| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 100mg | |||
| Other Sizes |
| Targets |
Alpha/beta-hydrolase domain containing 6 (ABHD6) (IC50 = 0.8 – 1.7 nM, gel-based ABPP; IC50 = 3.9 – 15.1 nM, 2-AG hydrolysis assay)
Diacylglycerol lipase β (DAGLβ) (~50% inhibition at 1 µM). [1] |
|---|---|
| ln Vitro |
In gel-based competitive activity-based protein profiling (ABPP) assays using the tailored probe HT-01 on Neuro2A cell membrane proteomes, KT203 (compound 20) inhibited endogenous ABHD6 with high potency. [1]
In 2-arachidonoylglycerol (2-AG) substrate hydrolysis assays using recombinant mouse ABHD6 overexpressed in HEK293T cell membranes, KT203 inhibited ABHD6 activity. [1] Gel-based competitive ABPP using the broad-spectrum serine hydrolase (SH) probe FP-rhodamine on mouse brain membrane proteomes showed that KT203 at 1 µM selectively inhibited ABHD6 without observable off-targets against other SHs, except for a modest (~50%) inhibition of DAGLβ. At 10 µM, off-target activity against FAAH and DAGLβ was observed. [1] Quantitative mass spectrometry-based ABPP-SILAC analysis in Neuro2A cells treated with 3 nM KT203 for 4 hours showed that it blocked >90% of ABHD6 activity with negligible cross-reactivity (<50% inhibition) against more than 50 other SHs detected in the proteome. [1] |
| ln Vivo |
Mice treated intraperitoneally (i.p.) with KT203 at 1 mg/kg for 4 hours showed near-complete blockade of ABHD6 activity in the liver, as measured by gel-based competitive ABPP using the HT-01 probe. At lower doses (0.5 and 0.1 mg/kg), KT203 maintained strong inhibition (~80%) of liver ABHD6. [1]
In contrast to its effect in the liver, KT203 showed negligible inhibition of ABHD6 activity in the brain at all tested doses (0.1, 0.5, and 1 mg/kg, i.p.), indicating peripherally-restricted activity. [1] KT203 displayed good selectivity in vivo. In the liver, it showed little cross-reactivity against numerous carboxylesterase (CES) enzymes, which are common off-targets for serine hydrolase inhibitors. In the brain and liver, gel-based ABPP with the FP-rhodamine probe revealed minimal off-target activity. [1] |
| Enzyme Assay |
The activity of ABHD6 was determined using a 2-AG hydrolysis assay. Membrane lysates from HEK293T cells overexpressing recombinant mouse ABHD6 were diluted in assay buffer (PBS with 0.05% Triton X-100). The lysates were pre-treated with DMSO or compound for 30 minutes at 37°C. The reaction was initiated by adding 2-AG substrate (final concentration 100 µM) and incubated for 30 minutes at 37°C. The reaction was quenched by adding a chloroform:methanol mixture (2:1 v/v) containing an internal standard (pentadecanoic acid). After vortexing and centrifugation, the organic phase was analyzed by LC-MS to quantify the release of arachidonic acid, the hydrolysis product. [1]
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| Cell Assay |
For in situ potency measurement, Neuro2A cells were treated with varying concentrations of KT203 in serum-free media for 4 hours at 37°C. Cells were then lysed, and membrane proteomes were prepared. The proteomes were subjected to gel-based competitive ABPP analysis by labeling with the HT-01 probe (1 µM, 30 min, 37°C). After SDS-PAGE and in-gel fluorescence scanning, the percentage of remaining ABHD6 activity was quantified using image analysis software to determine the IC50 value. [1]
For proteome-wide selectivity profiling in cells (ABPP-SILAC), Neuro2A cells were cultured in "light" or "heavy" SILAC media. "Heavy" cells were treated with 3 nM KT203 for 4 hours, while "light" cells were treated with DMSO. Cells were harvested, lysed, and membrane/soluble proteomes were isolated. Proteomes were labeled with FP-biotin (10 µM, 2 hours) to enrich serine hydrolases. Light and heavy proteomes were mixed 1:1, and biotinylated proteins were captured using avidin beads. Proteins were digested on-bead with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The heavy/light ratio for peptides corresponding to individual serine hydrolases was calculated to determine the extent of inhibition. [1] |
| Animal Protocol |
For in vivo efficacy and selectivity studies, C57Bl/6 mice were injected intraperitoneally (i.p.) with KT203. The compound was formulated in an 18:1:1 (v/v/v) solution of saline/ethanol/PEG40 (ethoxylated castor oil) and administered at a volume of 10 µL per gram of body weight. Mice were treated with varying doses of KT203 (0.1, 0.5, or 1 mg/kg). After 4 hours, the mice were anesthetized and euthanized. Brain and liver tissues were collected, homogenized in PBS, and subjected to differential centrifugation to isolate membrane fractions. The membrane proteomes were then analyzed by gel-based competitive ABPP using the HT-01 and FP-rhodamine activity-based probes to assess ABHD6 inhibition and overall serine hydrolase selectivity. [1]
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| References | |
| Additional Infomation |
KT203 (compound 20) is an optimized irreversible inhibitor belonging to the (2-substituted)-piperidinyl-1,2,3-triazolurea class of compounds, which was initially developed as a chemical probe for ABHD6. [1]
Its development was achieved through structure-activity relationship (SAR) studies, which focused on introducing polar substituents onto the triazole biphenyl group and modifying the piperidine substituent. KT203 is characterized by a 2-benzyl group on the piperidine ring and a carboxylic acid substituent at the 3-position of the distal benzene ring of the triazole biphenyl group. [1] KT203 is a highly efficient and selective ABHD6 inactivator, and is expected to irreversibly inhibit the enzyme via a serine nucleophile at the active site of carbamate. [1] A key feature of KT203 is that its in vivo activity is limited to the periphery. While it effectively inhibits ABHD6 in the liver, its activity in the brain is negligible, likely due to its carboxylic acid group reducing its permeability in the central nervous system (CNS). This property makes it a suitable probe for pairing with the brain osmotic inhibitor KT182 (compound 9) to differentiate the central and peripheral functions of ABHD6 in animal models. [1] The development of KT203 addresses the need for an optimized tool to study ABHD6 function. ABHD6 is a serine hydrolase that hydrolyzes the endocannabinoid 2-arachidonic acid glycerol (2-AG) and may play a role in neuroinflammation and neuroprotection. [1] |
| Molecular Formula |
C28H26N4O3
|
|---|---|
| Molecular Weight |
466.531046390533
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| Exact Mass |
466.2
|
| CAS # |
1402612-64-9
|
| PubChem CID |
53364510
|
| Appearance |
White to yellow solid powder
|
| LogP |
5.3
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
35
|
| Complexity |
719
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1(C2=CC=C(C3=CN(C(N4CCCCC4CC4=CC=CC=C4)=O)N=N3)C=C2)=CC=CC(C(O)=O)=C1
|
| InChi Key |
SSSCOJOXPDDHOO-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C28H26N4O3/c33-27(34)24-10-6-9-23(18-24)21-12-14-22(15-13-21)26-19-32(30-29-26)28(35)31-16-5-4-11-25(31)17-20-7-2-1-3-8-20/h1-3,6-10,12-15,18-19,25H,4-5,11,16-17H2,(H,33,34)
|
| Chemical Name |
3-[4-[1-(2-benzylpiperidine-1-carbonyl)triazol-4-yl]phenyl]benzoic acid
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~214.35 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.36 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.5 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1435 mL | 10.7174 mL | 21.4348 mL | |
| 5 mM | 0.4287 mL | 2.1435 mL | 4.2870 mL | |
| 10 mM | 0.2143 mL | 1.0717 mL | 2.1435 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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