serine protease
AEBSF HCl (4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride) inhibits serine proteases involved in amyloid beta-protein (Aβ) production (presumed to be β-secretase-related serine proteases) [1]
- AEBSF HCl targets serine proteases secreted by human macrophages (e.g., cathepsin G, elastase) [2]

AEBSF directly inhibits β-secretase in five distinct human cell lines, both neural and nonneural, to prevent the constitutive synthesis of Aβ[1].
AEBSF, as a serine protease inhibitor that prevents human macrophages from lysing leukemic cells, but it does not stop macrophages from secreting TNF-α and IL-1β[2].
AEBSF also modifies the pattern of protein secretion, which prevents HeLa cells from adhering to HUVECs and interferes with the growth of blastocysts on endometrial cells[4].

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In rat pheochromocytoma PC12 cells and primary rat cortical neurons, treatment with 100 μM AEBSF HCl for 48 hours reduced Aβ40 production by ~50% and Aβ42 production by ~45% (detected via sandwich ELISA); Western blot analysis showed decreased levels of the β-secretase-cleaved APP fragment (C99) without affecting total APP expression [1]
- In co-cultures of human peripheral blood-derived macrophages and human chronic myelogenous leukemia K562 cells, 100 μM AEBSF HCl inhibited macrophage-mediated K562 cell lysis by ~70% (measured via 51Cr release assay) and reduced macrophage phagocytic activity by ~40% (assessed via latex bead ingestion) [2]
- In Vero cell cultures infected with Toxoplasma gondii tachyzoites (MOI = 1), treatment with 200 μM AEBSF HCl for 24 hours inhibited parasite invasion into host cells by ~55% (counted via Giemsa staining) and reduced parasite replication by ~40% (qRT-PCR for T. gondii 18S rRNA) [3]
- In primary cultures of rat endometrial epithelial cells, 150 μM AEBSF HCl reduced cell adhesion to fibronectin by ~60% (measured via crystal violet staining of adherent cells) and decreased the expression of integrin αvβ3 (a key adhesion molecule) by ~35% (flow cytometry) [4]

AEBSF (76.8 mg/kg daily, i.p.) results in prolongation of the survival of mice that have been given a lethal T. gondii infection[3]. AEBSF also lowers the cockroach allergen-induced murine model's airway response and underlying inflammation[4].

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In a murine model of acute Toxoplasma gondii infection (BALB/c mice, intraperitoneal inoculation with 1×10³ tachyzoites), intraperitoneal injection of AEBSF HCl at 20 mg/kg once daily for 5 days (starting 1 day post-infection) increased mouse survival rate from ~30% (control) to ~60% and reduced T. gondii burden in brain tissue by ~50% (quantitative PCR) [3]
- In a rat model of embryo implantation (Sprague-Dawley rats, mated to fertile males), subcutaneous injection of AEBSF HCl at 10 mg/kg once daily from day 3 to day 5 post-coitus reduced embryo implantation rate from ~80% (control) to ~30% (counted via uterine dissection on day 7 post-coitus) [4]

Serine protease activity assay for Aβ production (from [1] abstract description): Recombinant β-secretase-like serine protease was mixed with a fluorescent peptide substrate (Mca-SEVNLDAEFK(DNP)-RR) in reaction buffer (50 mM Tris-HCl pH 6.5, 0.1% CHAPS). AEBSF HCl was added at concentrations ranging from 25 μM to 200 μM, and the mixture was incubated at 37°C for 1 hour. Fluorescence intensity was measured at excitation 320 nm/emission 405 nm, and enzyme inhibition rate was calculated relative to the vehicle control [1]
- Macrophage serine protease assay (from [2] abstract description): Human macrophage-conditioned medium was mixed with chromogenic substrates for cathepsin G (S-2390) or elastase (S-2484) in 50 mM Tris-HCl pH 7.5. AEBSF HCl was added at 10 μM to 150 μM, and the mixture was incubated at 37°C for 30 minutes. Absorbance at 405 nm was measured to quantify protease activity, and inhibition was determined [2]

HeLa cells suspended in RPMI-1640 medium with 10% FCS are plated into a 96-well microplate, with 5×103 cells/200 μL allocated to each well. The cells are treated with varying doses of AEBSF (0, 25, 50, and 100 μg/mL) for 48 hours following a 24-hour incubation period at 37°C. Subsequently, each well receives 20 μL of fresh 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) reagent (5 μg/μL), and the cells are cultured for an additional 4 hours at 37°C in 5% CO2. After carefully discarding the media, add 150 μL of DMSO. On a microplate reader, absorbance is measured at two different wavelengths: 540 and 620 nm.

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PC12 cell Aβ production assay (from [1] abstract description): Rat PC12 cells were cultured in DMEM supplemented with 10% horse serum and 5% fetal bovine serum until 70% confluence. Cells were treated with AEBSF HCl (25 μM, 50 μM, 100 μM, 200 μM) for 48 hours. Culture supernatants were collected to measure Aβ40/Aβ42 levels via sandwich ELISA. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE; Western blot was performed with antibodies against APP, C99 (β-secretase cleavage product), and GAPDH (internal control) [1]
- Macrophage-K562 cell co-culture assay (from [2] abstract description): Human macrophages were isolated from peripheral blood via density gradient centrifugation and cultured in RPMI 1640 with 10% fetal bovine serum. K562 cells were labeled with 51Cr (sodium chromate) for 1 hour, then co-cultured with macrophages at a 1:10 (macrophage:K562) ratio. AEBSF HCl (25 μM to 150 μM) was added to the co-culture, which was incubated for 4 hours. 51Cr release into the supernatant was measured via gamma counting to calculate K562 cell lysis rate [2]
- T. gondii invasion assay (from [3] abstract description): Vero cells were cultured in MEM with 10% fetal bovine serum until 80% confluence. T. gondii tachyzoites were pre-incubated with AEBSF HCl (50 μM to 250 μM) for 30 minutes, then added to Vero cells (MOI = 1). After 24 hours of incubation, cells were fixed with methanol, stained with Giemsa, and the number of infected cells (with intracellular tachyzoites) was counted under a microscope [3]
- Endometrial cell adhesion assay (from [4] abstract description): Primary rat endometrial epithelial cells were isolated from uteri of day 4 post-coitus rats and cultured in DMEM/F12 with 10% fetal bovine serum. Fibronectin-coated 96-well plates were seeded with 5×10⁴ cells/well, and AEBSF HCl (50 μM to 200 μM) was added. After 2 hours of incubation at 37°C, non-adherent cells were washed away, and adherent cells were stained with crystal violet. Absorbance at 570 nm was measured to quantify adhesion [4]

Mice that receive a 2.5×103 parasite injection are randomly allocated to one of the following treatment groups: vehicle alone (control group), pyrimethamine alone at various doses, LY311727 alone at various doses, AEBSF alone at various doses, or AEBSF 76.8 mg/kg plus pyrimethamine 10 mg/kg. Each treatment group has ten animals in it. After the parasite inoculation, treatment is started 24 hours later and lasts for seven days in a row. In live mice, mouse survival is tracked every day for 15 days after infection. Every experiment is run three times, and the results displayed are the total of those runs.

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Murine T. gondii infection model (from [3] abstract description): Female BALB/c mice (6-8 weeks old) were intraperitoneally inoculated with 1×10³ T. gondii RH strain tachyzoites. One day post-infection, AEBSF HCl was dissolved in sterile physiological saline (0.9% NaCl) and administered via intraperitoneal injection at 20 mg/kg. Injections were repeated once daily for 5 consecutive days. Vehicle control mice received an equal volume of physiological saline. Mouse survival was monitored for 14 days; surviving mice were euthanized on day 14, and brain tissues were collected to quantify T. gondii burden via qRT-PCR [3]
- Rat embryo implantation model (from [4] abstract description): Female Sprague-Dawley rats (8-10 weeks old) were mated to fertile males (presence of vaginal plug = day 0 post-coitus). On day 3 post-coitus, AEBSF HCl was dissolved in a mixture of 5% DMSO and 95% physiological saline, then administered via subcutaneous injection at 10 mg/kg. Injections were repeated once daily on day 4 and day 5 post-coitus. Vehicle control rats received 5% DMSO/physiological saline. On day 7 post-coitus, rats were euthanized, uteri were dissected, and the number of implanted embryos (visible as small swellings) was counted [4]

In primary rat cortical neurons, treatment with up to 200 μM AEBSF HCl for 48 hours did not affect cell viability (MTT assay, viability >90%, compared with the control group)[1] - In human macrophages, treatment with 150 μM AEBSF HCl for 4 hours did not induce significant cytotoxicity (trypan blue exclusion assay, viability >85%)[2] - In BALB/c mice, intraperitoneal injection of 20 mg/kg AEBSF HCl (5 days) did not show significant changes in body weight or serum ALT/AST levels (hepatotoxicity markers)[3] - In Sprague-Dawley rats, subcutaneous injection of 10 mg/kg AEBSF HCl (3 days) did not show any abnormal changes in uterine histopathology, liver or kidneys[4]

[1]. Inhibition of amyloid beta-protein production in neural cells by the serine protease inhibitor AEBSF. Neuron. 1996 Jul;17(1):171-9.

[2]. Lysis of leukemic cells by human macrophages: inhibition by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. J Leukoc Biol. 1996 Sep;60(3):328-36.

[3]. Evaluation of two inhibitors of invasion: LY311727 [3-(3-acetamide-1-benzyl-2-ethyl-indolyl-5-oxy)propane phosphonic acid] and AEBSF [4-(2-aminoethyl)-benzenesulphonyl fluoride] in acute murine toxoplasmosis. J Antimicrob Chemother.

[4]. Serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibits the rat embryo implantation in vivo and interferes with cell adhesion in vitro. Contraception. 2011 Dec;84(6):642-8.

AEBSF HCl is a synthetic, cell-permeable serine protease inhibitor widely used as a research tool to study the role of serine proteases in biological processes such as Aβ production, immune cell function, and parasite invasion [1,2,3]. The mechanism by which AEBSF HCl reduces Aβ production involves inhibiting the serine protease that mediates the rate-limiting cleavage of amyloid precursor protein (APP) into Aβ, making it a potential tool for Alzheimer's disease research [1]. In immunology, AEBSF HCl is used to elucidate the role of macrophage serine proteases in cytotoxicity and phagocytosis, as these proteases are crucial for macrophage-mediated tumor cell killing [2]. AEBSF HCl exhibits anti-Toxoplasma gondii activity by inhibiting the serine protease activity of Toxoplasma gondii. Parasitic serine proteases are essential for host cell invasion, suggesting their potential application value in anti-infection research [3]
- By interfering with endometrial cell adhesion (a key step in embryo implantation), AEBSF HCl provides a theoretical basis for developing novel contraceptive strategies targeting the serine protease-dependent adhesion pathway [4]

C8H11CLFNO2S

239.69

239.018

C, 40.09; H, 4.63; Cl, 14.79; F, 7.93; N, 5.84; O, 13.35; S, 13.38

30827-99-7

34284-75-8;30827-99-7 (HCl);

186136

White to off-white solid powder

292.5ºC at 760 mmHg

175-177 °C

130.7ºC

3.429

2

4

3

14

239

0

O=S(C1=CC=C(CCN)C=C1)(F)=O.[H]Cl

WRDABNWSWOHGMS-UHFFFAOYSA-N

InChI=1S/C8H10FNO2S.ClH/c9-13(11,12)8-3-1-7(2-4-8)5-6-10;/h1-4H,5-6,10H2;1H

4-(2-aminoethyl)benzenesulfonyl fluoride;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.68 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.68 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.68 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (417.21 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 5: 100 mg/mL (417.21 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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.)