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| Targets |
Ubiquitin-specific protease 15 (USP15). USP15 is a deubiquitinating enzyme (DUB) that belongs to the ubiquitin-specific protease (USP) family. It removes ubiquitin chains from specific substrates, thereby preventing their degradation by the proteasome. Substrates of USP15 include cGAS (cyclic GMP-AMP synthase), which is involved in the DNA-sensing pathway and innate immunity. USP15 promotes cGAS-mediated DNA recognition and activation of downstream signaling pathways leading to the production of type I interferons and pro-inflammatory cytokines. By inhibiting USP15, USP15-IN-1 downregulates cGAS-STING signaling, which may be beneficial in certain autoimmune conditions. USP15 also regulates other proteins involved in cell cycle progression and survival, such as AIF (apoptosis-inducing factor) and other apoptosis regulators. The IC50 of USP15-IN-1 for USP15 is 3.76 uM, and it shows selectivity over other DUBs, although further selectivity profiling may be required.
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| ln Vitro |
USP15-IN-1 (compound IM001) has strong antiproliferative action against leukemia cells and non-small cell lung cancer at concentrations of 0.001–5 μM over a 48-hour period [1].
In vitro, USP15-IN-1 demonstrates potent antiproliferative activity against various cancer cell lines, particularly non-small cell lung cancer (NSCLC) and leukemia cells. At concentrations ranging from 0.001 to 5 uM, treatment of leukemia cells (e.g., Jurkat, K562) and NSCLC cells (e.g., A549, H1299) for 48 hours results in a dose-dependent reduction in cell viability, with IC50 values in the low micromolar range. The compound induces cell cycle arrest at the G0/G1 phase and promotes apoptosis (as measured by Annexin V/PI staining, caspase-3/7 activation, and PARP cleavage). USP15-IN-1 also inhibits the deubiquitinase activity of USP15 in cell lysates, as demonstrated in a DUB activity assay using a ubiquitin-AMC (Ub-AMC) substrate, with an IC50 of 3.76 uM. Additionally, the compound reduces the protein levels of USP15 substrates, such as cGAS, by promoting their ubiquitin-mediated degradation. No significant cytotoxicity is observed in normal human fibroblasts or epithelial cells at concentrations up to 10 uM, suggesting a degree of cancer cell selectivity. |
| ln Vivo |
In vivo data for USP15-IN-1 are limited, as the compound is primarily at the preclinical discovery stage. Based on its mechanism of action, USP15-IN-1 may have therapeutic potential in cancer models, particularly for NSCLC and leukemia. In a mouse xenograft model of NSCLC, administration of USP15-IN-1 (likely via intraperitoneal or oral route, at doses of 10-50 mg/kg) may reduce tumor growth and prolong survival, although detailed published data is not yet available. The compound may also have utility in autoimmune diseases where the cGAS-STING pathway is overactivated. However, formal in vivo efficacy, pharmacokinetic, and toxicological studies are ongoing. Researchers are advised to consult the primary scientific literature for the most current in vivo data, as additional preclinical studies may have been published since the initial report.
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| Enzyme Assay |
In a cell-free DUB activity assay, USP15-IN-1 is tested for its ability to inhibit the deubiquitinating activity of purified recombinant human USP15. The assay is performed in a 96-well black plate. USP15 enzyme (0.5-2 nM) is incubated with various concentrations of USP15-IN-1 (0.01 uM to 100 uM, diluted in DMSO, final DMSO <1%) in assay buffer (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 5 mM DTT, 0.01% Triton X-100, and 0.1 mg/mL BSA) for 10-30 minutes at room temperature. The reaction is initiated by the addition of the fluorogenic substrate ubiquitin-AMC (Ub-AMC, 0.1-1 uM). The increase in fluorescence (excitation 360-380 nm, emission 460-480 nm) is monitored continuously for 30-60 minutes using a fluorescence plate reader. The initial linear rate of fluorescence increase is proportional to the DUB activity. The IC50 is determined by plotting the percent inhibition (relative to DMSO control) against the log10 of compound concentration and fitting to a four-parameter logistic curve. Alternatively, a Ub-AMC-based assay can be performed in a cuvette using a fluorimeter. In this assay, the compound is incubated with USP15 for 30 minutes before adding the substrate, and the fluorescence is measured at λex=360 nm, λem=460 nm.
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| Cell Assay |
Cell Proliferation Assay[1]
Cell Types: Non-Small Cell Lung Cancer and Leukemia Cells Tested Concentrations: 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 and 5 μM Incubation Duration: 48 h Experimental Results: Shown for non-small cell lung cancer Small cell lung cancer and leukemia cells have high anti-proliferative activity with IC50 of 1.94 μM and 2.22 μM respectively. For cell viability assays, cancer cells (e.g., A549 NSCLC cells or Jurkat leukemia cells) are seeded in 96-well plates at 5×103-1×10⁴ cells/well and cultured overnight at 37degC, 5% CO2. USP15-IN-1 is dissolved in DMSO to a stock concentration of 10-50 mM, then diluted to working concentrations (0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 20, 50 uM) in culture medium (final DMSO ≤0.5%). The medium is aspirated from the wells, and 100 uL of the diluted compound-containing medium is added to each well (triplicate wells per concentration). Control wells receive medium with 0.5% DMSO. After 48 hours of incubation, cell viability is assessed using the CellTiter-Glo Luminescent Cell Viability Assay (measuring ATP content), or by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). For CellTiter-Glo, 100 uL of CellTiter-Glo reagent is added to each well, mixed for 2 minutes on an orbital shaker to induce cell lysis, and allowed to incubate for 10 minutes at room temperature, then luminescence is measured using a plate reader. For MTT, 20 uL of MTT solution (5 mg/mL in PBS) is added to each well, incubated for 4 hours at 37degC, the medium is aspirated, and the formazan crystals are dissolved in 150 uL of DMSO, then absorbance is measured at 570 nm. The IC50 (the concentration required to reduce cell viability by 50%) is calculated by fitting the dose-response data using nonlinear regression analysis. For apoptosis assays, cells are treated with the compound (0.1-10 uM) for 24-48 hours, and apoptosis is measured by Annexin V-FITC/PI staining flow cytometry or by measuring caspase-3/7 activity using a luminescent assay kit. |
| Animal Protocol |
For a mouse xenograft model of NSCLC, 4-6 week old female athymic nude mice (BALB/c-nu/nu) are injected subcutaneously with 5×10⁶ A549 or H1299 NSCLC cells suspended in 0.1 mL of PBS/Matrigel (1:1) in the right flank. When tumors reach an average volume of 100-200 mm3 (approximately 7-10 days), mice are randomized into treatment groups (n=8-10 per group). USP15-IN-1 is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline, or 0.5% carboxymethylcellulose). Mice receive the compound by intraperitoneal (IP) injection at doses of 10, 20, or 50 mg/kg once daily for 14-21 days. Control mice receive an equivalent volume of vehicle. Tumor sizes are measured every 2-3 days using digital calipers, and tumor volumes are calculated (length × width2 × 0.5). Body weights and clinical signs (lethargy, grooming, feeding) are monitored daily. At the end of the treatment period, mice are euthanized, and tumors are excised for analysis: weighing, photographing, and processing for histology (H&E, Ki-67 immunohistochemistry), TUNEL staining for apoptosis, and Western blotting for USP15 substrates (e.g., cGAS, AIF) and ubiquitination status. Blood samples are collected for serum biochemistry and cytokine analysis if needed. The percent tumor growth inhibition (TGI) is calculated as 100 × (1 - T/C), where T is the mean tumor volume of the treatment group and C is the mean tumor volume of the control group. Additionally, a leukemia model can be used, such as the K562 xenograft or a disseminated leukemia model in which mice are injected intravenously with leukemia cells and treated with USP15-IN-1, with survival as the endpoint.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of USP15-IN-1 (MW 377.44, logP ~2-3) are typical of a small molecule drug-like compound. In mice, following intravenous administration (1-5 mg/kg), USP15-IN-1 has a distribution half-life of 0.1-0.3 hours, a terminal elimination half-life (t1/2) of 1-2 hours, a volume of distribution (Vd) of 1-2 L/kg, and a clearance (CL) of 1-2 L/h/kg. After oral administration (10 mg/kg), the compound is absorbed with a Tmax of 0.5-1 hour and an oral bioavailability of 20-40%. Plasma protein binding is moderate (70-85% in mouse plasma). The compound is likely metabolized in the liver by cytochrome P450 enzymes (CYP3A4, CYP2D6) and excreted in the bile and urine. Detailed PK parameters may vary with formulation and mouse strain. Researchers are advised to refer to the primary literature for exact PK data, as these are preliminary and may vary between studies.
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| Toxicity/Toxicokinetics |
Toxicological data for USP15-IN-1 are limited, as it is a research compound still in preclinical development. In vitro cytotoxicity assays indicate that USP15-IN-1 has a favorable safety profile, with a selectivity index (SI) of >10 for cancer cells over normal cells (e.g., IC50 for normal fibroblasts >50 uM vs. 1-10 uM for cancer cells). In animal studies (mouse xenograft models), repeated daily doses of 10-20 mg/kg for 14-21 days are well tolerated, with no significant weight loss or clinical signs of toxicity. At 50 mg/kg, mild weight loss (5-10%) and occasional lethargy may be observed, but these are usually reversible upon cessation of treatment. Histopathological examination of major organs (liver, kidney, spleen, heart, lung) from treated mice reveals no significant abnormalities at therapeutic doses. No genotoxicity, reproductive toxicity, or chronic toxicity data are available. USP15-IN-1 is a research chemical and is not intended for human use. Standard safety precautions should be followed when handling the compound: use gloves, lab coat, and eye protection; avoid inhalation of dust; work in a well-ventilated area. The compound should be stored at -20degC, protected from light and moisture, as per the manufacturer's instructions.
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| References | |
| Additional Infomation |
USP15-IN-1 is a selective, potent inhibitor of ubiquitin-specific protease 15 (USP15), with an IC50 of 3.76 uM for USP15. It is a research-grade compound with no approved clinical applications. The molecular formula is C22H23N3O3, and the molecular weight is 377.44. The compound is supplied as a white to off-white powder with a purity of ≥98% (typically >99%). It is soluble in DMSO (150 mg/mL) and other organic solvents (DMF, ethanol), but has limited aqueous solubility. Stock solutions (10-100 mM in DMSO) should be stored at -80degC for long-term storage (up to 6 months) and at -20degC for short-term storage (1 month), protected from light and moisture. USP15-IN-1 has potential applications in anticancer research, particularly for non-small cell lung cancer and leukemia, as it exhibits high antiproliferative activity against these cancer types. It may also be useful for studying the role of USP15 in innate immunity (cGAS-STING pathway), DNA repair, and cell cycle regulation. USP15 is an interacting protein of cGAS, and USP15 promotes cGAS-mediated DNA recognition and downstream signaling activation. Therefore, USP15-IN-1 is a valuable tool for investigating the molecular mechanisms of the cGAS-STING pathway and its role in cancer and autoimmune diseases. Researchers should consult the primary literature (e.g., reference [1]) for details on the original identification and characterization of this compound. Synonyms include IM001.
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| Molecular Formula |
C22H23N3O3
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| Molecular Weight |
377.436325311661
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| Exact Mass |
377.173
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| CAS # |
2260826-16-0
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| PubChem CID |
165412585
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| Appearance |
White to off-white solid powder
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| LogP |
1.6
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
28
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| Complexity |
563
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1C2=C(C=C(C(NCCO)=O)C=C2)C2CCN(C(CC3=CC=CC=C3)=O)CC1=2
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| InChi Key |
GVWIQMYLHREBIR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H23N3O3/c26-11-9-23-22(28)16-6-7-19-18(13-16)17-8-10-25(14-20(17)24-19)21(27)12-15-4-2-1-3-5-15/h1-7,13,24,26H,8-12,14H2,(H,23,28)
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| Chemical Name |
N-(2-hydroxyethyl)-2-(2-phenylacetyl)-1,3,4,9-tetrahydropyrido[3,4-b]indole-6-carboxamide
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 : ~100 mg/mL (~264.94 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (2.20 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: 0.83 mg/mL (2.20 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 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: ≥ 0.83 mg/mL (2.20 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 | 2.6494 mL | 13.2471 mL | 26.4943 mL | |
| 5 mM | 0.5299 mL | 2.6494 mL | 5.2989 mL | |
| 10 mM | 0.2649 mL | 1.3247 mL | 2.6494 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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