| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
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Purity: ≥98%
| Targets |
Gasdermin D[1].
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|---|---|
| ln Vitro |
LDC7559 (1 and 5 μM) dramatically reduces the lethal effect of human and mouse GSDMD NT domains transfected into HEK293T cells and decreases the release of IL-1β upon inflammasome activation [3]. Rather than interfering with the activation and cleavage mechanism, LDC7559 acts directly by preventing the GSDMD NT domain from functioning [3].
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| Cell Assay |
Transfection of GSDMD constructs in HEK293T cells [2]
HEK293T cells were seeded the day before transfection at a density of 5 × 105 cells/ml. Transfection was done in Opti-MEM using 2 μg of DNA and 7 μl of Lipofectamine 2000 for a six-well plate or respective scaling for other surface areas. LDC7559 or caspase inhibitors were added 2 hours after transfection, and lactate dehydrogenase (LDH) release of cells was measured 16 hours after transfection. Incubation of HEK293T lysates with recombinant proteases[2] HEK293T cell lysate was harvested 16 hours after transfection by changing the medium of a six well to 200 μl of Opti-MEM containing 0.05% (v/v) Triton X-100 and scraping cells off the bottom of the well. After 10 min of centrifugation at full speed and 4°C in a tabletop centrifuge, supernatants were used for cleavage assays. Fifty microliters of lysate were incubated with 10 mU of purified human NE, 2 U of recombinant caspase-4, 4 μg of purified CG (RP-77525), or 6.25 μg of recombinant PR3 (C628) for the indicated time points at 25°C on a shaking incubator. Reactions were stopped by directly adding 50 μl of 2× SDS loading buffer (at 98°C) containing 200 mM DTT and by immediately boiling samples before loading on SDS–polyacrylamide gel electrophoresis gels. |
| Animal Protocol |
Live imaging of neutrophils [2]
Cells were resuspended in an assay medium supplemented with 500 nM SYTOX Green and 2.5 μM DRAQ5 and seeded at a density of 5 × 105 cells per well into μ-Slide 8 Well ibiTreat dishes (ibidi). After LDC7559 treatment and NET induction, images were collected at 2-min intervals on a Leica TCS SP8 confocal microscope equipped with a climate chamber at 37°C and with a Leica HC PL APO 20×/0.75 IMM CORR CS2 objective using glycerol immersion. The recording of DRAQ5 (cell-permeable DNA dye) was used to track individual nuclei over time and to determine nuclear area. The recording of SYTOX Green (cell-impermeable dye) was used to determine permeability of cells. Bright-field recording was added for a video representation of the data. Automated algorithms were used to determine the time point of permeabilization and to categorize nuclei into “non-expanded,” “fully expanded” (filling the entire cell), or “NET” (extending beyond the maximal area of a cell). The results were then mapped back to a video representation of the data and manually inspected for accuracy before they were used for further analysis. The complete image analysis pipeline consisting of ImageJ and R scripts will be described elsewhere in detail. |
| References |
[1]. Spel L, et al. Gasdermin D opens the way for NETs. Nat Rev Rheumatol. 2018 Dec;14(12):690-692.
[2]. Sollberger G, et al. Gasdermin D plays a vital role in the generation of neutrophil extracellular traps. Sci Immunol. 2018 Aug 24;3(26). pii: eaar6689. [3]. Ankit Pandeya, et al. Gasdermin D (GSDMD) as a New Target for the Treatment of Infection. Medchemcomm. 2019 Apr 4;10(5):660-667. |
| Molecular Formula |
C20H19N3O3
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|---|---|
| Molecular Weight |
349.38
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| Exact Mass |
349.142639
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| Elemental Analysis |
C, 68.75; H, 5.48; N, 12.03; O, 13.74
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| CAS # |
2407782-01-6
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| Appearance |
White to light yellow solid
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| LogP |
2.25
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| SMILES |
O1C2C=C(C=CC=2CN2C(=CC(C3C=CC=CC=3OC)=N2)C1)NC(C)=O
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| InChi Key |
IFOIINXSFCQLOV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H19N3O3/c1-13(24)21-15-8-7-14-11-23-16(12-26-20(14)9-15)10-18(22-23)17-5-3-4-6-19(17)25-2/h3-10H,11-12H2,1-2H3,(H,21,24)
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| Chemical Name |
N-(2-(2-Methoxyphenyl)-4H,10H-benzo[f]pyrazolo[5,1-c][1,4]oxazepin-7-yl)acetamide
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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 : 100 mg/mL (286.22 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.95 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 20.8 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: ≥ 2.08 mg/mL (5.95 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 20.8 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: ≥ 2.08 mg/mL (5.95 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 | 2.8622 mL | 14.3111 mL | 28.6221 mL | |
| 5 mM | 0.5724 mL | 2.8622 mL | 5.7244 mL | |
| 10 mM | 0.2862 mL | 1.4311 mL | 2.8622 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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