clathrin

HeLa cells treated with Clathrin-IN-1 (Pitstops 2) prior to incubation exhibit a dose-dependent reduction in Tf uptake, with an IC50 value of 12–5 μM. When 30 μM Clathrin-IN-1 was applied, Tf endocytosis was totally inhibited. After one to three hours of drug washout, the HeLa cells' clathrin-IN-1-induced inhibition of Tf endocytosis was fully restored. The Tf uptake IC50 in U2OS cells is 9.7 μM. Additionally, Pitstop 2 strongly inhibits the absorption of EGF[1]. In HeLa cells, clathrin-IN-1 (Pitstops 2) effectively and selectively decreased HIV-1 infectivity by more than 90%[1]. Pitstop-induced clathrin TD function suppression abruptly disrupts HIV entry, receptor-mediated endocytosis, and synaptic vesicle recycling. The durations of clathrin coat components, such as FCHo, clathrin, and dynamin, dramatically increase during endocytosis inhibition, indicating that clathrin TD governs coated pit dynamics[1].

ELISA-Based Clathrin Inhibitor Binding Assay [2]
The clathrin inhibitor assay was based on that of our previous report. Bacterially expressed recombinant purified His6-tagged amphiphysin 1 (amino acids 250–578) in screening buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM DTT, 1 mM PMSF) was added to a 384 well ELISA plate (high-binding PS Microplate) and bound to the plastic for 1 h at room temperature. Nonspecific binding was reduced by overnight incubation with 50 μL blocking buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM PMSF, 2% BSA, 2.5% skim milk) at 4 °C. Following extensive washes (with 20 mM HEPES, pH 7.4, 50 mM NaCl, 0.05% Tween 20), chemical compounds diluted in DMSO (10 μL) were added and incubated together with bacterially expressed recombinant GST-tagged clathrin heavy chain TD (amino acids 1–364) for 1 h at room temperature in screening buffer. After three washes, horseradish peroxidase-coupled anti-GST antibodies were added in screening buffer, and the plate was incubated for 15 min at room temperature. Following additional washes, 50 μL of TMB (3,3′,5,5′-tetramethylbenzidine) chromogenic substrate of horseradish peroxidase was added, and the plate was incubated for 20 min before the reaction was terminated by adding 50 μL of 1 N sulfuric acid to produce a yellow color. The amount of bound protein was determined by spectrophotometric measurement in a plate reader. Relative binding was calculated as a percentage of DMSO control.

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Dynamin GTPase Assay [2]
Native dynamin I was purified from sheep brain by extraction from the peripheral membrane fraction of whole brain and affinity purification on GST-Amph2-SH3-sepharose as previously described. The GTPase assay was based on colorimetric detection of phosphate release from GTP using the Malachite Green method as previously described. except that the GTPase assay buffer contained 5 mM Tris-HCl, 10 mM NaCl, 2 mM Mg2+, pH 7.4, 1 μg/mL leupeptin, 0.1 mM PMSF, and 0.3 mM GTP. Dynamin (20 nM) activity was stimulated by 4 μg/mL sonicated phosphatidylserine in the presence of test compounds for 30 min at 37 °C.

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Screening Procedure [1]
Seventeen thousand small molecules (100 μM) from the central open access technology platform of the ChemBioNet hosted by FMP (http://fmp-berlin.info/screening_unit.html) were screened using an automated platform for their ability to perturb clathrin TD-amphiphysin association using an ELISA-based assay in a 384-well format (see below for details). Forty-eight initial hits (>80% inhibition) were selected. Ten of these hits could be validated by dose-response analysis (at 3–300 μM) and structural hit clustering. Hits were verified. Two lead compounds were selected based on the absence of off-target effects and inhibition of transferrin endocytosis. Focused libraries synthesized based on these leads were re-screened as above resulting in the identification of pitstops 1 and 2.

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ELISA-Based Binding Assay [1]
Purified His6-tagged protein was diluted into screening buffer (20 mM HEPES [pH 7.4], 50 mM NaCl, 1 mM DTT, 1 mM PMSF), added to a 384-well ELISA plate (high-binding PS Microplate) and bound for 1 hr at RT. Nonspecific binding was blocked by incubation in 50 μl blocking buffer (20 mM HEPES [pH 7.4], 50 mM NaCl, 1 mM DTT, 1 mM PMSF, 2% BSA, 2.5% milk) overnight at 4°C. Following extensive washes with 20 mM HEPES (pH 7.4), 50 mM NaCl, 0.05% Tween 20, chemical compounds diluted in DMSO (10 μl) were added and incubated together with GST-tagged protein for 1 hr at RT in screening buffer. After three washes, HRP-coupled anti-GST antibodies were added in screening buffer and the plate was incubated for 15 min at RT. Following additional washes, bound protein was determined by photometric measurement in a plate reader at 450 nm using TMB as a substrate.

Cell-Cycle Analysis by Flow Cytometry [1]
Cells (5 × 105) were grown in 10 cm dishes. Following inhibitor treatment, cells (floating and adherent) were collected and single-cell suspensions were fixed in 80% ice-cold ethanol at −20°C for at least 16 hr. Cells were stained with propidium iodide and the cell cycle was analyzed as described previously (Joshi et al., 2010). Cell cycle profiles were acquired with a FACS Canto Flow Cytometer using FACS Diva software (v5.0.1) at 488 nm. Cell cycle profiles were analyzed using FlowJo software (v7.1).

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Trypan Blue Exclusion Assay [1]
Cells were seeded in 10 cm dishes (1 × 105 cells/dish). On day 0 (24 hr after seeding), cells in triplicate were treated in the presence or absence of pitstops at concentrations of 1, 3, 10, and 30 μM. After 20 hr, the cell number and viability were measured using a Vi-CELL XR cell viability analyzer as previously described (Joshi et al., 2010).

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Lactate Dehydrogenase Toxicity Assay [1]
Toxicity was assayed by determination of lactate dehydrogenase (LDH) activity. HeLa cells were seeded in 96-well plates. Asynchronously growing cells were treated in the presence or absence of pitstops at the indicated concentration for 8h. The supernatant (50 μl) was added to 100 μl of LDH assay reagent (Sigma-Aldrich) and the reaction was allowed to develop for 20 min. Absorbance was measured at 490 nm and 690 nm (plate background absorbance). Values were normalized to drug/media background value and toxicity was calculated as a % of a 100% lysed cell control.

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MTT Assay [1]
Growth inhibitory assays were carried out as described previously.4,5 Cells in logarithmic growth were transferred to 96-well plates (100 μl medium/well) at a density of 2500 cells/well for HeLa, HT29, H460, A431, and DU145 cells, 3000 cells/well for SW480, 3500 cells/well for MCF7, BE2-C and SJ-G2, and 2000 cells/well for A2780. On day 0 (24 hr after plating), cells in duplicate were treated with or without pitstops. After 72 hr drug exposure, cytotoxicity and growth inhibitory effects were evaluated using the MTT (3-[4,5-dimethyltiazol-2-yl] 2,5-diphenyl-tetrazolium bromide) assay. GI50 values were calculated from the MTT dose response curve from three independent experiments, each performed in duplicate. GI50 is the drug concentration at which cell growth is inhibited by 50% based on the difference between the optical density values on day 0 and those at the end of drug exposure.

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CME Assay (Texas red-Tf Uptake) [2]
Tf uptake was analyzed in U2OS cells based on methods previously described. Cells were exposed to test inhibitors (from 1 to 300 μM) or vehicle for 30 min prior to addition of 4 μg/mL Tf-A594 for 8 min at 37 °C. Quantitative analysis of the inhibition of TxR-Tf endocytosis in U2OS cells was performed on large numbers of cells by an automated acquisition and analysis system. The average number of cells for each data point was ∼1200. IC50 values were calculated using Graphpad Prism v5 and data were expressed as mean ±95% confidence interval (CI) for three wells and ∼1200 cells.

[1]. Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell. 2011 Aug 5;146(3):471-84.

[2]. Development of 1,8-naphthalimides as clathrin inhibitors. J Med Chem. 2014 Jan 9;57(1):131-43.

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.[1]

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Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.[2]

C19H13N2NAO5S

404.37

404.044

C, 56.44; H, 3.24; N, 6.93; Na, 5.69; O, 19.78; S, 7.93

1332879-51-2

Pitstop 2;1419320-73-2

71483522

Typically exists as solid at room temperature

1

6

3

28

716

0

S(C1C=C2C=CC=C3C(N(CC4C=CC(=CC=4)N)C(C(C=1)=C32)=O)=O)(=O)(=O)[O-].[Na+]

MKPPGLKJTZAXNE-UHFFFAOYSA-M

InChI=1S/C19H14N2O5S.Na/c20-13-6-4-11(5-7-13)10-21-18(22)15-3-1-2-12-8-14(27(24,25)26)9-16(17(12)15)19(21)23;/h1-9H,10,20H2,(H,24,25,26);/q;+1/p-1

sodium;2-[(4-aminophenyl)methyl]-1,3-dioxobenzo[de]isoquinoline-5-sulfonate

ClathrinIN1; pitstop 1; 1332879-51-2; sodium;2-[(4-aminophenyl)methyl]-1,3-dioxobenzo[de]isoquinoline-5-sulfonate; Clathrin inhibitor 1; GLXC-04355; sodium 2-(4-aminobenzyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-sulfonate; Clathrin IN 1; Clathrin-IN-1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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