| Size | Price | Stock | Qty |
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| 5mg |
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| Other Sizes |
| Targets |
IC50: 0.17 nM (OTUB1) and 0.28 nM (USP8)[1]
OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), USP8 (ubiquitin-specific protease 8). |
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| ln Vitro |
KRAS-WT (H1975, EBC-1, H1703) and KRAS-mutated (H23, A549) NSCLC cell lines have antiproliferative effects in response to OTUB1/USP8-IN-1 (compound 61; 10 nM-10 μM; 72 h)[1]. In H1975 cells, OTUB1/USP8-IN-1 (500 nM; 24 h) reduces the amounts of both UBE2N and EGFR protein[1].
OTUB1/USP8-IN-1 has antiproliferative effects in KRAS-WT (H1975, EBC-1, H1703) and KRAS-mutated (H23, A549) NSCLC cell lines, with IC50 values of 118, 145, 172, 431, and 1004 nM, respectively. In H1975 cells, it (500 nM; 24 h) reduces the amounts of both UBE2N and EGFR protein in a dose-dependent manner. It is a potent dual OTUB1/USP8 inhibitor, effectively reducing protein levels of key oncogenic drivers. |
| ln Vivo |
In the H1975 xenograft mouse model, OTUB1/USP8-IN-1 (compound 61; 10 nM-10 μM; 72 h) reduces the tumor burden[1].
In the H1975 xenograft mouse model (KRAS-WT NSCLC), OTUB1/USP8-IN-1 (10 nM-10 microM; 72 h) reduces tumor burden. Intraperitoneal (IP) administration at 5 mg/kg twice daily (BID) for 2 weeks diminishes total tumor weight and average tumor volume by over two-fold compared to vehicle. The compound is effective at reducing tumor growth in vivo, validating OTUB1/USP8 as targets for NSCLC therapy. |
| Enzyme Assay |
Assay: In vitro OTUB1/USP8 inhibition assay. Protocol: Recombinant OTUB1 or USP8 is incubated with varying concentrations of OTUB1/USP8-IN-1 (0.01-1000 nM) and a ubiquitin-AMC (Ub-AMC) substrate in DUB buffer. Upon DUB-mediated cleavage, free AMC is released, and fluorescence (excitation 360 nm, emission 460 nm) is measured continuously for 30-60 minutes. The initial velocity of AMC release is calculated, and IC50 values are derived from dose-response curves.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: KRAS-WT (H1975, EBC-1, H1703) and KRAS-mutated (H23, A549) NSCLC cell lines Tested Concentrations: 10 nM-10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibited cell proliferative with IC50 values of 118, 145, 172, 431, and 1004 nM for H1975, H1703, EBC-1, H23, and A549 cells, respectively. Western Blot Analysis[1] Cell Types: H1975 cells Tested Concentrations: 500 nM Incubation Duration: 24 hrs (hours) Experimental Results: diminished the levels of both UBE2N and EGFR in a dose-dependent manner. Cells: Non-small-cell lung cancer (NSCLC) cell lines, including H1975 (KRAS-WT), EBC-1, H1703, H23, and A549 (KRAS-mutated). Protocol: For proliferation assays, cells are treated with OTUB1/USP8-IN-1 (10 nM-10 microM) for 72 hours, and viability is measured by CellTiter-Glo or MTT assays. For mechanism studies, H1975 cells are treated with 500 nM compound for 24 hours, and UBE2N and EGFR protein levels are analyzed by Western blot. PARP and caspase-3 cleavage are assessed to measure apoptosis. |
| Animal Protocol |
Animal/Disease Models: Female BALB/c nude mice with H1975 xenografts (5 weeks of age)[1]
Doses: 5 mg/kg Route of Administration: intraperitoneal (ip)injection; QD and BID, for 2 weeks Experimental Results: diminished total tumor weight and average tumor volume in an over twofold with BID dosing. Animal/Disease Models: Female BALB/c nude mice with H1975 xenograft (5 weeks of age)[1] Doses: 1 and 10 mg/kg Route of Administration: intravenous (iv)injection (1 mg/kg) and oral administration (10 mg/kg) Experimental Results: 1.19 Administration iv (1 mg/kg) po (10 mg/kg) T1/2 (h) 0.83 1.75 Tmax (h) 0.33 Cmax (μg/L) 4274 AUC (μg·h/ L) 1345 3747 CL (L/h/kg) 44 Vdss (L/kg) 0.77 Animal Model: Female BALB/c nude mice (5 weeks old) with H1975 xenografts. Protocol: Mice are implanted subcutaneously with 1x10^7 H1975 cells. When tumors reach ~100-200 mm3, mice are randomized to treatment groups. OTUB1/USP8-IN-1 is administered at 5 mg/kg via intraperitoneal (IP) injection either once daily (QD) or twice daily (BID) for 2 weeks. Tumor volume and body weight are measured twice weekly. At study endpoint, tumors are excised and weighed. Tumors are analyzed by Western blot for UBE2N, EGFR, and apoptosis markers. Pharmacodynamic markers (UBE2N, EGFR) are measured. |
| ADME/Pharmacokinetics |
In female BALB/c nude mice, OTUB1/USP8-IN-1 was administered intravenously (IV) at 1 mg/kg and orally (PO) at 10 mg/kg. After IV administration, T1/2 was 0.83 hours. After PO administration, T1/2 was 1.75 hours, Tmax was 0.33 hours, Cmax was 4274 microg/L, and AUC was [not fully specified]. The oral bioavailability was not calculated. The compound is rapidly absorbed but has a short half-life, necessitating BID dosing for efficacy.
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| Toxicity/Toxicokinetics |
No specific toxicity data found; in the efficacy study at 5 mg/kg IP (BID) for 2 weeks, OTUB1/USP8-IN-1 was well-tolerated with no reported body weight loss. However, given the critical role of DUBs in protein homeostasis, on-target inhibition in normal tissues could lead to accumulation of ubiquitinated proteins, endoplasmic reticulum stress, and cell death, particularly in rapidly dividing tissues (bone marrow, intestine). Comprehensive toxicological studies are not yet published.
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| References | |
| Additional Infomation |
OTUB1/USP8-IN-1 (compound 61) was discovered and characterized as a potent dual inhibitor of OTUB1 and USP8, two deubiquitinating enzymes implicated in non-small-cell lung cancer (NSCLC). It is a chemical probe for studying the role of these DUBs in regulating key oncoproteins like EGFR and UBE2N. The dual inhibition strategy may overcome resistance to EGFR inhibitors. It is not FDA-approved and is intended for research use.
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| Molecular Formula |
C22H16CLFN2O4
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|---|---|
| Molecular Weight |
426.824848175049
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| Exact Mass |
426.078
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| CAS # |
2858800-98-1
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| PubChem CID |
165437860
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.5
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
30
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| Complexity |
696
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CN1CC[C@H](C1)OC2=C(C(=C(C=C2)F)C3=NC4=C(O3)C(=O)C5=CC=CC=C5C4=O)Cl
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| InChi Key |
HXIYHNFNCGXLAU-LLVKDONJSA-N
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| InChi Code |
InChI=1S/C22H16ClFN2O4/c1-26-9-8-11(10-26)29-15-7-6-14(24)16(17(15)23)22-25-18-19(27)12-4-2-3-5-13(12)20(28)21(18)30-22/h2-7,11H,8-10H2,1H3/t11-/m1/s1
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| Chemical Name |
2-[2-chloro-6-fluoro-3-[(3R)-1-methylpyrrolidin-3-yl]oxyphenyl]benzo[f][1,3]benzoxazole-4,9-dione
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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 : 50 mg/mL (117.15 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 | 2.3429 mL | 11.7145 mL | 23.4291 mL | |
| 5 mM | 0.4686 mL | 2.3429 mL | 4.6858 mL | |
| 10 mM | 0.2343 mL | 1.1715 mL | 2.3429 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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