Figure 1 |. Roseoflavin mononucleotide at the center of a medicinal chemistry optimization strategy that led to the discovery of synthetic analogs with potent activity and selectivity.

(A) Chemical structure of 5FDQD (5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]- quinoxaline-1,3(2H,5H)-dione), a potent inhibitor of the FMN riboswitch. Color-coding for functional groups introduced during SAR study: orange, negatively charged/polar group; tan, hydrophobic group. IC₅₀, half maximal inhibitory concentration as measured by in-line probing; EC₅₀, half maximal effective concentration in transcription termination assays.³¹ Note that all values for IC₅₀ and EC₅₀ in subsequent figures are given in units of μM. (B) Secondary structure of the FMN riboswitch showing sequence and structure conservation among bacteria. Residues that interact with flavin-bearing ligands in crystal structures are circled in pink.^{30, 33} The list of the six joining regions is indicated, with numbering for B. subtilis and F. nucleatum (in parenthesis). (C) Dissecting functional positions 8 and 10 of roseoflavin mononucleotide (RoFMN) over the course of a structure-activity relationship (SAR) study of the FMN riboswitch. Color-coding for functional groups: red, left unaltered during SAR study; green, primary focus of SAR study; blue, secondary focus. IC₅₀ and EC₅₀ calculated as for 5FDQD (see Methods; Tables S1and S2; Figure S1). (D) Comparative banding pattern of SHAPE chemical probing within the J6/1 joining region, which serves as an indicator for ligand binding.³⁰ [MgCl₂] tested were: 0, 0.1, 0.5, 2.0 and 15.0 mM. Arrowheads: residues of interest within J6/1. The gels were aligned in SAFA^53–54 (full unaltered SHAPE gels shown in Figure S2).