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| Targets |
6RK73 targets ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme that removes ubiquitin from protein substrates and plays critical roles in regulating protein degradation and cellular signaling. UCHL1 is implicated in the progression of several cancers, including breast cancer, where it promotes metastasis through the TGF-β signaling pathway. 6RK73 acts as a covalent and irreversible inhibitor, forming a stable bond with the active site cysteine residue of UCHL1. The compound shows remarkable selectivity for UCHL1 over UCHL3 (236 µM vs 0.23 µM), a 1,000-fold difference in potency. This high selectivity makes 6RK73 an ideal tool for dissecting UCHL1-specific functions.
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| ln Vitro |
In MDA-MB-436 cells, 6RK73 (5 μM; 1-3 h) treatment resulted in significant suppression of TGFβ-induced pSMAD2 and pSMAD3, along with a decrease in TβRI and total SMAD protein levels [1]. 6RK73 (5 μM; 24
In vitro, 6RK73 inhibits UCHL1 with an IC₅₀ of 0.23 µM while showing negligible activity against UCHL3 (IC₅₀ = 236 µM), demonstrating exceptional selectivity. In MDA-MB-436 breast cancer cells, treatment with 5 µM 6RK73 for 1-3 hours significantly suppresses TGFβ-induced phosphorylation of SMAD2 and SMAD3 (pSMAD2/pSMAD3), and decreases TβRI and total SMAD protein levels. Treatment with 5 µM 6RK73 for 24-48 hours significantly impairs cell migration compared to DMSO controls. The compound effectively suppresses UCHL1-driven signaling and demonstrates strong anti-metastatic activity in breast cancer cells. |
| ln Vivo |
In zebrafish, 6RK73 effectively inhibits culture media leakage [1].
In vivo, 6RK73 demonstrates potent inhibition of breast cancer metastasis in zebrafish models. The compound significantly impairs breast cancer cell extravasation, indicating strong anti-metastatic activity. In MDA-MB-436 breast cancer cells, 6RK73 reduces TGFβ-induced signaling and cell migration. These findings position 6RK73 as a valuable tool for studying UCHL1 biology and deubiquitinase-targeted therapy in metastatic breast cancer. The compound's ability to inhibit metastasis in vivo supports the therapeutic potential of targeting UCHL1 in cancer. Detailed pharmacokinetic parameters for 6RK73 in mammalian systems require further investigation from primary research publications. |
| Enzyme Assay |
In vitro enzyme/receptor binding (non-cellular) assays for 6RK73 involve measuring inhibition of UCHL1 deubiquitinating activity using purified recombinant enzyme. The assay typically uses a fluorogenic substrate such as ubiquitin-AMC (ubiquitin-7-amino-4-methylcoumarin), which releases fluorescent AMC upon cleavage by UCHL1. The compound is incubated with purified UCHL1 enzyme and substrate in assay buffer (e.g., 50 mM Tris-HCl, pH 7.5, with 0.5 mM EDTA and 1 mM DTT). Fluorescence is measured over time (excitation 380 nm, emission 460 nm), and IC₅₀ values are calculated from concentration-response curves. Selectivity against UCHL3 is assessed using the same assay format with purified UCHL3 enzyme.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: MDA-MB-436 cells Tested Concentrations: 5 μM Incubation Duration: 24, 48 -48 h) The results demonstrated that the migration in MDA-MB-436 cells was Dramatically slower than that of DMSO [1]. Hour Experimental Results: Migration speed was Dramatically slower than DMSO control group Western Blot analysis [1] Cell Types: MDA-MB-436 Cell Tested Concentrations: 5 µM Incubation Duration: 1, 2, 3 hrs (hours) Experimental Results: demonstrated strong resistance to TGFβ-induced pSMAD2 inhibition and pSMAD3, as well as a reduction in TβRI and total SMAD protein levels. In vitro cellular experiments with 6RK73 are performed using MDA-MB-436 breast cancer cells. Cells are cultured in appropriate medium (e.g., DMEM with 10% FBS) and treated with 5 µM 6RK73 for 1-3 hours to assess signaling pathway modulation, or for 24-48 hours to evaluate cell migration. TGFβ-induced SMAD2 and SMAD3 phosphorylation is measured by Western blot analysis using phospho-specific antibodies. TβRI and total SMAD protein levels are also assessed by immunoblotting. Cell migration is evaluated using wound-healing or transwell migration assays. Cells are maintained at 37°C in 5% CO₂ with appropriate media supplements. |
| Animal Protocol |
In vivo animal studies with 6RK73 have been conducted in zebrafish models to assess anti-metastatic activity. Breast cancer cells (e.g., MDA-MB-436) are labeled with fluorescent markers and injected into zebrafish embryos. 6RK73 is administered to the zebrafish at appropriate concentrations, and cancer cell extravasation (spread from blood vessels into tissues) is monitored by fluorescence microscopy. The compound demonstrates significant inhibition of breast cancer extravasation, indicating potent anti-metastatic effects. This model enables rapid in vivo assessment of anti-metastatic activity and provides insights into UCHL1's role in cancer dissemination.
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| ADME/Pharmacokinetics |
Comprehensive pharmacokinetic data for 6RK73 are not extensively reported in the available literature. The compound has a molecular weight of 307.37 g/mol, suggesting reasonable potential for oral bioavailability and tissue penetration. It is soluble in DMSO at 112.5 mg/mL and can be formulated for in vivo administration using vehicles such as 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline at 4 mg/mL. As a covalent inhibitor, 6RK73 may exhibit prolonged target engagement due to irreversible binding to UCHL1. Detailed pharmacokinetic parameters including half-life, clearance, and bioavailability require further investigation from primary research publications.
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| Toxicity/Toxicokinetics |
Toxicological information for 6RK73 indicates that the compound is classified as toxic and contains a pharmaceutically active ingredient. Handling should only be performed by personnel trained and familiar with handling of potent active pharmaceutical ingredients. Under fire conditions, the compound may decompose and emit toxic fumes. Runoff from fire control or dilution water may cause environmental pollution. The compound should be stored appropriately and handled with caution using personal protective equipment including gloves and safety goggles. As an investigational compound, 6RK73 is not approved for human therapeutic use and is intended for research purposes only.
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| References | |
| Additional Infomation |
6RK73 (CAS 1895050-66-4) is a covalent, irreversible, and highly specific inhibitor of UCHL1 with an IC₅₀ of 0.23 µM. The compound has molecular formula C₁₃H₁₇N₅O₂S and molecular weight 307.37 g/mol. It shows exceptional selectivity with almost no inhibition of UCHL3 (IC₅₀ = 236 µM). 6RK73 specifically inhibits UCHL1 activity in breast cancer and suppresses TGFβ-induced signaling, cell migration, and metastasis. The compound is a valuable tool for studying UCHL1 biology, deubiquitinase-targeted therapy, and metastatic breast cancer research. It is supplied as a solid with purity ≥95% and is soluble in DMSO. Storage recommendations include -20°C in dry conditions. 6RK73 is for research use only and not approved for therapeutic applications.
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| Molecular Formula |
C13H17N5O2S
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| Molecular Weight |
307.371380567551
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| Exact Mass |
307.11
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| CAS # |
1895050-66-4
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| PubChem CID |
121248200
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| Appearance |
Off-white to light yellow solid powder
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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 |
3
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| Heavy Atom Count |
21
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| Complexity |
434
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| Defined Atom Stereocenter Count |
1
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| SMILES |
S1C(=NC=C1N1CCOCC1)NC([C@@H]1CN(C#N)CC1)=O
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| InChi Key |
ONIHSXKWNFYNNS-JTQLQIEISA-N
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| InChi Code |
InChI=1S/C13H17N5O2S/c14-9-17-2-1-10(8-17)12(19)16-13-15-7-11(21-13)18-3-5-20-6-4-18/h7,10H,1-6,8H2,(H,15,16,19)/t10-/m0/s1
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| Chemical Name |
(3S)-1-cyano-N-(5-morpholin-4-yl-1,3-thiazol-2-yl)pyrrolidine-3-carboxamide
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| Synonyms |
6RK73; 6RK73
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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 : ~125 mg/mL (~406.68 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.77 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 20.8 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: ≥ 2.08 mg/mL (6.77 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 20.8 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: ≥ 2.08 mg/mL (6.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2534 mL | 16.2670 mL | 32.5341 mL | |
| 5 mM | 0.6507 mL | 3.2534 mL | 6.5068 mL | |
| 10 mM | 0.3253 mL | 1.6267 mL | 3.2534 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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