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Selective small-molecule inhibition of an RNA structural element

Abstract

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

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Figure 1: Genetic and pharmacological inhibition of riboflavin biosynthesis.
Figure 2: FMN riboswitch and ribocilR mutation mapping.
Figure 3: X-ray crystal structure of ribocil bound to the F. nucleatum FMN riboswitch.
Figure 4: In vivo activity of ribocil in a murine systemic infection model of E. coli.

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Primary accessions

Protein Data Bank

Data deposits

X-ray structure data have been deposited in the Protein Data Bank under accession code 5C45.

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Acknowledgements

We thank T. Silhavy for critical reading of the manuscript and providing constructive comments. We also thank the IMCA staff for making the beam line available to us. The X-ray diffraction data were collected by Shamrock (Woodridge, Illinois) and we thank G.Ranieri, J. Carter and R. Walter for collecting the data. We thank L.-K. Zhang (Merck) for helping with HRMS analysis. K. Devito (Merck) and L. E. Smith (Merck) are also thanked for providing cytotoxicity analysis.

Author information

Authors and Affiliations

Authors

Contributions

T.R. conceived the project; T.R., J.A.H., H.W., T.O.F., C.J.B., A.M.G., J.C.M., T.M., A.N., D.C., J.W., C.G.G., R.Z., P.R.S., C.J.G. and H.T. designed experiments; H.W., J.A.H., T.O.F., C.J.B., L.X., A.M.G., J.C.M., T.M., A.V., N.M., C.M.B., P.A.M., D.C., E.X., P.Z., D.R., R.E.P., S.S.W., B.S., R.d-J., W.P., M.A.P., J.W., D.R., J.C., H.F., performed experiments; T.R. and J.A.H. wrote the manuscript, and all authors analysed data and contributed to editing the manuscript.

Corresponding author

Correspondence to Terry Roemer.

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Competing interests

All authors are employees of Merck & Co. and may own stock in the company.

Extended data figures and tables

Extended Data Figure 1 Enzymatic steps responsible for riboflavin, FMN, and FAD biosynthesis in E. coli.

a, Enzyme names are shown above reaction arrows. RibA is a GTP cyclohydrolase II, RibB is a (3S)-3,4-dihydroxy-2-butanone 4-phasphate synthase. RibDG is a bifunctional enzyme encoding 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate deaminase and 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate reductase activity. RibH is a lumazine synthase. RibE is a riboflavin synthase. RibFC is another bifunctional enzyme encoding flaviokinase and FAD synthetase activity. Note, one molecule of GTP and two molecules of ribulose 5-phosphate are required to make one molecule of riboflavin. I, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′ phosphate; II, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate; III, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate; IV, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; V, (3S)-3,4-dihydroxy-2-butanone 4-phosphate; VI, 6,6-dimethyl-8-D-ribityllumazine. Note two molecules of VI dismutate (step 7) to give IV and riboflavin. Note also that the phosphatase converting III to IV (step 4) is not known. Adapted from Pedrolli et al.25. b, HPLC-based quantitative analysis of riboflavin, FMN, and FAD levels in E. coli strain MB5746 following genetic inactivation of riboflavin biosynthesis (ΔribA or ΔribB) or ribocil drug treatment. Wild-type strain MB5746 and isogenic ΔribA or ΔribB strains were grown overnight in CAMHB media containing 10 µM riboflavin, washed in CAMHB media lacking riboflavin, diluted (1:50) and grown for an additional 20 h before harvesting cells and HPLC analysis of cell lysates as described in the Methods. In parallel, overnight cultures of MB5746 grown in CAMHB were diluted (1:50) and treated with DMSO as a control or 20 μM ribocil, grown for an additional 20 h and analysed as above (middle panel). Data are presented as the mean of two technical repeats and are representative of two independent experiments. Riboflavin, FMN and FAD depletion levels are listed as a percentage relative to wild-type controls (right panel). c, HPLC-based quantitative analysis of riboflavin levels in 19 independent E. coli ribocilR mutant isolates versus the isogenic parent strain, MB5746. Overnight cultures of each strain were diluted 1:50 in CAMHB and treated with 10 µM ribocil or mock treated (1% DMSO). After growth at 37 °C with agitation for 20 h, cell lysates were prepared and riboflavin levels were quantitated as described in the Methods. Data are the mean of two technical repeats (error bars indicate range) and is representative of two independent experiments.

Extended Data Figure 2 E. coli FMN riboswitch-regulated reporter gene expression and ribocil-mediated inhibition and isolation of ribocilR mutations mapping to A. baumannii and P. aeruginosa FMN riboswitches.

a, GFP reporter constructs under the control of the intact E. coli ribB promoter and FMN riboswitch (EcPro-EcFMN–GFP), or replaced with the A. baumannii FMN riboswitch (EcPro-AbFMN–GFP) or P. aeruginosa FMN riboswitch (EcPro-PaFMN–GFP) are maintained in E. coli ribocilR mutant, M5 (Fig. 1e). Note, E. coli ribB upstream promoter sequence (EcPro) was fused to A. baumannii and P. aeruginosa FMN elements (AbFMN and PaFMN, respectively) to facilitate sufficient baseline expression in E. coli MB5746 host cells. b, Introduction of FMN riboswitch reporter plasmids into a ribocilR mutant strain background (M5) enables ribocil dose-dependent inhibition of GFP expression without inhibiting cell growth. Dose-response curve for 16 technical repeats of ribocil-mediated inhibition of EcPro-EcFMN–GFP expression is shown. c, Tabulated EC50 values (±s.d.) of ribocil required to inhibit EcPro-EcFMN–GFP, EcPro-AbFMN–GFP and EcPro-PaFMN–GFP expression. Data for EcPro-EcFMN–GFP are from eight independent experiments each with two technical repeats, whereas data for EcPro-AbFMN–GFP and EcPro-PaFMN–GFP are from two independent experiments each with four technical repeats. d, Schematic summary of E. coli recombineered ribB locus, EcPro-AbFMN-ribB, in which the endogenous FMN riboswitch is replaced with the A. baumannii FMN riboswitch. Below, predicted secondary structure of the A. baumannii FMN riboswitch and ribocilR mutations highlighted in red. e, Schematic summary of E. coli recombineered ribB locus, EcPro-PaFMN-ribB, in which the E. coli FMN riboswitch is replaced by the P. aeruginosa FMN riboswitch. Below, predicted secondary structure of the P. aeruginosa FMN riboswitch and ribocilR mutations highlighted in red.

Extended Data Figure 3 Ribocil mutant analysis; Ribocil:FMN competition binding and Ribocil–analogue binding studies.

a, Wild-type FMN and mutant constructs responsive to ribocil are displayed with a curve fit. Fluorescence was measured using 405 nm excitation and 510 nm emission and data represent the mean of two independent experiments (error bars indicate range). EC50, the maximum cell-density-adjusted fluorescence signal observed with no ribocil addition, and the maximum per cent inhibition of fluorescence for each construct is listed in the accompanying table. b, Wild-type RNA aptamer samples were separated on 1.2% agarose gels and visualized by ethidium bromide staining. c, Binding affinity for ribocil. Binding affinity to the E. coli FMN riboswitch aptamer is determined from ribocil dose-dependent competition against a fixed concentration of FMN (60 nM) and a fixed concentration of E. coli FMN riboswitch aptamer (150 nM). Shown is a representative example of ribocil competition data. Mean affinity (±s.d.) from four independent experiments for ribocil, ribocil-A, ribocil-B and ribocil-C is reported in the table (panel e of this figure). d, Chemical structure of ribocil-A, ribocil-B and ribocil-C. e, Steady-state Kd, binding kinetics, riboflavin biosynthesis inhibition and minimum inhibitory concentration (MIC) in E. coli MB5746 for ribocil-A, ribocil-B and ribocil-C. Binding kinetics were determined using a competition method employing a fixed amount of FMN (60 nM) and E. coli FMN riboswitch (48 nM). FMN binding affinity and kinetics are determined by fluorescence quenching experiments (see Methods). Given the tight-binding conditions, both FMN and ribocil affinity values represent an upper limit. As a result, the koff values may also be an upper limit, and/or the kon values may be a lower limit. Inhibition of riboflavin biosynthesis after treatment of E. coli MB5746 with ribocil, ribocil-A, ribocil-B and ribocil-C for 20 h as described in Methods. Data for ribocil are the mean (±s.d.) of three independent experiments, for ribocil-B the mean (±s.d.) of four independent experiments and for ribocil-C the mean (error bars indicate range) of two independent experiments. MIC of ribocil, ribocil-A, ribocil-B and ribocil-C against E. coli MB5746 determined by the broth microdilution method.

Extended Data Figure 4 Electron density omit map of F. nucleatum impX FMN aptamer–ribocil co-crystal structure.

a, The electron density difference map (see Methods for details regarding its calculation) is shown as a grid contoured at 3.0σ level. The refined structure of the ligand is shown as sticks, methyls in slate blue, the aptamer as lines with the methyl coloured in orange, cations as spheres coloured in purple. b, Same figure as a but rotated to bring the electron density around the planar 6-thiophenyl-pyrimidonyl horizontal and perpendicular to the figure plane. The best fit of the (S)-isomer which maintains the pyrimidinyl in its electron density, stacking against G62, and inserts the 6-thiophenyl-pyrimidonyl between A48 and A85 is shown (see Methods). The 6-thiophenyl-pyrimidonyl is clearly slanted compared to the density and the planes of the A48 or A85 bases. c, Superposition of the X-ray co-structures of the F. nucleatum riboswitch aptamer with either FMN (PDB entry 3F2Q) or ribocil. After superposition using the phosphorus atoms of the bases in the immediate vicinity of the ligand, the RNA is represented as lines in cyan and orange for the co-structures with FMN and ribocil, respectively, and as sticks for the ligand, in cyan and slate blue, respectively. The number for the key bases interacting with either FMN or ribocil is indicated.

Extended Data Figure 5 Homology model of the predicted ribocil binding site within the E. coli FMN aptamer.

a, The homology model was constructed using program mutate_bases of the 3DNA package53 using the F. nucleatum impX riboswitch aptamer X-ray structure as the template and the FMN aptamer alignment of E. coli, F. nucleatum, P. aeruginosa and A. baumannii. Only two bases differ (in red labels) compared to F. nucleatum FMN riboswitch, G66A and A95U. b, A full-length homology model of the E. coli FMN aptamer and mapping of ribocilR mutations. Location of the ribocilR mutants C33U, G37U, G93U, C100U, C111U and Δ94–102 are mapped on the E. coli homology model of the ribocil-bound E. coli FMN riboswitch aptamer. In the model, all nucleotide insertions in the E. coli sequence not found in F. nucleatum were not modelled in the resulting crystal structure. There are 34 base changes among the 111 nucleotide modelled. A119 is removed for clarity. Nucleotides C33, G37, G93, C100 and C111 are coloured (green) and bases deleted in Δ94–102 are highlighted (red). G96 (red) which makes direct contact with ribocil (blue) in the wild-type aptamer is deleted in Δ94–102. c, Alignment of bacterial riboswitch sequences from RFAM54. E. coli aptamer nucleotide G96, which is equivalent to nucleotide G62 in the F. nucleatum aptamer, is indicated with an asterisk. Black shading represents identical and grey is similar using default consensus settings with the BOXCHADE program (http://sourceforge.net/projects/boxshade/).

Extended Data Figure 6 Ribocil, roseoflavin and ribocil-C cross-resistance to E. coli ribocilR mutants.

a, 5 μl of ribocil (1.3 mM), roseoflavin (31 mM) and the negative control, novobiocin (2.5 mM), were spotted (twofold dilution series) on the surface of a CAMH plate (a) or CAMH plate plus 20 μM riboflavin (b) and seeded with E. coli MB5746. ci, Same as a but plates seeded with the indicated ribocilR mutants. Note, whereas the growth-inhibiting activity of ribocil is completely suppressed by riboflavin, roseoflavin activity is only partially suppressed and ribocilR mutants are cross-resistant to roseoflavin and display similar level of suppression as riboflavin supplementation. The figure is representative of two independent experiments. j, Ribocil-C inhibits the FMN riboswitch and is cross-resistant to ribocilR mutations. 5 μl of ribocil (1.3 mM), ribocil-C (153 μM) and the negative control, novobiocin (2.5 mM), were spotted (twofold dilution series) on the surface of CAMH plates seeded with the above described strains and/or riboflavin supplement as shown in ai. The figure is representative of three independent experiments. RF, riboflavin.

Extended Data Figure 7 Bioactivity of riboflavin analogues are not suppressed by riboflavin supplements.

a, Left plates; 5 μl of ribocil (1.3 mM), roseoflavin (31 mM), C002 (2 mM), C003 (2 mM), C004 (2 mM), C005 (2 mM), C006 (2 mM), C007 (2 mM), C008 (2 mM), and the negative control, Chiron 90 (9 μM), were spotted (twofold dilution series) on the surface of a CAMH plate seeded with E. coli MB5746. Right plates; same as left plates but supplemented with 20 μM riboflavin. Chemical structures are shown. The figure is representative of two independent experiments. b, Inhibition of flavin synthesis by COO6. Flavin levels were determined by HPLC after C006 treatment of MB5746. All data are from a single 11-point dose titration and is representative of two independent experiments.

Extended Data Figure 8 In vivo activity of ribocil-C in a murine systemic infection model of E. coli.

a, DBA/2J mice were infected i.p. with E. coli strain MB5746 (5.0 × 105 CFU per mouse, a tenfold higher inoculum than that used in Fig. 4) and treated by subcutaneous injection with ribocil-C or ciprofloxacin at the indicated doses (mg kg−1) three times over a 24 h infection period. Spleens were aseptically collected from five mice per group and the reduction of log[CFU per g spleen tissue] was calculated on the basis of bacterial burden in spleens of the vehicle-treated (10% DMSO) control group. Data represents the average CFU per g spleen ± s.e.m. One-way ANOVA with Dunnett’s multiple comparison test demonstrates statistically significant (*P < 0.05, ***P < 0.001) log[CFU per g spleen] reductions in the 60 and 120 mg kg−1 ribocil-C treatment groups and ciprofloxacin control. b, The CFU data from each mouse plotted as individual points, solid bar and error bars represent the average CFU per g spleen ±s.e.m. One-way ANOVA with Dunnett’s multiple comparison test demonstrates statistically significant (*P < 0.05, ***P < 0.001) log[CFU per g spleen] reductions in the 60 and 120 mg kg−1 ribocil-C treatment groups and ciprofloxacin control.

Extended Data Table 1 Frequency of resistance (FOR) determination and microbiological activity summary
Extended Data Table 2 X-ray crystal data collection and refinement statistics

Supplementary information

Supplementary Information

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Location of the ribocilR mutants C33U, G37U, G93U, C100U, C111U and Δ94-102 mapped on the E. coli homology model of the ribocil-bound E. coli FMN riboswitch aptamer.

Nucleotides C33, G37, G93, C100 and C111 are colored (green) and bases deleted in Δ94-102 are highlighted (red). G96 (red), which makes direct contact with ribocil (blue) in the wild-type aptamer is deleted in Δ94-102. (MPG 28184 kb)

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Howe, J., Wang, H., Fischmann, T. et al. Selective small-molecule inhibition of an RNA structural element. Nature 526, 672–677 (2015). https://doi.org/10.1038/nature15542

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