resorufin ester probes with varying acyl chain lengths to improve the fluorogenic assay used to monitor CapV (from Acinetobacter baumannii and Vibrio cholerae) activation. While resorufin butyrate and pentanoate exhibited substantial cGAMP-independent hydrolysis, resorufin octanoate and decanoate provided an optimal balance between catalytic turnover and activation-dependent discrimination, displaying over 5-fold and approximately 10-fold increases in hydrolysis, respectively, upon 3',3'-cGAMP activation. Using resorufin octanoate as a fluorogenic reporter, we systematically investigated the effects of cyclic dinucleotide structural modifications on CapV activation. Phosphorothioate analogues with nonbridging sulfur (exo-S linkages) retained partial agonist activity, whereas the corresponding bridging 5'-phosphorothioester (endo-S) analogues was inactive, highlighting the importance of the native phosphodiester moiety in activating CapV. Replacement of the ribose 2'-hydroxyl groups with fluorine preserved CapV activation, whereas 2'-deoxy substitutions reduced activity, with modifications on the adenosine moiety having a greater detrimental effect than those on guanosine. These findings using the fluorogenic probes were validated using phospholipid hydrolysis and activity-based protein labeling assays. Molecular docking and 100 ns molecular dynamics simulations revealed that the observed activity trends arise from differential disruption of key hydrogen-bonding interactions and ligand conformations required for productive CapV activation. Together, these fluorogenic probes provide a robust, high-throughput-compatible platform for studying CapV activation and defining cyclic dinucleotide structure-activity relationships.