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| Targets |
UCHL1 50 nM (IC50)
UCHL1 (ubiquitin C-terminal hydrolase L1). GK13S is a selective ligand and inhibitor of the deubiquitinase UCHL1 (also known as PGP9.5). UCHL1 is a member of the ubiquitin-specific protease family responsible for hydrolyzing ubiquitin from small adducts and recycling free ubiquitin monomers. GK13S binds to UCHL1 and inhibits its enzymatic activity, leading to reduced hydrolysis of ubiquitin chains, accumulation of polyubiquitinated proteins, and depletion of free monoubiquitin. This results in altered ubiquitin homeostasis, disruption of protein degradation pathways, and modulation of cell survival, proliferation, and differentiation. GK13S is a cell-permeable inhibitor that can be used to investigate the role of UCHL1 in cancer, neurodegenerative disorders (Parkinson's disease, Alzheimer's disease), and other pathologies. |
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| ln Vitro |
GK13S (0-1 μM, 1 hour) inhibits recombinant UCHL1 with an IC50 of 50 nM[1]. HEK293 cells' intracellular UCHL1 is inhibited by GK13S (1–10 μM, 24 hours)[1]. HEK293 cell growth is unaffected by GK13S (5 μM, 72 h) [1]. U-87 MG cells' ubiquitin levels are decreased by GK13S (5 μM, 48 hours) [1].
In vitro, GK13S potently inhibits recombinant UCHL1 activity with an IC50 of 50 nM. The compound inhibits cellular UCHL1 activity in HEK293 cells at concentrations of 1-10 uM. GK13S treatment reduces levels of monoubiquitin in human glioblastoma U87-MG cells, confirming target engagement and inhibition of the deubiquitinase activity that normally generates free ubiquitin. The compound does not substantially inhibit other deubiquitinases at low concentrations, indicating selectivity. GK13S contains an Alkyne group for click chemistry-based labeling, allowing visualization of probe binding and identification of potential off-target proteins through pull-down and mass spectrometry experiments. GK13S can also be used to study the functional consequences of UCHL1 inhibition in cancer cell proliferation, migration, invasion, and resistance to apoptosis. |
| ln Vivo |
No specific in vivo data are available for GK13S. As a chemogenomic probe, GK13S is primarily used in cell-based assays to study UCHL1 function. However, based on its cell-permeable and target-specific properties, GK13S could theoretically be used in animal models to assess UCHL1's role in disease progression, such as in xenograft mouse models of glioblastoma for cancer studies, or in neurodegenerative disease models. A typical protocol would involve intraperitoneal or intravenous administration at doses of 10-50 mg/kg. However, GK13S is not an approved drug and is strictly for research use. In vivo studies would require validation of target engagement and assessment of potential off-target effects using the inactive control probe GK16S.
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| Enzyme Assay |
For direct binding assays, GK13S can be used in a competitive binding format. Recombinant UCHL1 protein (10-50 nM) is incubated with a fluorescent ubiquitin-AMC substrate (0.5 uM) in assay buffer (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 5 mM DTT, 0.05% Tween-20) and varying concentrations of GK13S (0.1-1000 nM) for 30-60 minutes at 37degC. The release of free AMC is monitored by fluorescence (excitation 380 nm, emission 460 nm). The IC50 is calculated from the inhibition curve using a four-parameter logistic model. Alternatively, surface plasmon resonance (SPR) can be used: UCHL1 is immobilized on a sensor chip, and GK13S is flowed over at increasing concentrations (0.1-500 nM) to measure binding kinetics (KD). The alkyne group of GK13S does not interfere with binding to UCHL1.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: HEK293 cell Tested Concentrations: 1, 5, 10 μM Incubation Duration: 24 h Experimental Results: Nearly completely inhibits UCHL1 at 1 μM. For cellular assays, HEK293 or U87-MG glioblastoma cells are seeded in 6-well plates (5 × 10^5 cells/well) or 96-well plates (1 × 10^4 cells/well) and grown overnight. GK13S is added at concentrations of 0.1-10 uM (final DMSO concentration ≤0.1%) and incubated for 24-72 hours. For assessment of UCHL1 inhibition, cells are lysed in 0.5% NP-40 or RIPA buffer, and lysates (10-20 ug protein) are incubated with 0.5 uM ubiquitin-AMC substrate in assay buffer at 37degC. Fluorescence is measured over 30-60 minutes to determine residual UCHL1 activity. For monoubiquitin detection, lysates are subjected to Western blotting using an anti-ubiquitin antibody (e.g., FK2 clone) which detects monoubiquitin. For cell viability, an MTT or CellTiter-Glo assay is performed after 48-72 hours of treatment. For click chemistry labeling, cells are treated with GK13S for 2-4 hours, then lysed, and cell lysates are reacted with TAMRA-azide or biotin-azide under Cu(I) catalysis to visualize probe-protein adducts by in-gel fluorescence or pull-down. |
| Animal Protocol |
For in vivo studies, a typical protocol is not available for GK13S. For a generic UCHL1 inhibitor, adult male C57BL/6 mice (6-8 weeks old) would be administered GK13S intraperitoneally at 10-30 mg/kg daily for 5-14 days. Compound would be formulated in 10% DMSO, 40% PEG400, 5% Tween 80 in water. Blood would be collected for PK analysis, and tissues (brain, liver, kidney, tumor) would be harvested for UCHL1 activity measurement using ubiquitin-AMC assay and for monoubiquitin Western blotting. Tumor xenograft models: nude mice implanted with U87-MG cells would be treated with GK13S when tumors reach 100-150 mm3. Tumor volume and body weight would be monitored every 2-3 days. However, GK13S is primarily a research tool for in vitro studies, and its in vivo use has not been extensively reported.
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| ADME/Pharmacokinetics |
No specific pharmacokinetic data are available for GK13S. As a small-molecule UCHL1 inhibitor (MW 390.44 g/mol), GK13S is expected to be cell-permeable and likely to have moderate oral bioavailability. The compound contains an alkyne group, which may undergo metabolism, but detailed PK studies have not been published. For a typical small-molecule probe, the plasma half-life in mice after intraperitoneal administration is 1-4 hours. GK13S is not intended for therapeutic applications; its use is limited to research settings. The compound is soluble in DMSO, which is typically used as the stock solution for in vitro assays.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for GK13S. As an inhibitor of UCHL1, a deubiquitinase critical for neuronal survival and cancer progression, prolonged inhibition of UCHL1 may have functional consequences. However, GK13S is generally used at concentrations up to 10 uM in vitro and has not been reported to cause acute cytotoxicity in cell viability assays at these concentrations. In animals, the potential toxicities of UCHL1 inhibition are not well characterized. No genotoxicity, organ toxicity, or carcinogenicity has been reported for GK13S. The compound is for research use only and not for human or therapeutic applications. Standard laboratory safety precautions should be followed.
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| References | |
| Additional Infomation |
UCHL1 is a deubiquitinating enzyme highly expressed in neurons (comprising 1-2% of total brain protein) and in certain cancers (e.g., glioblastoma, lung cancer, prostate cancer). UCHL1 functions to hydrolyze ubiquitin from small adducts and to maintain the free ubiquitin pool, which is essential for ubiquitin-dependent protein degradation. Dysregulation of UCHL1 is implicated in Parkinson's disease (where UCHL1 mutations cause decreased activity) and in cancer (where UCHL1 is often overexpressed and promotes cell survival). GK13S is a first-in-class selective small-molecule inhibitor of UCHL1, discovered through a chemogenomics approach, with an IC50 of 50 nM. The compound contains an Alkyne group, enabling click chemistry applications for target engagement studies. For chemogenomic validation, the inactive control probe GK16S (which does not inhibit UCHL1 but binds off-target proteins) is used alongside GK13S to confirm UCHL1-dependent phenotypes. GK13S is for research use only.
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| Molecular Formula |
C21H22N6O2
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| Molecular Weight |
390.44
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| Appearance |
Off-white to light yellow solid powder
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 2.5612 mL | 12.8061 mL | 25.6121 mL | |
| 5 mM | 0.5122 mL | 2.5612 mL | 5.1224 mL | |
| 10 mM | 0.2561 mL | 1.2806 mL | 2.5612 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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