Pitstop 2d
Based on 1 Customer Validation
Pitstop 2d is a clathrin (Clathrin) terminal domain (TD) inhibitor with an IC50 of 1.7 μM. Pitstop 2d occupies the Clathrin box binding site within the clathrin TD, blocking the binding of endocytic protein ligands such as Epsin to clathrin. Pitstop 2d inhibits clathrin-mediated endocytosis (Endocytosis) and the cellular entry of vesicular stomatitis virus (VSV). Pitstop 2d has low cytotoxicity, does not disrupt the nuclear permeability barrier, and does not inhibit clathrin-independent endocytosis. Pitstop 2d can be used in studies related to endocytosis and vesicular stomatitis virus infection.
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- Pureté : 97.11%
- Formule: C26H18N2O4S2
- Masse moléculaire:486.56
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Stockage:
4°C, sealed storage, away from moisture
* In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
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Activité biologique
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Chemical Information
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Solvant et solubilité
- Protocole
- Pureté et documentation
- Références
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Help & FAQs
Activité biologique
Description
IC50 & Target
IC50: <2 μM (Clathrin TD)[1]
In Vitro
Pitstop 2d potently inhibits the interaction between purified clathrin terminal domain and amphiphysin B/C domain in cell-free ELISA assays, with an IC50 of 1.7 μM[1].
Pitstop 2d (25-100 μM; 8-20 h) exhibits only extremely low cytotoxicity in HEK293 and Cos7 cells[1].
Pitstop 2d (30 μM; 15 min) does not disrupt the nuclear permeability barrier in HeLa cells[1].
Pitstop 2d (13-45 μM; 15 min preincubation followed by 15 min transferrin uptake; 4 h washout) inhibits clathrin-mediated transferrin endocytosis in HeLa, HEK293T and Cos7 cells, with an IC50 of 13 μM in HeLa cells, and its inhibitory effect is reversible after washout[1].
Pitstop 2d significantly reduces the proportion of dynamic clathrin-coated pits in Cos7 cells expressing eGFP-clathrin light chain α, shifting CCP dynamics toward long-lived structures[1].
Pitstop 2d interferes with clathrin dynamics in the perinuclear region of Cos7 cells expressing eGFP-clathrin light chain α, reducing the maximum fluorescence recovery rate after photobleaching to 37.6%[1].
Pitstop 2d (45 μM; 15 min preincubation followed by 60 min dextran uptake) does not inhibit fluid-phase endocytosis of Alexa488-labeled dextran in HeLa cells[1].
Pitstop 2d binds to the clathrin box site of the purified clathrin terminal domain in an extended conformation, forming a specific and stable interaction[1].
Pitstop 2d (100 μM; 1 h) reduces the binding level between clathrin and Epsin 1 in Cos7 cells expressing eGFP-clathrin light chain α, while increasing the binding level between clathrin and SNX9, but has no effect on most other early-acting endocytic proteins[1].
Pitstop 2d (15 min) increases the total number of clathrin-coated structures on the plasma membrane of BS-C-1 cells, causes the accumulation of shallow clathrin-coated pits, but does not alter the diameter of free clathrin-coated vesicles[1].
Pitstop 2d (20 μM; 15 min preincubation followed by 1 h virus infection) inhibits over 60% of VSV entry into Vero-E6 cells, confirming that clathrin-mediated endocytosis (CME) is the primary pathway for efficient VSV cellular entry[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.
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Cell Line:HEK293, Cos7 cells
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Concentration:25, 50 and 100 μM
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Incubation Time:8 and 20 h
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Result:Exhibited low cytotoxicity, with minimal LDH release detected across all tested concentrations and incubation times, in contrast to the high cytotoxicity of Pitstop 2.
Quantified cytotoxicity as 1.53% relative to maximal LDH release induced by Triton X-100.
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Cell Line:Cos7 cells expressing eGFP-clathrin light chain α
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Concentration:100 μM
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Incubation Time:1 h
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Result:Reduced the binding level of clathrin to Epsin 1.
Increased the binding level of clathrin to SNX9.
Had no effect on most other early-acting endocytic proteins.
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Cell Line:HeLa cells
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Concentration:30 μM
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Incubation Time:15 min
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Result:Did not disrupt the nuclear permeability barrier
Chemical Information
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Appearance Solid
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Masse moléculaire 486.56
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Formule C26H18N2O4S2
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Color White to yellow
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SMILES
O=S(C1=CC2=C(C=C1)C=CC=C2)(NC(S/C3=C\C4=C(C=CC=C4)OC5=CC=CC=C5)=NC3=O)=O
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Livraison
Room temperature in continental US; may vary elsewhere.
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Stockage
4°C, sealed storage, away from moisture
* In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)
Solvant et solubilité
In Vitro:
DMSO : 100 mg/mL (205.52 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
In Vivo:
Select the appropriate dissolution method based on your experimental animal and administration route.
- For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
- To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for In Vivo experiments, it is recommended to prepare freshly and use it on the same day.
- The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.
Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
Solubility: ≥ 2.5 mg/mL (5.14 mM); Clear solution
This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.
Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:
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Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
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%DMSO +
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
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%+
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+%Tween-80 + +
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%Saline +
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Working solution concentration: 0.22 mg/mL
Method for preparing stock solution: mg drug dissolved in μL DMSO. Stock solution concentration: mg/mL. * In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)
1. Take μL DMSO stock solution;
2. Add μL .
μL , mix evenly;
3. Then add μL Tween 80, mix evenly;
4. Then add μL
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Protocole
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Research Protocol for Infectious Diseases
Infectious-disease experiments test how pathogens interact with host barriers, innate immune receptors, inflammatory signaling, pathogen replication, and tissue injury; pattern-recognition receptors such as TLRs, RIG-I-like receptors, NOD-like receptors, and inflammasomes detect microbial molecules and activate NF-κB, interferon, and cytokine responses. The central hypothesis is that infection severity reflects the balance between pathogen burden and host response: protective inflammation restricts pathogen growth, whereas excessive or mislocalized inflammation contributes to tissue damage and disease phenotype. Unresolved questions include which host pathways are protective versus pathogenic, why some infection models fail to translate to human disease, and which combined readouts best predict clinically relevant infection outcomes.
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Cell Cytotoxicity Assay
Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.
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Mammalian live/dead viability and cytotoxicity staining
Live/dead viability and cytotoxicity staining assays are based on the simultaneous detection of intracellular esterase activity in metabolically active (viable) cells and membrane integrity loss in non-viable cells. In commonly used dual-staining approaches, membrane-permeant fluorogenic substrates are converted by intracellular esterases into fluorescent products in live cells, while impermeant DNA-binding dyes selectively enter cells with compromised plasma membranes and label nucleic acids in dead or dying cells, enabling discrimination between viable and non-viable populations by fluorescence microscopy or flow cytometry.
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Transepithelial/transendothelial electrical resistance assay
TEER measures electrical resistance across epithelial or endothelial monolayers cultured on permeable supports, and the readout reflects ionic conductance through the cell barrier, especially the paracellular pathway regulated by junctional integrity. TEER can be measured without destroying the monolayer and is commonly used before or during transport, permeability, barrier-disruption, and barrier-maturation experiments. TEER values are influenced by biological maturation and technical conditions; reported factors include temperature, medium formulation, passage number, electrode geometry, membrane properties, and junctional length during early monolayer maturation. Therefore, TEER should be interpreted with blank-insert subtraction, area normalization, repeated readings, and, when possible, orthogonal barrier readouts such as FITC-dextran flux or tight-junction staining.
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Cell Viability Determination by MTT Colorimetric Assay
The following protocol uses the MTT colorimetric assay as a classic literature-established method for assessing cell viability/metabolic activity in cultured mammalian cells. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by metabolically active cells to a colored formazan product; the amount of formazan is quantified spectrophotometrically and provides an indirect measure of metabolically active viable cells. Importantly, MTT reduction reflects cellular oxidoreductase/metabolic activity rather than an absolute direct count of living cells, so changes in cellular metabolism can alter the signal independently of cell number.
Pureté et documentation
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Fiche technique (283 KB)
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SDS (252 KB)
- English - EN (252 KB)
- Français - FR (252 KB)
- Deutsch - DE (252 KB)
- Norwegian - NO (252 KB)
- Español - ES (252 KB)
- Swedish - SV (252 KB)
- Italian - IT (252 KB)
- Korean - KR (252 KB)
- Portuguese - PT (252 KB)
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Instruction de manipulation (2659 KB)
Références
Complete Stock Solution Preparation Table
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
| Optional Solvent | Concentration Solvent Mass | 1 mg | 5 mg | 10 mg | 25 mg |
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| DMSO | 1 mM | 2.0552 mL | 10.2762 mL | 20.5524 mL | 51.3811 mL |
| 5 mM | 0.4110 mL | 2.0552 mL | 4.1105 mL | 10.2762 mL | |
| 10 mM | 0.2055 mL | 1.0276 mL | 2.0552 mL | 5.1381 mL | |
| 15 mM | 0.1370 mL | 0.6851 mL | 1.3702 mL | 3.4254 mL | |
| 20 mM | 0.1028 mL | 0.5138 mL | 1.0276 mL | 2.5691 mL | |
| 25 mM | 0.0822 mL | 0.4110 mL | 0.8221 mL | 2.0552 mL | |
| 30 mM | 0.0685 mL | 0.3425 mL | 0.6851 mL | 1.7127 mL | |
| 40 mM | 0.0514 mL | 0.2569 mL | 0.5138 mL | 1.2845 mL | |
| 50 mM | 0.0411 mL | 0.2055 mL | 0.4110 mL | 1.0276 mL | |
| 60 mM | 0.0343 mL | 0.1713 mL | 0.3425 mL | 0.8564 mL | |
| 80 mM | 0.0257 mL | 0.1285 mL | 0.2569 mL | 0.6423 mL | |
| 100 mM | 0.0206 mL | 0.1028 mL | 0.2055 mL | 0.5138 mL |
Pitstop 2d Related Classifications
HY-186018 Related Classifications
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Keywords
- Pitstop 2d
- Clathrin
- VSV
- clathrin inhibitor
- clathrin terminal domain
- clathrin-mediated endocytosis
- CME
- endocytosis inhibitor
- Pitstop 2 derivative
- epsin
- SNX9
- transferrin uptake
- VSV entry
- vesicular stomatitis virus
- low cytotoxicity
- nuclear permeability barrier
- Cos7 cells
- HeLa cells
- clathrin-coated pits
- protein-protein interaction inhibitor
- Inhibitor
- inhibitor
- inhibit