Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interactions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interactions without the need to purify the protein when performed in cells with low background activity. This method, differential radial capillary action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein–ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through capillary action. We show here that DRaCALA allows detection of specific interactions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli , demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interaction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interactions in a systematic and comprehensive approach.

Small molecules that can attenuate bacterial toxin production or biofilm formation have the potential to solve the bacteria resistance problem. Although several molecules, which inhibit bacterial cell-to-cell communication (quorum sensing), biofilm formation and toxin production, have been discovered, there is a paucity of US FDA-approved drugs that target these processes. Here, we review the current understanding of quorum sensing in important pathogens such as Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus and provide examples of experimental molecules that can inhibit both known and unknown targets in bacterial virulence factor production and biofilm formation. Structural data for protein targets that are involved in both quorum sensing and cyclic diguanylic acid signaling are needed to aid the development of molecules with drug-like properties in order to target bacterial virulence factors production and biofilm formation.

Significance: The development of small-molecule therapeutics that act by targeting defined DNA or RNA sequences associated with human disease remains a challenge. RG-7916, a small-molecule drug candidate for the treatment of spinal muscular atrophy (SMA), selectively regulates the alternative splicing (AS) of the SMN2 gene. Herein, we show that SMN-C2 and -C3, close analogs of RG-7916, act by binding SMN2 pre-mRNA and thereby increasing the affinity of the RNA binding proteins far upstream element binding protein 1 (FUBP1) and KH-type splicing regulatory protein (KHSRP) to the SMN2 pre-mRNA complex. These results suggest that nucleic acid targeted small molecules may have untapped potential for modulating disease processes at the level of pre-mRNA splicing.