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| 25mg |
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| 250mg |
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| Other Sizes |
Purity: ≥98%
| Targets |
Target: Ubiquitin-proteasome system (UPS) upstream of the 20S proteasome (inhibits ubiquitin-dependent protein degradation without affecting 20S proteasome catalytic activities) [1]
HeLa cervical cancer cell viability IC50 = 2.0 μM (as determined by XTT assay) [1] |
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| ln Vitro |
Treatment with RAMB4 (0-30 µM; 48 hours) led to a dose-dependent reduction in the viability of HPV16-positive SiHa and Caski cells and HPV-39-positive ME180 cervical cancer cell lines, respectively [1]. With an IC50 value of 2 µM, RAMB4 dose-dependently lowers the viability of exponentially developing HeLa cervical cancer cells [1].
In Vitro: RAMB4 (PTP1B-IN-9) treatment (5 μM for 6 hours) in HeLa cells caused accumulation of poly-ubiquitinated proteins, with GAPDH-normalized levels up to 3-fold higher than mock-treated cells [1]. RAMB4 (PTP1B-IN-9) at concentrations up to 10 μM failed to inhibit the chymotrypsin-like, trypsin-like, or peptidyl-glutamyl peptide hydrolyzing-like activities of purified 20S proteasomes [1]. RAMB4 (PTP1B-IN-9) treatment (10 μM for 8 hours) in HeLa cells caused accumulation of Hsp90 (approximately 2-fold increase by densitometry) [1]. RAMB4 (PTP1B-IN-9) produced a dose-dependent reduction in viability of HPV16-positive SiHa and CaSki cells and HPV39-positive ME180 cervical cancer cell lines, with minimal effects on primary human keratinocytes when tested at 0.1, 1, 5, 10, and 20 μM over 48 hours [1]. |
| Enzyme Assay |
Enzyme Assay: Purified 20S proteasomes were pre-treated with or without RAMB4 (PTP1B-IN-9) at escalating doses up to 10 μM for 30 minutes at 37°C. After treatment, fluorogenic substrates specific for three catalytic activities were added: Suc-LLVY-AMC for chymotrypsin-like activity, Boc-LRR-AMC for trypsin-like activity, and Ac-YVAD-AMC for peptidyl-glutamyl peptide hydrolyzing-like activity. The reaction was carried out at 37°C for 45 minutes in buffer containing 50 mM TRIS-HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT (final volume 100 μL). The reaction was quenched with 1% SDS, and fluorescence was measured with excitation at 380 nm and emission at 440 nm. Unlike Bortezomib, RAMB4 (PTP1B-IN-9) failed to inhibit any of the three proteasomal activities when tested up to 10 μM [1].
For measurement of proteasomal activity in living cells, exponentially growing CaSki cervical cancer cells were treated with RAMB4 (PTP1B-IN-9) at concentrations up to 20 μM for 4 hours. Cell lysates were prepared in NP-40 lysis buffer (0.1% NP-40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT). Residual chymotrypsin-like activity was determined by adding Suc-LLVY-Glo substrate and measuring luminescence. RAMB4 (PTP1B-IN-9) treatment failed to inhibit proteasomal activity when tested up to 20 μM [1]. |
| Cell Assay |
Cell Viability Assay[1]
Cell Types: Keratinocytes, SiHa, CaSki and ME180 Cell Tested Concentrations: 5, 10, 15, 20, 25, 30 µM Incubation Duration: 48 hrs (hours) Experimental Results: Dose-dependent decrease in HPV16-positive viability SiHa and Caski cells and HPV-39-positive ME180 cervical cancer cell lines respectively had minimal effects on the viability of primary human keratinocytes, with IC50 similar to HeLa cells. Cell Assay: Cell viability was determined by XTT assay. Cervical cancer cells (HeLa, SiHa, CaSki, ME180) or primary human keratinocytes were seeded at 1,000 cells per well in 96-well plates and treated with RAMB4 (PTP1B-IN-9) at specified concentrations (0.1, 1, 5, 10, 20 μM) for 48 hours. After treatment, cells were incubated with XTT labeling mixture for 4 hours. Formazan dye was quantified by measuring absorbance at 450 nm using a spectrophotometric plate reader. RAMB4 (PTP1B-IN-9) reduced viability of cervical cancer cells in a dose-dependent manner but had minimal effect on keratinocytes [1]. For immunoblot analysis of ubiquitinated proteins, HeLa cells were treated with 5 μM RAMB4 (PTP1B-IN-9) for 6 hours. Total cellular protein (10-20 μg) was separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-ubiquitin antibody. GAPDH was used as loading control. RAMB4 (PTP1B-IN-9) treatment resulted in clear accumulation of poly-ubiquitinated proteins compared to mock-treated cells [1]. For immunoblot analysis of Hsp90, HeLa cells were treated with 10 μM RAMB4 (PTP1B-IN-9) for 8 hours, and Hsp90 expression levels were detected by Western blot with anti-Hsp90 antibody, normalized to GAPDH. RAMB4 (PTP1B-IN-9) induced approximately 2-fold accumulation of Hsp90 [1]. For the 4XUbiquitin-Luciferase degron assay, HeLa cells were transfected with Ub-FL or control CMV-FL plasmid. Transfected cells were treated with RAMB4 (PTP1B-IN-9) at 20 μM for 6 hours, and luciferase activity was measured. RAMB4 (PTP1B-IN-9) induced weaker stabilization of the Ub-FL reporter compared to Bortezomib or RA-1 [1]. |
| References | |
| Additional Infomation |
Additional Info: RAMB4 (PTP1B-IN-9) is a chalcone-based derivative lacking amino acid components, containing an α,β-unsaturated carbonyl system which is proposed as the molecular determinant for inhibition of ubiquitin-mediated protein degradation upstream of the proteasome. It selectively kills cervical cancer cells (HPV-positive) without affecting normal keratinocytes, regardless of HPV genotype (HPV16, HPV18, HPV39). The mechanism involves accumulation of poly-ubiquitinated proteins without direct inhibition of 20S proteasome catalytic activities, leading to unfolded protein responses including aggresome formation and Hsp90 stabilization. RAMB4 (PTP1B-IN-9) does not inhibit chymotrypsin-like, trypsin-like, or PGPH-like activities of purified 20S proteasomes at concentrations up to 10 μM, distinguishing it from Bortezomib [1].
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| Molecular Formula |
C19H13CL4NO
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|---|---|
| Molecular Weight |
413.119
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| Exact Mass |
410.975
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| Elemental Analysis |
C, 55.24; H, 3.17; Cl, 34.32; N, 3.39; O, 3.87
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| CAS # |
145888-79-5
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| Related CAS # |
(E,E)-RAMB4;919091-61-5
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| PubChem CID |
5388806
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
6.268
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
25
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| Complexity |
516
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C\1NC/C(=C\C2=CC(=C(C=C2)Cl)Cl)/C(=O)/C1=C/C3=CC(=C(C=C3)Cl)Cl
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| InChi Key |
GJPXGFGIFQWUOC-ACFHMISVSA-N
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| InChi Code |
InChI=1S/C19H13Cl4NO/c20-15-3-1-11(7-17(15)22)5-13-9-24-10-14(19(13)25)6-12-2-4-16(21)18(23)8-12/h1-8,24H,9-10H2/b13-5+,14-6+
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| Chemical Name |
(3E,5E)-3,5-Bis(3,4-dichlorobenzylidene)piperidin-4-one
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| Synonyms |
PTP1B-IN-9 PTP1B-IN 9 PTP1B-IN9 RAMB4 RAMB-4 RAMB 4
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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 : ~5 mg/mL (~12.10 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4206 mL | 12.1030 mL | 24.2060 mL | |
| 5 mM | 0.4841 mL | 2.4206 mL | 4.8412 mL | |
| 10 mM | 0.2421 mL | 1.2103 mL | 2.4206 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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