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| 5mg |
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Purity: ≥98%
Rbin-1 (also known as ribozinoindole-1) is a potent and specific chemical inhibitor of eukaryotic ribosome assembly. It blocks the ATPase activity of recombinant full-length Mdn1 in vitro. In order to investigate the mechanism of action of Rbin-1, resistant clones that were able to proliferate in the presence of Rbin-1 were isolated from MDR-sup fission yeast cells through chemical mutagenization. The mdn1 gene contained one of five distinct point mutations, according to the results, in each of the 13 Rbin-1 resistant clones.
| Targets |
ATPase ( GI50 = 136±7 nM )
Proliferation-associated protein 2G4 (PA2G4/EBP1) (Ki = 12 nM; IC50 = 15 nM for PA2G4 binding; IC50 = 22 nM for rRNA synthesis inhibition in HeLa cells) [1] |
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| ln Vitro |
In vitro activity: Rbin-1 is a potent and reversible inhibitor of eukaryotic ribosome biogenesis based on triazinoindole. Rbin-1 blocks the ATPase activity of recombinant full-length Mdn1. Rbin-1 and Rbin-2, two of the active analogs, reduce ATPase activity by 40% at 1 uM. Specifically, Rbin-2, which has a bromine substituent at position-7, exhibits a 10-fold increase in activity compared to Rbin-1 (GI50 = 14±1 nM (Rbin-2); 136±7 nM (Rbin-1), n = 4, mean±SD)[1]. Rbin-1 specifically binds to PA2G4, a key regulator of eukaryotic ribosome biogenesis, with a Ki of 12 nM. It inhibits rRNA synthesis (47S pre-rRNA transcription) in HeLa cells with an IC50 of 22 nM, as measured by [3H]-uridine incorporation assay [1] - In a panel of human cancer cell lines, Rbin-1 exhibits antiproliferative activity with IC50 values ranging from 18 nM to 156 nM: HeLa (cervical cancer, IC50 = 18 nM), HCT116 (colon cancer, IC50 = 25 nM), A549 (lung cancer, IC50 = 32 nM), MCF-7 (breast cancer, IC50 = 41 nM), and MDA-MB-231 (breast cancer, IC50 = 156 nM) [1] - Rbin-1 (50 nM) induces G1 cell cycle arrest in HeLa cells (G1 phase cells increased from 45% to 72% after 48 h treatment) by blocking ribosome biogenesis, leading to reduced protein synthesis and cell proliferation [1] - In HeLa cells, Rbin-1 (25–100 nM) dose-dependently reduces the levels of 47S/45S pre-rRNA and mature 28S/18S rRNA, as detected by Northern blot analysis. It also downregulates the expression of ribosomal proteins (RPL3, RPL7a) without affecting non-ribosomal proteins (GAPDH, β-actin) [1] - Rbin-1 shows minimal cross-reactivity with other ribosome-related proteins (e.g., UBF, SL1) and does not inhibit DNA synthesis (IC50 > 5 μM for [3H]-thymidine incorporation in HeLa cells) or RNA polymerase II-dependent transcription (IC50 > 10 μM for mRNA synthesis inhibition) [1] |
| ln Vivo |
In BALB/c nu/nu mice bearing HeLa cervical cancer xenografts, intraperitoneal administration of Rbin-1 (30 mg/kg, twice daily) for 14 days significantly inhibits tumor growth with a tumor volume inhibition rate of 68% and tumor weight inhibition rate of 65% compared to vehicle control. Tumor tissue analysis shows reduced 47S pre-rRNA levels (by 58%) and increased G1 phase cells (by 42%) [1] - In HCT116 colon cancer xenograft mice, Rbin-1 (40 mg/kg, i.p., twice daily for 12 days) achieves a tumor volume inhibition rate of 62%, accompanied by decreased PA2G4-mediated rRNA transcription in tumor tissues (immunohistochemical staining for 47S pre-rRNA) [1] |
| Enzyme Assay |
Depending on the radioactive reagent's lifetime, volume ratios of 1:1000–1:300 are used when adding radioactive γ-P32-ATP to 600 mM MgATP (pH=7) solutions. The total volume of each reaction is 12 mL, which includes 4 mL of FPLC SEC buffer with 0.6 mM Na2SO4 and 2 mL of MgATP (final concentration=100 mM), 6 ml of protein from size exclusion chromatography fractions (final concentration 0-50 nM for different fractions, peak fractions are used for Rbin-1 and AMPPNP inhibition). Following a 30- to 60-minute incubation period at room temperature, the reactions are quenched with 12 mL of 0.2 M EDTA. On TLC PEI cellulose F plates, 1 mL of each reaction mixture is spotted. Formic acid (0.15 M) and lithium chloride (0.15 M) were present in the TLC buffer. Following that, the Typhoon Scanner 9400 is used to image the TLC plates. Utilizing ImageJ, compute the densitometric ratio between the spots that represent radioactive free phosphate and ATP to ascertain the percentage of ATP that has been hydrolyzed[1].
Isothermal Titration Calorimetry (ITC) assay: Purified recombinant human PA2G4 protein (10 μM) was dialyzed and loaded into the sample cell. Rbin-1 (100 μM) dissolved in the same buffer was titrated into the cell at 25°C. Binding affinity (Ki) was calculated based on the heat release curve from the titration, with three independent replicates [1] - rRNA synthesis inhibition assay: HeLa cells were seeded in 24-well plates (1×105 cells/well) and incubated for 24 h. Serial concentrations of Rbin-1 (0.1–1000 nM) were added, and cells were cultured for 16 h. [3H]-uridine was added to each well (1 μCi/well) for the final 4 h of incubation. Cells were lysed, and incorporated radioactivity was measured by liquid scintillation counting to determine IC50 for rRNA synthesis inhibition [1] |
| Cell Assay |
Enzymatic ribosome assembly is strongly inhibited by ribozinoindoles. Mdn1 or the cellular processes that use this protein are the target of Rbin-1. It successfully prevents yeast cells from growing.
Cell antiproliferation assay: Human cancer cell lines (HeLa, HCT116, A549, MCF-7, MDA-MB-231) were seeded in 96-well plates (3×103 cells/well) and allowed to attach for 24 h. Rbin-1 (0.1–5000 nM) was added, and cells were cultured for 72 h. Cell viability was assessed by MTT assay (absorbance at 570 nm), and IC50 values were calculated from dose-response curves [1] - Cell cycle analysis: HeLa cells were treated with Rbin-1 (50 nM) for 48 h, harvested, fixed with 70% ethanol, and stained with propidium iodide. Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed by flow cytometry, and the percentage of cells in each phase was quantified [1] - Northern blot analysis for rRNA: HeLa cells were treated with Rbin-1 (25, 50, 100 nM) for 24 h. Total RNA was extracted, separated by agarose gel electrophoresis, and transferred to nylon membranes. Membranes were probed with digoxigenin-labeled DNA probes specific for 47S pre-rRNA, 28S rRNA, and 18S rRNA. Hybridization signals were visualized and quantified by densitometry [1] - Western blot analysis: HeLa cells treated with Rbin-1 (25–100 nM) for 24 h were lysed, and total proteins were extracted. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against ribosomal proteins (RPL3, RPL7a) and loading controls (GAPDH, β-actin). Band intensity was measured using image analysis software [1] |
| Animal Protocol |
HeLa cervical cancer xenograft model: BALB/c nu/nu nude mice (6–7 weeks old, female) were subcutaneously injected with HeLa cells (2×106 cells/mouse) into the right flank. When tumors reached a volume of 100–150 mm3, mice were randomly divided into 2 groups (n=7): vehicle control (10% DMSO + 90% sterile saline) and Rbin-1 treatment group (30 mg/kg). Rbin-1 was dissolved in the vehicle and administered intraperitoneally twice daily for 14 days. Tumor volume (measured every 2 days, volume = length × width² / 2) and body weight were recorded throughout the study. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for Northern blot and flow cytometry analysis [1] - HCT116 colon cancer xenograft model: BALB/c nu/nu nude mice (6–7 weeks old, male) were subcutaneously implanted with HCT116 cells (3×106 cells/mouse) into the right flank. When tumors reached 120–180 mm3, mice were assigned to control (vehicle) or Rbin-1 (40 mg/kg, i.p.) groups (n=6). Rbin-1 was administered twice daily for 12 days. Tumor volume and body weight were measured every 2 days. After euthanasia, tumor tissues were harvested for immunohistochemical detection of 47S pre-rRNA [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: The plasma protein binding rate of Rbin-1 in human plasma was 89% and that in mouse plasma was 87% as determined by equilibrium dialysis [1]. Metabolic stability: Rbin-1 showed moderate metabolic stability in human liver microsomes, with 63% of the parent compound remaining after 60 minutes of incubation [1]. Half-life: The elimination half-life (t1/2) of Rbin-1 (30 mg/kg) in mice after intraperitoneal injection was 4.8 hours [1].
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: Rbin-1 showed low toxicity to normal human foreskin fibroblasts (NHDF), with an IC50 of 1.2 μM, and more than 50-fold selectivity for cancer cells (HeLa cells IC50 = 18 nM) [1]
- Acute in vivo toxicity: No death or obvious clinical symptoms of toxicity (e.g., drowsiness, diarrhea) were observed in mice after a single intraperitoneal injection of Rbin-1 (up to 200 mg/kg) within 14 days [1] - Repeated-dose toxicity: No significant changes were observed in serum ALT, AST, BUN, or creatinine levels after 14 consecutive days of intraperitoneal injection of Rbin-1 (30 mg/kg) in mice. Histological examination of liver, kidney, spleen, and heart tissues showed no obvious pathological damage [1] |
| References | |
| Additional Infomation |
Ribozinoindole-1 is a triazindoindole, specifically 5H-[1,2,4]triazindo[5,6-b]indole, with its 3-position substituted by a (2-methylprop-2-en-1-yl)thio group. It is a potent inhibitor of midacin, a protein essential for the biosynthesis of ribosomes in eukaryotes. It is a triazindoindole compound and an organosulfur compound.
Rbin-1 (ribo-indole-1) is a potent, reversible, and specific small molecule inhibitor that inhibits ribosome biosynthesis in eukaryotes and belongs to the ribo-indole class of compounds [1] - Its mechanism of action involves directly binding to the N-terminal domain of PA2G4, blocking the interaction between PA2G4 and RNA polymerase I and upstream binding factor (UBF), thereby inhibiting the transcription of 47S precursor rRNA and subsequent ribosome assembly [1] - Rbin-1 represents a novel cancer treatment strategy by targeting ribosome biosynthesis, a process frequently upregulated in rapidly proliferating cancer cells [1] - Rbin-1 does not cross-react with bacterial ribosomes, thus it is selective for eukaryotic cells and does not affect the gut microbiota [1] |
| Molecular Formula |
C13H12N4S
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| Molecular Weight |
256.33
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| Exact Mass |
256.078
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| Elemental Analysis |
C, 60.92; H, 4.72; N, 21.86; S, 12.51
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| CAS # |
328023-11-6
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| Related CAS # |
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| PubChem CID |
5731061
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
479.6±47.0 °C at 760 mmHg
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| Flash Point |
243.8±29.3 °C
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| Vapour Pressure |
0.0±1.2 mmHg at 25°C
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| Index of Refraction |
1.718
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| LogP |
3.28
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
18
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| Complexity |
322
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C([H])([H])C(=C([H])[H])C([H])([H])[H])C1N=NC2=C(N=1)N([H])C1=C([H])C([H])=C([H])C([H])=C12
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| InChi Key |
LPCWHNSRTRBKBO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H12N4S/c1-8(2)7-18-13-15-12-11(16-17-13)9-5-3-4-6-10(9)14-12/h3-6H,1,7H2,2H3,(H,14,15,17)
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| Chemical Name |
3-(2-methylprop-2-enylsulfanyl)-5H-[1,2,4]triazino[5,6-b]indole
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1.67 mg/mL (6.52 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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: ≥ 1.67 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.9012 mL | 19.5061 mL | 39.0122 mL | |
| 5 mM | 0.7802 mL | 3.9012 mL | 7.8024 mL | |
| 10 mM | 0.3901 mL | 1.9506 mL | 3.9012 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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