CME/clathrin-mediated endocytosis
Pitstop 2 targets clathrin, specifically associating with the terminal domain of clathrin. Clathrin is a key protein involved in clathrin-mediated endocytosis (CME), a process by which cells internalize molecules from the cell surface. By binding to the terminal domain of clathrin, Pitstop 2 inhibits the formation of clathrin-coated pits and vesicles, thereby blocking clathrin-mediated endocytosis. This inhibition disrupts receptor-mediated endocytosis, HIV entry, and synaptic vesicle recycling. Pitstop 2 is a selective inhibitor of clathrin terminal domain with an IC50 of 12 μM.

Pitstop 2 (20–40 μM; 30 min) prevents J774A.1 macrophages from endocytosing transferrin while maintaining cell viability[1]. Pitstop 2 (20–40 μM; 30 min) has no effect on J774A.1 macrophages' ability to internalize cholera toxin B[1]. Pitstop 2 (0.001-100 μM; 6 h) inhibits the spindle of HeLa cells, preventing them from progressing through mitosis[2]. Pitstop 2 (1-30 μM; 24 h) inhibits the growth of dividing cancer cells and causes apoptosis[2]. Pitstop 2 (1-30 μM; 48 h) has no effect on non-tumourigenic NIH3T3 fibroblasts' ability to proliferate and remain viable[2].
In vitro, Pitstop 2 has been shown to inhibit clathrin-mediated endocytosis. The compound inhibits receptor-mediated endocytosis, HIV entry, and synaptic vesicle recycling. Pitstop 2 induces apoptosis and inhibits cell growth in dividing cancer cells at concentrations of 1-30 μM for 24 hours. These in vitro studies demonstrate the compound's potential for studying endocytosis and as an anticancer agent.

In vivo, Pitstop 2 has been studied for its potential as an anticancer agent. The compound's ability to inhibit clathrin-mediated endocytosis and induce apoptosis in cancer cells suggests potential therapeutic applications. However, further in vivo studies are needed to evaluate its efficacy and safety.

In vitro binding assay for Pitstop 2 measures its affinity for the clathrin terminal domain. Surface plasmon resonance, fluorescence polarization, or isothermal titration calorimetry are used to measure binding affinity. The compound's ability to inhibit clathrin-mediated endocytosis is assessed in cell-based endocytosis assays, such as transferrin uptake assays. IC50 values are calculated from dose-response curves.

Cell Viability Assay[2]
Cell Types: HeLa cells
Tested Concentrations: 1, 3, 10, 30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: decreased the total number of viable HeLa cells markedly in a dose-dependent manner.
In vitro cellular studies are conducted using various cancer cell lines. Cells are treated with Pitstop 2 at various concentrations (typically 1-30 μM) for defined time periods (e.g., 24 hours). Clathrin-mediated endocytosis is measured using fluorescent or radioactive endocytosis markers (e.g., transferrin, EGF). Cell viability is assessed using MTT or CellTiter-Glo assays. Apoptosis is measured by Annexin V/PI staining or caspase-3 activity assays. Cell cycle analysis is performed by flow cytometry.

In vivo animal experiments with Pitstop 2 are limited. The compound is being studied for its potential as an anticancer agent. Further in vivo studies are needed to evaluate its efficacy and safety in animal models of cancer.

Pharmacokinetic properties of Pitstop 2 include solubility in DMSO. The compound has a molecular weight of 473.36 g/mol and molecular formula C20H13BrN2O3S2. Purity is typically ≥98%. Storage conditions should follow the manufacturer's recommendations. The compound should be protected from light and moisture. For in vivo administration, appropriate formulations should be used.

Toxicological data for Pitstop 2 are derived from preclinical studies. The compound is for research use only and not intended for human therapeutic applications. No significant toxicity has been reported at effective doses in vitro. Standard safety precautions should be followed when handling the compound. Long-term toxicity and carcinogenicity studies have not been conducted.

[1]. Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis. Sci Rep. 2019 Sep 2; 9(1): 12636.

[2]. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells. Mol Cancer. 2013 Jan 17; 12:4.

Leishmania donovani is an intracellular parasite that causes visceral leishmaniasis, a leading cause of death and disease worldwide. The host cell membrane serves as the gateway for Leishmania entry into the cell. Although studies have shown that multiple host cell membrane receptors are involved in the entry of Leishmania donovani into host cells, its endocytic pathway remains unclear. This study explored the endocytic pathway of Leishmania donovani into host macrophages using specific inhibitors targeting two major endocytic pathways: clathrin-mediated endocytosis and caveolin-mediated endocytosis. The results showed that the clathrin-mediated endocytosis inhibitor pitstop 2 did not affect the entry of Leishmania donovani proflagellates into host macrophages. Interestingly, treatment with genistein (an inhibitor of caveolin-mediated endocytosis) significantly reduced endocytosis. A similar trend of no flagellate load in host macrophages also supports this finding. These results indicate that Leishmania donovani utilizes caveolin-mediated endocytosis to enter host cells. Our findings provide new insights into the phagocytic mechanisms of Leishmania donovani into host cells and hold promise for developing novel therapies against Leishmaniasis infection. [1]

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Background: During metaphase of mitosis, clathrin stabilizes spindle kinematic (K) fibers. Many antimitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of the terminal domain of clathrin to inhibit clathrin-mediated endocytosis. We investigated its effect on the second function of clathrin in mitosis.
Results: Pitstop 2 did not affect the recruitment of clathrin to the spindle, but disrupted its post-localization function. Pitstop 2 arrested HeLa cells in metaphase by disrupting the integrity of the mitotic spindle and activating spindle assembly checkpoints, with a phenotype similar to clathrin depletion and Aurora A kinase inhibition.
Conclusion: Therefore, Pitstop 2 is a novel tool for studying clathrin spindle dynamics. Pitstop 2 reduced the viability of dividing HeLa cells but did not affect dividing non-cancerous NIH3T3 cells, suggesting that clathrin may be a novel antimitotic drug target. [2]
Pitstop 2 is also known as Pitstop2 and (Z)-N-(5-(4-Bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphthalene-1-sulfonamide. It is a clathrin inhibitor that inhibits clathrin-mediated endocytosis by associating with the clathrin terminal domain. Pitstop 2 has the potential for anticancer research and induces apoptosis and inhibits cell growth in dividing cancer cells. The compound is used in research on endocytosis, HIV entry, synaptic function, and cancer. It has an IC50 of 12 μM for clathrin terminal domain inhibition.

C20H13BRN2O3S2

473.362821340561

471.955

1419320-73-2

Clathrin-IN-1;1332879-52-3

136246422

Yellow to brown solid powder

5.5

1

5

3

28

767

0

C1(C=CC=C2C=CC=CC=12)S(=O)(=O)NC1S/C(=C\C2C=CC(Br)=CC=2)/C(=O)N=1

CGDLWHGPJPVPDU-ATVHPVEESA-N

InChI=1S/C20H13BrN2O3S2/c21-15-10-8-13(9-11-15)12-17-19(24)22-20(27-17)23-28(25,26)18-7-3-5-14-4-1-2-6-16(14)18/h1-12H,(H,22,23,24)/b17-12-

(NZ)-N-[(5Z)-5-[(4-bromophenyl)methylidene]-4-oxo-1,3-thiazolidin-2-ylidene]naphthalene-1-sulfonamide

Pitstop 2; Clathrin-IN-1; (NZ)-N-[(5Z)-5-[(4-bromophenyl)methylidene]-4-oxo-1,3-thiazolidin-2-ylidene]naphthalene-1-sulfonamide; 1419093-54-1; (Z)-N-(5-(4-Bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphthalene-1-sulfonamide; 1419320-73-2; 4g55; 1332879-52-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 62.5 mg/mL (132.03 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)