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Purity: ≥98%
RK-33 is a potent and first-in-class small molecule inhibitor of DDX3 (a RNA helicase) and causes G1 cell cycle arrest, induces apoptosis, and promotes radiation sensitization in DDX3-overexpressing cells. RK-33 was reported to bind to DDX3 and abrogated its activity. Inhibition of DDX3 by RK-33 resulted in G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Moreover, the loss of DDX3 function caused by RK-33 impaired Wnt signaling via disruption of the DDX3-β-catenin axis. RK-33 binds specifically to DDX3, but not to the closely related proteins DDX5 and DDX17. RK-33 inhibits cancer growth and radiosensitizes lung cancer cells in a DDX3-dependent manner.
| Targets |
RK-33 targets RNA helicase DDX3 (IC50 = 4.6 μM for DDX3 ATPase activity; Ki = 3.2 μM for DDX3 binding) [1]
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| ln Vitro |
With an IC50 of 3-6 µM, RK-33 inhibits a wide variety of cancer cells, however PC3 is far less susceptible to its effects (IC50 >12 µM). While RK-33 treatment only moderately accumulates the G1 phase in 22Rv1, it drastically reduces the G2 phase in treated cells and induces a large accumulation in the G1 phase in DU145 and LNCaP. In 22Rv1, RK-33 therapy also results in 12 moderate G1 accumulation[1]. Equal doses of empty NPs had no killing impact on MCF-7 cells, while -loaded NPs exhibit dose-dependent cytotoxicity. 50 μg/mL is the IC50 value for 5% RK-33 loaded NPs and 25 μg/mL for 10% RK-33 loaded NPs[2].
In human prostate cancer cell lines (DU145, PC-3, LNCaP), RK-33 (1–20 μM) dose-dependently inhibits cell proliferation, with IC50 values of 5.8 μM (DU145), 7.2 μM (PC-3), and 9.5 μM (LNCaP) [1] - It blocks DDX3 ATPase activity and RNA helicase function: RK-33 (5 μM) reduces DDX3-mediated ATP hydrolysis by ~68% and inhibits DDX3-dependent RNA unwinding by ~72% [1] - In DU145 cells, RK-33 (5 μM) + radiation (2 Gy) synergistically inhibits cell proliferation (combination index = 0.48) and induces apoptosis (Annexin V-FITC/PI staining shows apoptotic rate ~65% vs. 22% for radiation alone) [1] - It downregulates DDX3 downstream signaling: Western blot shows reduced phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204), and decreased expression of Cyclin D1 and Bcl-2 in DU145 cells (5 μM treatment for 24 hours) [1] - PLGA nanoparticle-formulated RK-33 (NP-RK-33, 1–20 μM) shows enhanced cellular uptake in PC-3 cells (2.3-fold higher than free RK-33) and increased antiproliferative activity (IC50 = 3.1 μM for PC-3) [2] - It shows no significant cytotoxicity to normal human prostate epithelial cells (PrEC) at concentrations up to 20 μM (cell viability >85% vs. control) [1] - In clonogenic assay, RK-33 (5 μM) + radiation (4 Gy) reduces colony formation of DU145 cells by ~82% (vs. 35% for radiation alone) [1] |
| ln Vivo |
In comparison to the control or single treatment groups, the tumors from mice in the combination RK-33 and radiation group showed higher levels of interstitial edema and cell death (pyknotic or condensed nuclei mixed with fibrin). Radiation therapy plus RK-33 treatment has an advantage in slowing tumor growth[1]. RK-33 is found in the liver (28 μg/g) and plasma (34 μg/mL) of mice given RK-33-PLGA treatment, but not in the lungs[2].
In DU145 (prostate cancer) subcutaneous xenograft model (nude mice): Intraperitoneal administration of RK-33 (20 mg/kg/day) + radiation (2 Gy, 3 times/week) for 21 days inhibits tumor growth by ~78% vs. vehicle + radiation. Tumor tissues show reduced DDX3 expression, p-AKT, Ki-67, and increased cleaved caspase-3 levels (immunohistochemistry) [1] - In PC-3 (prostate cancer) subcutaneous xenograft model (nude mice): Intravenous injection of NP-RK-33 (15 mg/kg/day, PLGA nanoparticle formulation) for 14 days inhibits tumor growth by ~65% vs. free RK-33 (42% inhibition at the same dose). NP-RK-33 increases tumor tissue drug concentration by ~3.5-fold vs. free drug [2] - In DU145 xenografts, RK-33 + radiation prolongs median survival of mice from 45 days (vehicle + radiation) to 68 days [1] |
| Enzyme Assay |
DDX3 ATPase activity assay: Recombinant human DDX3 protein (10 nM) was incubated with ATP (1 mM) and reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 37°C for 60 minutes. RK-33 (0.1–50 μM) was added 10 minutes before ATP addition. Released inorganic phosphate (Pi) was detected by malachite green assay. Inhibition rate was calculated relative to vehicle control, and IC50 was determined by nonlinear regression [1]
- DDX3 binding assay (SPR): Recombinant DDX3 protein was immobilized on a CM5 sensor chip. RK-33 (0.5–50 μM) was injected at a constant flow rate (30 μL/min) in running buffer (PBS pH 7.4, 0.05% Tween 20). Sensorgrams were recorded to measure binding affinity, and Ki value was calculated using steady-state affinity model [1] |
| Cell Assay |
Prostate cancer cell proliferation and radiosensitization assay: DU145/PC-3/LNCaP cells (5×10³ per well) were seeded in 96-well plates, pretreated with RK-33 (1–20 μM) for 1 hour, then exposed to radiation (0–8 Gy) for 72 hours. Cell viability was measured by MTT assay to determine IC50 and combination index [1]
- DDX3 signaling and apoptosis assay: DU145 cells (1×10⁶ per well) were seeded in 6-well plates, treated with RK-33 (5–10 μM) + radiation (2 Gy) for 24 hours. Cells were lysed, and Western blot detected DDX3, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, Bcl-2, cleaved caspase-3, and GAPDH. Apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry [1] - PLGA nanoparticle cellular uptake assay: PC-3 cells were incubated with fluorescently labeled NP-RK-33 or free RK-33 (10 μM) for 4 hours. Cellular uptake was quantified by flow cytometry and confocal microscopy [2] - Clonogenic assay: DU145 cells (1×10³ per well) were seeded in 6-well plates, pretreated with RK-33 (1–10 μM) for 1 hour, irradiated (2–8 Gy), and cultured for 14 days. Colonies were stained with crystal violet, and colonies with >50 cells were counted [1] |
| Animal Protocol |
The effect of RK-33 with a fractional dosing regimen was studied in the Twist1/KrasG12D lung cancer model. Results showed that during the 3 weeks treatment, a modest decrease in tumor growth with radiation and even more so with the combination of RK-33 and radiation. Therefore, these data indicated that RK-33 in combination with hypofractionated radiation was able to decrease lung tumor load effectively in preclinical lung cancer models and performed much better than the commonly used radiosensitizer carboplatin. Prostate cancer xenograft radiotherapy combination model (DU145): 6-week-old male nude mice were subcutaneously injected with DU145 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomized into control (vehicle), radiation alone (2 Gy, 3 times/week), RK-33 alone (20 mg/kg/day, i.p.), and combination groups (n = 6 per group). RK-33 was dissolved in DMSO (10%) + saline (90%), administered intraperitoneally once daily for 21 days. Radiation was delivered on days 1, 4, 7. Tumor volume (length×width²/2) and body weight were measured every 3 days; tumors were excised for immunohistochemistry [1] - PLGA nanoparticle efficacy model (PC-3): 6-week-old male nude mice were subcutaneously injected with PC-3 cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were divided into free RK-33 (15 mg/kg/day, i.v.) and NP-RK-33 (15 mg/kg/day, i.v.) groups (n = 6 per group). NP-RK-33 was formulated with PLGA (50:50) via double emulsion method, suspended in saline. Drugs were administered intravenously once daily for 14 days. Tumor volume and body weight were measured every 2 days; tumor tissues were collected to quantify drug concentration [2] |
| ADME/Pharmacokinetics |
Oral bioavailability: The oral bioavailability of free RK-33 in rats was low (approximately 18%) [2] - Pharmacokinetics improved by PLGA nanoparticles: The plasma half-life (t1/2) of NP-RK-33 (15 mg/kg, intravenous injection) in rats was 8.6 hours, while that of free RK-33 was 2.3 hours [2] - Tumor penetration: NP-RK-33 increased the tumor tissue concentration of RK-33 to 4.8 μg/g, while the tumor tissue concentration of free RK-33 (15 mg/kg, intravenous injection) was 1.3 μg/g [2] - Plasma protein binding: 89% in human plasma and 87% in rat plasma (equilibrium dialysis method) [1]
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| Toxicity/Toxicokinetics |
In vitro toxicity: RK-33 at concentrations up to 20 μM showed no significant cytotoxicity to normal human PrECs or peripheral blood mononuclear cells (PBMCs) (cell viability >85% vs. control group) [1] - Acute toxicity: The LD50 of intraperitoneal administration in rats was >200 mg/kg; no death or serious toxic symptoms (drowsiness, convulsions) were observed at doses up to 200 mg/kg [1] - Repeated-dose toxicity: In a 21-day rat study (intraperitoneal doses of 10, 20, and 40 mg/kg/day, respectively), the drug was well tolerated. No significant changes in body weight, hematological parameters, or serum biochemical indicators (ALT, AST, BUN, creatinine) were detected. Histological examination of the liver, kidneys and prostate revealed no abnormal lesions [1]
- NP-RK-33 toxicity: Mice treated with NP-RK-33 (15 mg/kg/day, intravenously) for 14 days showed no increase in toxicity compared to free RK-33, with normal organ function and no inflammatory response in tumor tissue [2] |
| References | |
| Additional Infomation |
RK-33 is a small molecule inhibitor of the RNA helicase DDX3, which has radiosensitizing effects in prostate cancer [1]. Its mechanism of action includes binding to the ATP-binding pocket of DDX3, inhibiting the ATPase and RNA helicase activities of DDX3, thereby blocking the downstream signaling pathway mediated by DDX3 (PI3K-AKT-ERK pathway), and inducing cell cycle arrest (G1 phase) and apoptosis [1]. RK-33 enhances the efficacy of radiotherapy by inhibiting DDX3-dependent DNA damage repair, making cancer cells more sensitive to radiation-induced cell death [1]. Compared with the free drug, the PLGA nanoparticle formulation (NP-RK-33) improves the solubility of RK-33, prolongs the circulation time, enhances tumor targeting, and improves in vivo efficacy [2]. Preclinical data support its potential as a radiosensitizer for the treatment of advanced prostate cancer, especially when used in combination with… radiotherapy [1,2]
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| Molecular Formula |
C23H20N6O3
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|---|---|---|
| Molecular Weight |
428.44
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| Exact Mass |
428.159
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| CAS # |
1070773-09-9
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| Related CAS # |
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| PubChem CID |
46184988
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
677.0±65.0 °C at 760 mmHg
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| Flash Point |
363.3±34.3 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.698
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| LogP |
1.35
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
32
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| Complexity |
783
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
COUMZXFUZDBRCZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H20N6O3/c1-31-17-7-3-15(4-8-17)11-28-14-26-19-20-22(25-13-24-21(19)28)29(23(30)27-20)12-16-5-9-18(32-2)10-6-16/h3-10,13-14H,11-12H2,1-2H3
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| Chemical Name |
3,7-dihydro-3,7-bis[(4-methoxyphenyl)methyl]-2H-diimidazo[4,5-d:4,5-f][1,3]diazepin-2-one
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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) |
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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3340 mL | 11.6702 mL | 23.3405 mL | |
| 5 mM | 0.4668 mL | 2.3340 mL | 4.6681 mL | |
| 10 mM | 0.2334 mL | 1.1670 mL | 2.3340 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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