A STING-based fluorescent polarization assay for monitoring activities of cyclic dinucleotide metabolizing enzymes.

Cyclic dinucleoties, such as cGAMP, c-di-GMP and c-di-AMP, are fascinating second messengers with diverse roles in both prokaryotes and eukaryotes. Consequently there is a need for simple and inexpensive methods for profiling these compounds in biological media, monitoring their synthesis or degradation by enzymes and for identifying inhibitors of proteins that metabolize or bind to these dinucleotides. Since 2011, when we reported the first simple method to detect c-di-GMP (S. Nakayama, I. Kelsey, J. Wang, K. Roelofs, B. Stefane, Y. Luo, V. T. Lee and H. O. Sintim, J. Am. Chem. Soc., 2011, 133, 4856) or in 2014 when we revealed another surprisingly simple assay to detect c-di-AMP (J. Zhou, D. A. Sayre, Y. Zheng, H. Szmacinski and H. O. Sintim, Anal. Chem., 2014, 86, 2412), there have been efforts to develop assays to detect cyclic dinucleotides by others. However a unified and simple assay, which can be used for all cyclic dinucleotides is lacking. Here, we investigate STING binding by various fluorescein-labeled c-di-GMP, c-di-AMP and cGAMP, using fluorescent polarization (FP). Fluorescein-labeled c-di-GMP (F-c-di-GMP) was found to be the best binder of STING. This probe could be displaced by unlabeled cGAMP, c-di-AMP or c-di-GMP and hence it is a universal probe, which can be used to monitor all three dinucleotides. HPLC analysis was used to validate the new F-c-di-GMP-based FP assay.

Bacterial Cyclic Dinucleotides and the cGAS-cGAMP-STING Pathway: A Role in Periodontitis?

Host cells can recognize cytosolic double-stranded DNAs and endogenous second messengers as cyclic dinucleotides-including c-di-GMP, c-di-AMP, and cGAMP-of invading microbes via the critical and essential innate immune signaling adaptor molecule known as STING. This recognition activates the innate immune system and leads to the production of Type I interferons and proinflammatory cytokines. In this review, we (1) focus on the possible role of bacterial cyclic dinucleotides and the STING/TBK1/IRF3 pathway in the pathogenesis of periodontal disease and the regulation of periodontal immune response, and (2) review and discuss activators and inhibitors of the STING pathway as immune response regulators and their potential utility in the treatment of periodontitis. PubMed/Medline, Scopus, and Web of Science were searched with the terms "STING", "TBK 1", "IRF3", and "cGAS"-alone, or together with "periodontitis". Current studies produced evidence for using STING-pathway-targeting molecules as part of anticancer therapy, and as vaccine adjuvants against microbial infections; however, the role of the STING/TBK1/IRF3 pathway in periodontal disease pathogenesis is still undiscovered. Understanding the stimulation of the innate immune response by cyclic dinucleotides opens a new approach to host modulation therapies in periodontology.

Identification of a Mycobacterium tuberculosis Cyclic Dinucleotide Phosphodiesterase Inhibitor.

Immune cells sense bacteria-derived c-di-GMP and c-di-AMP as well as host-derived cGAMP, which is synthesized by cGAS upon binding to the pathogen's DNA, to mount an immunological response (cytokine production) via the STING-TBK1 pathway. Successful pathogens, such as Mycobacterium tuberculosis and group B streptococcus, harbor phosphodiesterases (PDEs) that can cleave bacterial c-di-AMP as well as host-derived cGAMP to blunt the host's response to infection. Selective inhibitors of bacterial cyclic dinucleotide (CDN) PDEs are needed as tool compounds to study the role(s) of CDN PDEs during infection and they could also become bona fide antivirulence compounds, but there is a paucity of such compounds. Using a high-throughput assay, we identified six inhibitors of MTB CDN PDE (CdnP). The most potent inhibitor, C82 with an IC50 of ∼18 µM, did not inhibit the enzymatic activities of three other bacterial CDN PDEs (Yybt, RocR, and GBS-CdnP), a viral CDN PDE (poxin) or mammalian ENPP1.