Atomic resolution mechanistic studies of ribocil: A highly selective unnatural ligand mimic of the E. coli FMN riboswitch

Abstract

Bacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway. Although ribocil is structurally distinct from FMN, ribocil functions as a potent and highly selective synthetic mimic of the natural ligand to repress riboswitch-mediated ribB gene expression and inhibit bacterial growth both in vitro and in vivo. Herein, we expand our analysis of ribocil; including mode of binding in the FMN binding pocket of the riboswitch, mechanisms of resistance and structure-activity relationship guided efforts to generate more potent analogs.

Keywords: Antibiotics; FMN riboswitch; RNA regulatory element; riboflavin.

Figure 1.

FMN riboswitch mechanism of action, ribocil chemical structures, and suppression of ribocil activity by riboflavin (A) Diagram of the FMN riboswitch including the 5′ mRNA aptamer with bound FMN and the 3′ expression platform which regulates expression of the downstream ribB gene open reading frame (blue). In the FMN ligand bound form (left panel) the FMN aptamer induces formation of the sequester loop in the expression platform that inhibits ribB expression (OFF) through early termination of transcription of the ribB ORF and sequestration of the Shine-Dalgarno ribosome binding sequence to prevent translation of fully transcribed ribB mRNAs. Alternatively, in the absence of FMN, the FMN aptamer adopts an alternative structural conformation (ON) that induces an anti-sequester loop in the expression platform enabling uninterrupted ribB expression (right panel). (B) Chemical structures of the ribocil enantiomers ribocil-A (R isomer), ribocil-B (S isomer) and of the ribocil analog ribocil-C (S isomer). (C) Anti-bacterial activity of ribocil A, B, C spotted on top of Mueller Hinton agar plates embedded with the E. coli strain MB5746 either in the absence (left panel) or presence (right panel) of riboflavin (20 μM). Compounds were suspended in DMSO and 5 μl was spotted after 2-fold dilutions starting at 512 μg/ml for ribocil A, B and novobiocin (negative control) and at 64 μg/ml for ribocil-C.

Figure 2.

Co-crystal structure of ribocil-D bound to the F. nucleatum FMN riboswitch. (A) Chemical structure of ribocil-D. (B) Electron density difference map of ribocil-D displayed as a grid at 3.0σ level. (C) Ribocil-D in the FMN binding site of the F. nucleatum riboswitch with RNA and ligand structures represented as sticks. Carbon atoms are colored slate blue for the ligand and orange for the RNA nucleotide bases. Solvent-accessible surface is represented as gray, with darker gradations representing surfaces facing up. Key bases in the binding site are labeled and key H-bond is indicated by the red dashed lines. The weak H-bond formed by a methyl group is indicated with a gray dashed line. (D) Overlay of the of the X-ray co-crystal structures of ribocil-D, which is represented as sticks colored slate blue, and ribocil-B. (PBD entry 5C45), which is represented as sticks colored cyan. The nucleotide number for RNA bases interacting with ribocil-D and ribocil-B is indicated.

Figure 3.

Analyzing binding differences of ribocil-A (non-binder) and ribocil-B (tight binder). (A) Structure models of ribocil-A and ribocil-B in E. coli FMN aptamer in 2 orientations, ribocil-B carbon atoms colored in light blue, ribocil-A in magenta, hydrogen atom at the chiral center of 2 compounds in white. (B) Energetic analysis of ribocil-A and ribocil-B binding toward E. coli riboswitch. Notable differences in binding energy are highlighted in red.

Figure 4.

Binding of FMN and ribocil-B, and ribocil-C to wild-type and ribocil^R mutant RNA apatmers. (A) Temperature dependent changes in absorbance for the wild-type riboswitch and mutants. (B) Fluorescence based analysis of binding of wild-type and mutant riboswitches to FMN. Solid lines represent the nonlinear least square analysis for direct FMN binding to riboswitches according to a quadratic equation. (C) Analysis of FMN competition binding by ribocil for wild-type and mutant riboswitches. For all panels in A, B and C, wild-type and mutant riboswitches are denoted with wild-type riboswitch (black), while mutants are shown in the following colors: C100U (red), C111U (green), C33U (blue), G37U (orange), G93U (purple), and Δ94–102 (cyan). (D) FMN-competitive binding of ribocil (black circles), ribocil A (blue diamonds), ribocil B (red squares), and ribocil C (green triangles) to the FMN riboswitch. Solid lines in C and D represent the nonlinear least squares analyses for the competition binding of each compound to the riboswitch aptamer against FMN according to a cubic equation fully describing the competition equilibria.

Figure 5.

Molecular modeling of ribocil^R mutants in the E. coli FMN riboswitch aptamer, ligand FMN carbon atoms in light green, ribocil carbon atoms in light blue. (A) The carbon atoms of all single nucleic acid mutants are colored in green, the deletion mutant del 94–102 colored in gray, (B) G93/U mutant. G93, its counterpart C67 and ligand binding neighbor G66 are displayed in stick, hydrogen bonds between G93 and C67 are highlighted in yellow dash line; (C) and (D) C33/U mutant. (C) Wild-type C33 and the hydrogen bond network with its 2 ligand binding neighbors G118 and G13, labeled with yellow dash lines; (D) Mutant U33-G118 mismatched base pair, U33 colored in green.

Figure 6.

SAR plan to discover more potent analogs of ribocil.