Search

c-di-AMP is an important bacterial second messenger found in Gram-positive and mycobacteria. c-di-AMP regulates myriads of processes in bacteria as well as immune response in higher organisms so interest in smallmolecules that would attenuate the activity of c-di-AMP metabolism enzymes is high. Herein, we report the first small molecule inhibitor of a c-di-AMP synthase, DisA, using a coralyne-based assay. [ABSTRACT FROM AUTHOR]

Cyclic diadenosine monophosphate (c-di-AMP) has emerged as an important dinucleotide that is involved in several processes in bacteria, including cell wall remodeling (and therefore resistance to antibiotics that target bacterial cell wall). Small molecules that target c-di-AMP metabolism enzymes have the potential to be used as antibiotics. Coralyne is known to form strong complexes with polyadenine containing eight or more adenine stretches but not with short polyadenine oligonucleotides. Using a panel of techniques (UV, both steady state fluorescence and fluorescence lifetime measurements, circular dichroism (CD), NMR, and Job plots), we demonstrate that c-di-AMP, which contains only two adenine bases is an exception to this rule and that it can form complexes with coralyne, even at low micromolar concentrations. Interestingly, pApA (the linear analog of c-di-AMP that also contains two adenines) or cyclic diguanylate (c-di-GMP, another nucleotide second messenger in bacteria) did not form any complex with coralyne. Unlike polyadenine, which forms a 2:1 complex with coralyne, c-di-AMP forms a higher order complex with coralyne (≥6:1). Additionally, whereas polyadenine reduces the fluorescence of coralyne when bound, c-di-AMP enhances the fluorescence of coralyne. We use the quenching property of halides to selectively quench the fluorescence of unbound coralyne but not that of coralyne bound to c-di-AMP. Using this simple selective quenching strategy, the assay could be used to monitor the synthesis of c-di-AMP by DisA or the degradation of c-di-AMP by YybT. Apart from the practical utility of this assay for c-di-AMP research, this work also demonstrates that, when administered to cells, intercalators might not only associate with polynucleotides, such as DNA or RNA, but also could associate with cyclic dinucleotides to disrupt or modulate signal transduction processes mediated by these nucleotides. [ABSTRACT FROM AUTHOR]

C-di-GMP, a cyclic guanine dinucleotide, has been shown to regulate biofilm formation as well as virulence gene expression in a variety of bacteria. Analogues of c-di-GMP have the potential to be used as chemical probes to study c-di-GMP signaling and could even become drug leads for the development of anti-biofilm compounds. Herein we report the synthesis and biophysical studies of a series of c-di-GMP analogues, which have both phosphate and sugar moieties simultaneously modified (called endo-S-c-di-GMP analogues). We used computational methods to predict the relative orientation of the guanine nucleobases in c-di-GMP and analogues. DOSY NMR of the endo-S-c-di-GMP series showed that the polymorphism of c-di-GMP can be tuned with conservative modifications to the phosphate and sugar moieties (conformational steering). Binding studies with Vc2 RNA (a class I c-di-GMP riboswitch) revealed that conservative modifications to the phosphate and 2'-positions of c-di-GMP dramatically affected binding to class I riboswitch. [ABSTRACT FROM AUTHOR]