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Purity: ≥98%
Ribocil is a novel, potenty and highly selective chemical modulator of bacterial riboflavin riboswitches. Ribocil strongly inhibits GFP expression, achieving a 50% effective concentration (EC50) of 0.3 μM. Ribocil is a highly specific bioactive synthetic mimic of FMN, which competes with the natural ligand to inhibit FMN riboswitch-mediated expression of ribB and inhibits bacterial growth. Ribocil-B demonstrates superior microbiological activity as compared to Ribocil-A (minimum inhibitory concentration (MIC) = 1 μg/ml versus MIC ≥ 64 μg/ml), inhibition of riboflavin synthesis (IC50 = 0.13 μM versus IC50 > 26 μM), and binding affinity to the E. coli FMN aptamer (Kd = 6.6 nM versus Kd ≥ 10,000 nM). 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.
| Targets |
Bacterial FMN riboswitch (a non-coding RNA structural element that regulates riboflavin biosynthesis). Specifically, ribocil competes with the natural ligand flavin mononucleotide (FMN) for binding to the FMN riboswitch aptamer domain (Kd = 16 nM for E. coli FMN aptamer). ribocil does not inhibit human flavoproteins or other essential proteins. [1]
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| ln Vitro |
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.
ribocil inhibits bacterial growth in Escherichia coli strain MB5746 (a sensitized strain with defective efflux and outer membrane permeability) with a minimum inhibitory concentration (MIC) of 2 µg/mL. Its growth inhibitory activity is completely suppressed by supplementation with 20 µM exogenous riboflavin. [1] Treatment of E. coli MB5746 with ribocil leads to a dose-dependent depletion of intracellular riboflavin, FMN, and FAD levels, mirroring the phenotype of riboflavin biosynthesis gene (ribA, ribB) deletion mutants. The half-maximal inhibitory concentration for riboflavin depletion (IC50) is 0.3 µM. [1] Using a GFP reporter gene under the control of the E. coli ribB promoter and FMN riboswitch sequence, ribocil inhibits GFP expression with an EC50 of 0.3 µM. This inhibition parallels its effect on riboflavin synthesis. [1] ribocil also inhibits GFP reporter expression regulated by orthologous FMN riboswitches from Pseudomonas aeruginosa and Acinetobacter baumannii, albeit with approximately 5-fold higher EC50 values compared to the E. coli riboswitch. [1] The (S)-enantiomer of ribocil (named ribocil-B) is the biologically active isomer, demonstrating superior activity (MIC = 1 µg/mL against E. coli MB5746, riboflavin synthesis IC50 = 0.13 µM, Kd = 6.6 nM for E. coli FMN aptamer) compared to the (R)-enantiomer (ribocil-A; MIC ≥ 64 µg/mL, IC50 > 26 µM, Kd ≈ 10,000 nM). [1] ribocil lacks cytotoxic activity against mammalian HeLa cells (EC50 ≈ 100 µM based on cell count and EdU incorporation assays). It also lacks antifungal activity against Saccharomyces cerevisiae and Candida albicans. [1] |
| ln Vivo |
In a murine systemic infection (septicaemia) model with E. coli MB5746, treatment with an (S)-enantiomer analogue of ribocil (ribocil-C, mechanistically equivalent) demonstrated dose-dependent efficacy. Subcutaneous administration of ribocil-C at 60 and 120 mg/kg (three doses over 24 hours) resulted in reductions of bacterial burden in the spleen by 1.87 log10 CFU/g and 3.29 log10 CFU/g, respectively, compared to vehicle-treated controls. No mortality or gross toxicity was observed at these doses. Similar efficacy was observed at a tenfold higher infectious inoculum. [1]
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| Enzyme Assay |
The binding affinity of ribocil to the FMN riboswitch was assessed using a fluorescence quenching assay. The purified E. coli FMN riboswitch aptamer RNA was first characterized for FMN binding (Kd = 1.2 nM). For competition binding, a titration series of ribocil was prepared in assay buffer. The assay mixture contained a fixed concentration of FMN ligand (60 nM) and the E. coli FMN aptamer (48 nM for general compounds, 150 nM specifically for ribocil titration). Fluorescence signal (excitation 455 nm, emission 525 nm) was monitored over time. The addition of ribocil restored FMN fluorescence in a dose-dependent manner, indicating direct competition. Steady-state binding data at 120 minutes were fitted to a competition binding model to derive the inhibition constant (Ki). The dissociation rate constant (koff) was derived by fitting kinetic data with numerical integration, and the association rate constant (kon) was calculated from Ki and koff. [1]
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| Cell Assay |
A phenotypic whole-cell screening assay was used to identify ribocil. E. coli strain MB5746 was grown in broth or on agar plates with or without supplementation of 10 µM riboflavin. Compound libraries were transferred to wells or spotted on agar. Hit compounds whose growth inhibitory activity was suppressed in the presence of riboflavin were selected for further study. This assay recapitulated the conditional essentiality of the riboflavin biosynthetic pathway. [1]
To quantify flavin depletion, E. coli MB5746 cultures were treated with a dilution series of ribocil or DMSO control. After incubation, cells were harvested, lysates were prepared, and levels of riboflavin, FMN, and FAD were quantified using HPLC analysis. [1] For the GFP reporter assay, reporter strains (harboring plasmids with GFP under control of various FMN riboswitches) were diluted in culture broth supplemented with an antibiotic. Compounds were serially diluted and added to the cultures in assay plates. After overnight growth, fluorescence and optical density were measured. Dose-response curves were generated to determine the concentration causing a 50% decrease in specific fluorescence signal (GFP EC50). [1] Mammalian cytotoxicity was assessed using a cell proliferation assay. HeLa cells were seeded in coated plates, treated with a dilution series of ribocil, and incubated with EdU for 24 hours. Total cell numbers and EdU incorporation were measured using a laser scanning cytometer to determine any cytotoxic or antiproliferative effects. [1] |
| Animal Protocol |
For the murine systemic infection model, DBA/2J mice were immunosuppressed with cyclophosphamide on days -4 and -1 before infection. On day 0, mice were infected via intraperitoneal injection with E. coli MB5746 suspended in mucin. Thirty minutes post-infection, treatment began. ribocil-C or the control antibiotic ciprofloxacin was administered by subcutaneous injection at specified doses (30, 60, or 120 mg/kg for ribocil-C; 0.5 mg/kg for ciprofloxacin) three times over a 24-hour period. A vehicle control group received DMSO. On day 1, mice were euthanized, spleens were aseptically removed, homogenized, serially diluted, and plated on agar to determine bacterial colony-forming units (CFU) per gram of spleen tissue. Statistical analysis was performed to compare bacterial burden reduction between treatment groups. [1]
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| Toxicity/Toxicokinetics |
In a mouse model of systemic infection, subcutaneous injection of up to 120 mg/kg of ribocil-C (three times over 24 hours) did not result in death or significant toxicity. [1] Ribocil at concentrations up to 100 µM was not cytotoxic to human HeLa cells. [1] No data on plasma protein binding, drug interactions, or organ-specific toxicity of ribocil were available in the literature provided. [1]
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| References | |
| Additional Infomation |
Ribocil is a highly selective synthetic small molecule that is an analog of the natural ligand FMN. It was discovered through phenotypic screening, which aims to find inhibitors whose activity can be inhibited by exogenous riboflavin. [1] Its main mechanism of action is to bind to the FMN riboswitch aptamer and compete with FMN, thereby locking the riboswitch in the "OFF" conformation. This leads to downregulation of transcription and translation of the downstream ribB gene, inhibiting de novo synthesis of riboflavin, which is essential for bacterial growth in an infectious environment. [1] Unlike the natural antibiotic roseflavin (a riboflavin analog), which inhibits multiple targets including the FMN riboswitch, FAD synthase, and human flavinase, ribocil has superior target selectivity and specifically acts on the bacterial FMN riboswitch. [1]
Resistance to ribocil stemmed from mutations in the FMN riboswitch, identified through whole-genome sequencing of resistant E. coli mutants. All resistant mutants exhibited base pair alterations localized to the riboswitch, confirming it as a resistance target. These mutants also showed cross-resistance to roseflavin. [1] This work demonstrates that non-coding RNA structural elements like riboswitches are effective targets for the synthesis of small molecule drugs. [1] |
| Molecular Formula |
C19H22N6OS
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|---|---|
| Molecular Weight |
382.482581615448
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| Exact Mass |
382.157
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| Elemental Analysis |
C, 59.66; H, 5.80; N, 21.97; O, 4.18; S, 8.38
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| CAS # |
1381289-58-2
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| Related CAS # |
Ribocil-C;1825355-56-3;Ribocil B;1825355-55-2;Ribocil-C Racemate;2309762-18-1
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| PubChem CID |
136881500
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| Appearance |
Solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
610.4±65.0 °C at 760 mmHg
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| Flash Point |
323.0±34.3 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.673
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| LogP |
0.99
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
27
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| Complexity |
601
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C=CC=C1C1=CC(NC(C2CN(CC3=CN=C(NC)N=C3)CCC2)=N1)=O
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| InChi Key |
ZSXCVAIJFUEGJR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H22N6OS/c1-20-19-21-9-13(10-22-19)11-25-6-2-4-14(12-25)18-23-15(8-17(26)24-18)16-5-3-7-27-16/h3,5,7-10,14H,2,4,6,11-12H2,1H3,(H,20,21,22)(H,23,24,26)
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| Chemical Name |
2-(1-((2-(methylamino)pyrimidin-5-yl)methyl)piperidin-3-yl)-6-(thiophen-2-yl)pyrimidin-4(3H)-one
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| Synonyms |
Ribocil;
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~12.5 mg/mL (~32.68 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (3.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.25 mg/mL (3.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 1.25 mg/mL (3.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 1.25 mg/mL (3.27 mM) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6145 mL | 13.0726 mL | 26.1452 mL | |
| 5 mM | 0.5229 mL | 2.6145 mL | 5.2290 mL | |
| 10 mM | 0.2615 mL | 1.3073 mL | 2.6145 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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