Validation and Development of an Escherichia coli Riboflavin Pathway Phenotypic Screen Hit as a Small-Molecule Ligand of the Flavin Mononucleotide Riboswitch.

A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50 = 0.3 µM), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of which are described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.

Cloning, expression, and pharmacological characterization of the GPR120 free fatty acid receptor from cynomolgus monkey: comparison with human GPR120 splice variants.

Activation of the GPCR GPR120 by free fatty acids has been reported to cause GLP-1 release in rodent intestine. One genetic sequence was reported for rodents, while two sequences were reported for human GPR120, BC101175 and NM_181745. A 1086 base pair sequence cloned from cynomolgus monkey colon cDNA has 85.1% and 83.4% homology with the mouse and rat GPR120 sequences, and 97.5% homology with the human BC101175 sequence. No splice variants of the cynomolgus monkey GPR120 receptor were found. Eight non-synonymous cSNPs were discovered with frequencies less than 4% in monkey samples tested. Real-time PCR demonstrated that, like the human, the highest GPR120 expression in cynomolgus monkey is in lung and colon. Studies measuring intracellular calcium release produced by free fatty acids and the small molecule GPR120 agonist GW9508 in cells expressing the cynomolgus monkey GPR120 receptor were compared to those expressing the human BC101175 splice variant. Long-chain free fatty acids produced the greatest response in cynomolgus monkey GPR120-expressing cells. GW9508 had similar efficacy at the cynomolgus monkey and at the BC101175 human GPR120 receptors. The cynomolgus monkey and the human GPR120 (BC101175) receptors have similar sequences and pharmacology. The possible significance of the alternate splice variant in human is discussed.