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| Targets |
Ribocil-C (R enantiomer) selectively targets bacterial FMN riboswitches, which are regulatory RNA elements located in the 5′-untranslated region of mRNA transcripts involved in riboflavin (vitamin B2) biosynthesis and uptake. By mimicking the natural ligand FMN, the compound binds with high affinity to the aptamer domain of the riboswitch, inducing a conformational change that prevents transcription or translation of downstream genes, including those encoding riboflavin synthase and other enzymes in the pathway. This riboswitch-mediated inhibition effectively shuts down de novo riboflavin synthesis, leading to riboflavin depletion and bacterial growth arrest. The R enantiomer exhibits superior binding affinity compared to the S enantiomer or the racemate. Selectivity for bacterial riboswitches over human RNA or protein targets is high, contributing to its favorable safety profile.
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| ln Vitro |
In vitro antibacterial assays demonstrate that Ribocil-C (R enantiomer) exhibits potent activity against a broad range of bacterial pathogens, including both Gram-positive (Staphylococcus aureus, including MRSA) and Gram-negative species. The minimum inhibitory concentration (MIC) values against susceptible strains typically range from 0.25-4 ug/mL. The compound shows dose-dependent inhibition of bacterial growth in broth microdilution assays. Mechanistically, Ribocil-C binding to the FMN riboswitch has been confirmed using in vitro transcription termination assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC), which reveal binding constants in the low nanomolar to micromolar range, depending on the specific riboswitch variant. The compound inhibits riboflavin biosynthesis and uptake in a concentration-dependent manner. No significant cross-resistance with conventional antibiotics has been observed, making it attractive for targeting resistant pathogens.
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| ln Vivo |
In vivo efficacy of Ribocil-C (R enantiomer) has been demonstrated in mouse models of bacterial infection, including systemic infection models using S. aureus and K. pneumoniae. Intraperitoneal or intravenous administration of Ribocil-C at doses of 10-50 mg/kg results in significant reduction of bacterial burden in blood and organs compared to vehicle-treated controls. Survival studies have shown improved survival rates in treated animals challenged with lethal doses of bacteria. The compound is effective against antibiotic-resistant strains, including MRSA and carbapenem-resistant Enterobacteriaceae. In pharmacokinetic/pharmacodynamic (PK/PD) studies, the therapeutic efficacy correlates with the extent and duration of riboswitch target engagement. However, detailed dose-finding studies and tissue distribution data are still being characterized.
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| Enzyme Assay |
A typical non-cellular binding assay for Ribocil-C involves surface plasmon resonance (SPR) to measure binding affinity to purified FMN riboswitch RNA. The RNA aptamer domain (typically 70-100 nucleotides) is synthesized in vitro by T7 RNA polymerase transcription, refolded by heating to 95degC for 3 min followed by slow cooling in binding buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2). The RNA is immobilized onto a sensor chip via biotin-streptavidin capture. Ribocil-C is injected at increasing concentrations (0.1 nM to 10 uM) at a flow rate of 50 uL/min for 120 sec, followed by dissociation for 300 sec. Sensorgrams are fitted using a 1:1 Langmuir binding model to determine ka (association rate constant), kd (dissociation rate constant), and KD (equilibrium dissociation constant). Competitive binding assays using FMN are performed to confirm the binding site. An isothermal titration calorimetry (ITC) method is also used, wherein RNA solution (10 uM) in the cell is titrated with Ribocil-C (100 uM) in the syringe to directly measure thermodynamic binding parameters.
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| Cell Assay |
In vitro cell-based assays for Ribocil-C are conducted using standard broth microdilution methods according to CLSI guidelines. Bacterial strains (S. aureus, K. pneumoniae, E. coli) are grown overnight in cation-adjusted Mueller-Hinton broth (MHB) at 37degC. The bacterial suspension is adjusted to 0.5 McFarland standard (~1-2 × 10⁸ CFU/mL) and diluted 100-fold in MHB to achieve a final inoculum of approximately 5 × 10⁵ CFU/well. Ribocil-C is serially diluted two-fold in 96-well plates (concentration range 0.125-64 ug/mL). Inoculum is added to each well, and plates are incubated at 37degC for 16-20 h. MIC is defined as the lowest concentration of Ribocil-C that inhibits visible bacterial growth. For time-kill kinetics, bacterial cultures are treated with the compound at various concentrations (0.5×, 1×, 2×, 4× MIC), and aliquots are plated at 0, 2, 4, 6, 8, and 24 h for CFU enumeration. Cytotoxicity is assessed using mammalian HEK293 or HepG2 cells cultured in DMEM with 10% FBS at 37degC, 5% CO2, with viability measured by MTT assay after 48 h exposure to Ribocil-C (0-100 uM).
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| Animal Protocol |
In vivo animal studies for Ribocil-C use female BALB/c or CD-1 mice (6-8 weeks old, 18-22 g). For systemic infection models, mice are inoculated intraperitoneally (IP) with 1-2 × 10⁷ CFU/mouse of S. aureus or K. pneumoniae suspended in 5% mucin to enhance virulence. Ribocil-C is administered intraperitoneally or intravenously at therapeutic doses (10, 25, 50 mg/kg) 1 h post-infection, then twice daily for 3-5 days. Control groups receive vehicle (PBS or saline) or a comparator antibiotic. Survival is monitored daily for 7-10 days. For tissue burden studies, mice are euthanized at predetermined time points (24 h, 48 h post-treatment), and spleens, livers, and kidneys are aseptically removed, weighed, homogenized, and plated on tryptic soy agar for CFU enumeration. Efficacy is evaluated as reduction in log10 CFU/organ relative to vehicle controls. Pharmacodynamic studies measure bacterial load reduction as a function of dose. All animal studies must be conducted in compliance with institutional animal care and use committee (IACUC) protocols.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of Ribocil-C (R enantiomer) are not extensively reported in the literature. As a small molecule inhibitor of bacterial riboswitches, the compound is expected to be metabolized by hepatic CYP450 enzymes, primarily CYP3A4, similar to the structurally distinct ribociclib (a CDK inhibitor). Ribociclib, the parent drug from which the name Ribocil-C is derived, is known to have moderate oral absorption (human bioavailability ~59%), extensive hepatic metabolism primarily via CYP3A4-mediated oxidation, and elimination predominantly by hepatic metabolism (~84% of total elimination), with minor contributions from renal excretion (~7%), intestinal excretion (~8%), and biliary elimination (~1%). However, these parameters should not be directly extrapolated to Ribocil-C without experimental confirmation. The compound may have limited oral bioavailability due to its molecular weight (419.51 g/mol) and potential P-glycoprotein substrate status. For research applications, the compound is typically formulated in DMSO or aqueous vehicle. Experimental PK data for Ribocil-C are limited.
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| Toxicity/Toxicokinetics |
Toxicological data for Ribocil-C (R enantiomer) in animals and humans are limited due to its use primarily as a research tool. Preliminary in vitro cytotoxicity studies using human cell lines (HEK293, HepG2) suggest low cytotoxicity at concentrations up to 50 uM, as measured by MTT or resazurin assays. The high selectivity of Ribocil-C for bacterial FMN riboswitches over human RNA or protein targets contributes to a potentially favorable safety profile. No systemic toxicity data from animal studies are available in the public literature. Standard genotoxicity assays (Ames test, in vitro micronucleus) have not been reported. Ribocil-C should be handled as a potentially bioactive research compound requiring appropriate safety precautions. It is for research use only and not intended for human therapeutic administration.
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| References |
[1]. Wang H, et al. Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch DisruptRiboflavin Homeostasis in an Infectious Setting. Cell Chem Biol. 2017 May 18;24(5):576-588.
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| Additional Infomation |
Ribocil-C (R enantiomer) is not approved for human use as a drug and is currently in preclinical research stages. No clinical trials have been registered for this compound. Its mechanism of action involves selective binding to the bacterial FMN riboswitch RNA, mimicking the natural ligand FMN, thereby repressing riboflavin biosynthesis and uptake, leading to bacterial growth arrest. This represents a novel antibacterial strategy targeting RNA rather than protein. The R enantiomer shows enhanced binding affinity and specificity compared to the racemate, making it a valuable tool for studying riboswitch biology and structure-activity relationships. The compound has potential applications in combating antibiotic-resistant bacterial infections. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C21H21N7OS
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| Exact Mass |
419.152
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| CAS # |
2177266-81-6
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| Related CAS # |
Ribocil-C;1825355-56-3;Ribocil-C Racemate;2309762-18-1
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| PubChem CID |
137217143
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
1
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
30
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| Complexity |
693
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1C[C@H](CN(C1)CC2=CN(C=N2)C3=NC=CC=N3)C4=NC(=CC(=O)N4)C5=CC=CS5
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| InChi Key |
UVDVCDUBJWYRJW-OAHLLOKOSA-N
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| InChi Code |
InChI=1S/C21H21N7OS/c29-19-10-17(18-5-2-9-30-18)25-20(26-19)15-4-1-8-27(11-15)12-16-13-28(14-24-16)21-22-6-3-7-23-21/h2-3,5-7,9-10,13-15H,1,4,8,11-12H2,(H,25,26,29)/t15-/m1/s1
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| Chemical Name |
2-[(3R)-1-[(1-pyrimidin-2-ylimidazol-4-yl)methyl]piperidin-3-yl]-4-thiophen-2-yl-1H-pyrimidin-6-one
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| HS Tariff Code |
2934.99.9001
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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: ≥ 6.7 mg/mL (15.97 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
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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