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| Other Sizes |
| ln Vitro |
KLH45 (25 nM, 4 hr) has no cross-reactivity with any of the over 40 identified serine hydrolases, with the exception of ABHD6, and completely inactivates DDHD2 in Neuro2A cells (>95% inhibition)[1]. In DDHD2-expressing cells supplemented with fatty acids, KLH45 (2 μM) enhances LD formation [2].
KLH45 (2 μM) selectively and completely inhibited the activity of wild-type DDHD2 in transfected COS-7 cells, as assessed by activity-based protein profiling (ABPP) with the DDHD2-directed probe HT-01. The structurally related control compound KLH40 showed no such inhibitory effect. [2] In oleic acid-supplemented COS-7 cells expressing wild-type DDHD2, treatment with KLH45 (2 μM, 17 h) reversed the suppressive effect of DDHD2 expression on lipid droplet (LD) accumulation. This was demonstrated by an increase in LD surface area and BODIPY 493/503 signal intensity per cell compared to cells treated with DMSO or the control compound KLH40. [2] Consistent with the LD imaging results, KLH45 (2 μM, 17 h) treatment in wild-type DDHD2-transfected COS-7 cells blocked the DDHD2-mediated decrease in triacylglycerol (TAG, specifically C18:1/C36:2) levels. It also blocked the DDHD2-mediated elevation of the TAG hydrolysis products diacylglycerol (DAG, specifically C18:1/C18:1), monoacylglycerol (MAG, specifically C18:1), and free fatty acid (FFA, specifically C18:1). Cells treated with KLH45 exhibited a lipid profile similar to that of mCherry-transfected control cells, whereas cells treated with DMSO or KLH40 maintained a lipid profile characteristic of active DDHD2 expression. [2] |
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| ln Vivo |
KLH45 (20 mg/kg; given every 12 hours) caused notable increases in a number of TAGs that accumulated over the course of four days in the brains of DDHD2−/− mice [1].
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| Enzyme Assay |
The inhibitory activity of KLH45 was assessed using an activity-based protein profiling (ABPP) method. Transfected COS-7 cells expressing wild-type DDHD2 were treated with KLH45 (2 μM) or the control compound KLH40 for 16 hours. Following this treatment, cell lysates were prepared and incubated with the DDHD2-directed activity-based probe HT-01 (1 μM, 30 min). The samples were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The degree of probe labeling, which correlates with enzyme activity, was visualized via in-gel fluorescence scanning. KLH45 treatment resulted in a complete loss of the HT-01 fluorescence signal corresponding to DDHD2, indicating complete inhibition of its activity. No such inhibition was observed with the control compound KLH40. [2]
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| Cell Assay |
Cell viability assay [2]
Cell Types: COS-7 cells. Tested Concentrations: 2μM. Incubation Duration: 16 hrs (hours). Experimental Results: Selective blocking of DDHD2 activity. KLH45 was evaluated in a cell-based assay for lipid droplet (LD) formation and lipid metabolism. COS-7 cells were transiently transfected with a construct expressing mCherry-tagged wild-type DDHD2. One hour prior to and during an overnight (16 h) incubation with oleic acid (200 μM) to induce LD formation, the cells were treated with KLH45 (2 μM), the control compound KLH40 (2 μM), or DMSO vehicle. The impact on LD accumulation was assessed by confocal microscopy after staining with BODIPY 493/503 (1 μg/mL) for LDs and Hoechst 33342 (0.1 μg/mL) for nuclei. Images were quantified to measure LD surface area and BODIPY 493/503 signal intensity per transfected cell (identified by mCherry signal). The effect on lipid metabolism was analyzed by targeted lipidomics, where cells from the same treatment groups were collected, and the levels of specific TAG, DAG, MAG, and FFA species (C18:1/C36:2 TAG, C18:1/C18:1 DAG, C18:1 MAG, and C18:1 FFA) were measured by liquid chromatography and mass spectrometry. [2] |
| Animal Protocol |
Animal/Disease Models: Mouse[1].
Doses: 5-40 mg/kg. Management: IP, once. Experimental Results: No altered brain signatures were shown. For subchronic in vivo efficacy studies, adult mice were administered KLH45 or the control compound KLH40 at a dose of 20 mg/kg. The compounds were delivered by intraperitoneal (i.p.) injection every 12 hours for a total of 4 days. A vehicle-treated control group was also included. After this treatment period, mice were euthanized, and brain and spinal cord tissues were collected for lipidomic analysis and competitive ABPP to assess target engagement. [1] For acute treatment studies, mice received a single dose of KLH45 or KLH40 (ranging from 5 to 40 mg/kg, i.p.) and were euthanized 4 hours post-injection. Brain tissue was collected to assess dose-dependent target engagement by gel-based competitive ABPP using the HT-01 probe. [1] For subchronic in vivo efficacy studies, adult mice were administered KLH45 or the control compound KLH40 at a dose of 20 mg/kg. The compounds were delivered by intraperitoneal (i.p.) injection every 12 hours for a total of 4 days. A vehicle-treated control group was also included. After this treatment period, mice were euthanized, and brain and spinal cord tissues were collected for lipidomic analysis and competitive ABPP to assess target engagement. [1] For acute treatment studies, mice received a single dose of KLH45 or KLH40 (ranging from 5 to 40 mg/kg, i.p.) and were euthanized 4 hours post-injection. Brain tissue was collected to assess dose-dependent target engagement by gel-based competitive ABPP using the HT-01 probe. [1] |
| References |
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| Molecular Formula |
C24H25F3N4O2
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|---|---|
| Molecular Weight |
458.476115942001
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| Exact Mass |
458.192
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| Elemental Analysis |
C, 62.87; H, 5.50; F, 12.43; N, 12.22; O, 6.98
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| CAS # |
1632236-44-2
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| PubChem CID |
126970670
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
561.0±60.0 °C at 760 mmHg
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| Flash Point |
293.1±32.9 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.591
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| LogP |
5.87
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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 |
6
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| Heavy Atom Count |
33
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| Complexity |
615
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1=CC(C2=CC=C(OC(F)(F)F)C=C2)=NN1C(N(C1CCCCC1)CCC1=CC=CC=C1)=O
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| InChi Key |
FYOYNRLSBYWAHL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H25F3N4O2/c25-24(26,27)33-21-13-11-19(12-14-21)22-17-28-31(29-22)23(32)30(20-9-5-2-6-10-20)16-15-18-7-3-1-4-8-18/h1,3-4,7-8,11-14,17,20H,2,5-6,9-10,15-16H2
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| Chemical Name |
N-cyclohexyl-N-(2-phenylethyl)-4-[4-(trifluoromethoxy)phenyl]triazole-2-carboxamide
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| Synonyms |
KLH45; KLH-45; KLH 45;
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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 : ~180 mg/mL (~392.60 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 4.5 mg/mL (9.82 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 45.0 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: ≥ 4.5 mg/mL (9.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 45.0 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: ≥ 4.5 mg/mL (9.82 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 | 2.1811 mL | 10.9056 mL | 21.8112 mL | |
| 5 mM | 0.4362 mL | 2.1811 mL | 4.3622 mL | |
| 10 mM | 0.2181 mL | 1.0906 mL | 2.1811 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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