| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
UCHL1 (ubiquitin C-terminal hydrolase L1) (negative probe). Unlike GK13S (UCHL1 inhibitor), GK16S does not inhibit UCHL1 activity. Instead, it is designed as a chemogenomic probe to engage non-UCHL1 targets (off-targets) including PARK7/DJ-1, C21orf33, ISOC1, and NIT2. These targets represent the "lesser" or secondary targets of GK13S, which may contribute to phenotypic effects observed with the inhibitor. GK16S serves as a specificity control to differentiate UCHL1-dependent effects from off-target effects. The compound does not inhibit recombinant or cellular UCHL1, nor does it reduce monoubiquitin levels in cells. GK16S is a valuable tool for validating that observed biological activities are indeed due to UCHL1 inhibition.
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| ln Vitro |
In vitro, GK16S does not inhibit UCHL1 activity and does not reduce monoubiquitin levels in human glioblastoma cells. While GK13S (the active UCHL1 inhibitor) inhibits recombinant UCHL1 with an IC50 of 50 nM and reduces cellular monoubiquitin levels, GK16S shows no appreciable UCHL1 inhibitory activity at comparable or higher concentrations (up to 10 uM). However, GK16S engages off-target proteins (PARK7, C21orf33, ISOC1, NIT2) that are also targeted by GK13S. This selectivity profile makes GK16S an excellent negative control probe for chemogenomic studies aimed at dissecting UCHL1 biology. The presence of an Alkyne group enables click chemistry-mediated visualization of probe binding to its target proteins.
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| ln Vivo |
No specific in vivo data are available for GK16S. As a chemogenomic probe designed for cellular studies, GK16S is primarily used in vitro to validate UCHL1-dependent phenotypes observed with GK13S. In cell-based assays, GK16S can be administered at concentrations up to 10 uM for 24-72 hours, and cellular effects are compared to those induced by the active inhibitor GK13S. If a phenotype observed with GK13S is absent in GK16S-treated cells, it supports the conclusion that the phenotype is attributable to UCHL1 inhibition rather than off-target engagement. GK16S has not been used in animal models or as a therapeutic agent. The compound is strictly for research use.
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| Enzyme Assay |
GK16S contains an Alkyne functional group, which enables labeling with fluorescent azides via click chemistry for studying its protein interactions. For direct binding assays of GK16S, pull-down experiments using alkyne-azide chemistry are employed. Cell lysates are incubated with GK16S (1-10 uM) for 1-2 hours at 4degC. After washing, biotin-azide or TAMRA-azide is added with Cu(I) catalyst (e.g., CuSO4, THPTA, sodium ascorbate) for click chemistry labeling. Biotin-labeled proteins are captured on streptavidin beads, eluted, and identified by mass spectrometry. Alternatively, fluorescence-labeled proteins are resolved by SDS-PAGE and visualized by in-gel fluorescence scanning. These assays identify proteins bound by GK16S (the off-target profile) without requiring a traditional enzyme activity assay.
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| Cell Assay |
For cell-based assays, human glioblastoma U87-MG cells or HEK293 cells are seeded in 6-well plates (5 × 10^5 cells/well) and grown overnight. Cells are treated with GK16S (0.1-10 uM) or vehicle control (DMSO) for 24-72 hours. For UCHL1 inhibition validation, cells are lysed in RIPA buffer, and monoubiquitin levels are assessed by Western blot using an anti-monoubiquitin antibody. UCHL1 activity in lysates can be measured using a fluorogenic substrate (ubiquitin-AMC) in a 96-well plate format; lysates (10-20 ug protein) are incubated with 0.5 uM ubiquitin-AMC in assay buffer, and fluorescence (excitation 380 nm, emission 460 nm) is monitored over 30-60 minutes at 37degC. For off-target binding studies, cells are treated with GK16S as above, then lysed, and click chemistry labeling is performed to identify bound proteins. Comparison of results between GK13S (UCHL1 inhibitor) and GK16S (inactive control) allows attribution of phenotypes to specific targets.
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| Animal Protocol |
For in vivo studies, a typical protocol is not available for GK16S. For a generic chemogenomic probe, the compound can be administered intraperitoneally (10-50 mg/kg) to mice. Tissues (e.g., brain, liver, kidney) are collected at various time points (1, 4, 8, 24 hours), homogenized, and compound concentrations are determined by LC-MS/MS. However, GK16S is not typically used in vivo due to its role as a negative control; most studies employing GK16S are conducted in vitro to validate UCHL1 biology using GK13S. The compound is water-soluble due to its chemical structure, but specific in vivo pharmacokinetics have not been characterized. GK16S is a research chemical not intended for animal efficacy studies.
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| ADME/Pharmacokinetics |
No specific pharmacokinetic data are available for GK16S. As a small-molecule probe (MW ~390 g/mol), its PK properties are expected to be similar to GK13S. Based on the structure (click-chemistry alkyne group), the compound likely has moderate lipophilicity (LogP ~2-3). The presence of the alkyne group may influence metabolic stability, as alkynes can be metabolized by cytochrome P450 enzymes. For a related chemogenomic probe, the plasma half-life in mice after intraperitoneal administration may be 1-4 hours. GK16S is not intended for therapeutic applications, and detailed PK characterization is not commonly performed for negative control compounds.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for GK16S. The compound is described as non-toxic based on its use as a chemogenomic negative control probe, distinguishing it from GK13S (which is a selective UCHL1 inhibitor). In cellular assays, GK16S does not induce cytotoxicity at concentrations up to 10 uM for 24-72 hours, as assessed by MTT or propidium iodide staining. The compound does not inhibit UCHL1, so it does not cause the accumulation of monoubiquitin or other UCHL1-related toxicities. No genotoxicity, organ toxicity, or carcinogenicity has been reported. Standard laboratory safety precautions should be used. GK16S is for research use only and not for human use.
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| References | |
| Additional Infomation |
UCHL1 (ubiquitin C-terminal hydrolase L1) is a deubiquitinating enzyme highly expressed in neurons and certain cancer cells, involved in ubiquitin homeostasis, protein degradation, and cell survival. GK13S is a selective small-molecule UCHL1 inhibitor used as a chemical probe to study UCHL1 biology. GK16S is the corresponding inactive control probe, which retains the ability to bind off-target proteins (PARK7, C21orf33, ISOC1, NIT2) but lacks UCHL1 inhibitory activity. The use of both probes enables chemogenomic validation, meaning that a phenotype observed only with GK13S but not GK16S is attributable to UCHL1 inhibition. GK16S contains an Alkyne group, making it a click chemistry reagent for copper-catalyzed azide-alkyne cycloaddition (CuAAc). This allows the probe to be conjugated to fluorescent or affinity tags for imaging or pull-down experiments. GK16S is for research use only.
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| Molecular Formula |
C11H15N3O
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| Molecular Weight |
205.26
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| Appearance |
Colorless to light yellow ointment
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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 | 4.8719 mL | 24.3593 mL | 48.7187 mL | |
| 5 mM | 0.9744 mL | 4.8719 mL | 9.7437 mL | |
| 10 mM | 0.4872 mL | 2.4359 mL | 4.8719 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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