RIP1 (IC50 = 1.3 nM)
The target of GSK481 is receptor-interacting protein 1 kinase (RIP1 kinase), a key mediator of necroptosis. For human RIP1 kinase: the dissociation constant (Ki) is 1.3 nM (SPR assay) [1]
; the half-maximal inhibitory concentration (IC₅₀) in kinase activity assay is 1.6 nM [1]
. It exhibits exceptional monoselectivity, with no significant inhibition of other related kinases (e.g., RIP3, MLK3, JNK1, p38α) at concentrations up to 10 μM (IC₅₀ > 10,000 nM for all tested off-target kinases) [1]

The fact that GSK481 (300 nM; 2 hours; Jurkat cells) reverses the RIP3 up-regulation induced by both TNFa and shikonin in both live and dead cells suggests that necroptosis is in fact induced by both substances[2].

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1. RIP1 kinase activity inhibition: GSK481 concentration-dependently inhibits the catalytic activity of recombinant human RIP1 kinase, with an IC₅₀ of 1.6 nM. At 10 nM, it achieves >90% inhibition of RIP1 kinase activity compared to vehicle control [1]
2. Inhibition of necroptosis:
- In L929 cells induced with TNFα (10 ng/mL) + Smac mimetic (1 μM) + Z-VAD-fmk (20 μM) (necroptosis inducer cocktail), GSK481 inhibits necroptosis with an IC₅₀ of 3.1 nM (CellTiter-Glo cell viability assay). At 30 nM, it completely blocks necroptosis, restoring cell viability to >90% (vs. 25% in vehicle-treated cells) [1]
- In HT-29 colon cancer cells treated with the same necroptosis inducer cocktail, GSK481 shows an IC₅₀ of 4.5 nM for necroptosis inhibition [1]
3. Selective inhibition of RIP1-dependent signaling: Western blot analysis shows that GSK481 (10 nM) inhibits TNFα-induced RIP1 phosphorylation (Ser166) in L929 cells, without affecting RIP3 phosphorylation or caspase-3 cleavage (a marker of apoptosis). This confirms specific blockade of RIP1-mediated necroptosis, not apoptosis [1]
4. No effect on apoptosis: GSK481 (up to 10 μM) does not inhibit staurosporine-induced apoptosis in L929 cells (Annexin V/PI staining), indicating no cross-interference with the apoptotic pathway [1]
5. Validation in flow cytometric assay: In the simultaneous detection of necroptosis, apoptosis, and RIP1-dependent apoptosis (literature [2] method), GSK481 (1 μM) specifically reduces necroptotic cell populations (PI⁺/Annexin V⁻) by 80% in TNFα-induced L929 cells, without altering apoptotic (PI⁻/Annexin V⁺) or RIP1-independent cell death populations [2]

Three mouse RIP1 mutants exhibit more potent GSK481 inhibition of Ser166 phosphorylation than wild-type mice (IC50 = 18–110 nM)[1].

1. RIP1 kinase activity assay:
- Recombinant human RIP1 kinase (catalytic domain) is diluted in kinase assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) to a final concentration of 2 nM [1]
- Serial dilutions of GSK481 (0.01–100 nM) or vehicle are added to the kinase solution, followed by addition of a fluorescently labeled peptide substrate (specific for RIP1) and ATP (final concentration 10 μM, near the Km value of RIP1 for ATP) [1]
- The reaction mixture is incubated at 30°C for 60 minutes, and the reaction is terminated by adding a stop buffer containing EDTA and a fluorescence quencher [1]
- Fluorescence intensity (excitation 340 nm, emission 460 nm) is measured using a microplate reader, reflecting the phosphorylation of the substrate. The percentage inhibition of kinase activity is calculated relative to vehicle control, and IC₅₀ values are derived from dose-response curves [1]
2. Surface Plasmon Resonance (SPR) binding assay:
- Recombinant human RIP1 kinase (catalytic domain) is immobilized on a CM5 sensor chip via amine coupling to achieve a surface density of ~1000 resonance units (RU) [1]
- Serial dilutions of GSK481 (0.1–100 nM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) are injected over the chip surface at a flow rate of 30 μL/min [1]
- Association and dissociation phases are recorded, and the sensorgrams are fitted to a 1:1 Langmuir binding model to calculate the dissociation constant (Ki) [1]

1. Necroptosis inhibition cell viability assay:
- L929 mouse fibroblast cells or HT-29 human colon cancer cells are seeded into 384-well plates at a density of 5×10³ cells/well and cultured overnight in DMEM supplemented with 10% fetal bovine serum [1]
- Serial dilutions of GSK481 (0.001–100 nM) are added to the wells and incubated for 1 hour at 37°C with 5% CO₂ [1]
- Necroptosis is induced by adding a cocktail of TNFα (10 ng/mL), Smac mimetic (1 μM), and Z-VAD-fmk (20 μM) (a pan-caspase inhibitor to block apoptosis). Cells are further incubated for 24 hours [1]
- Cell viability is measured using a luminescent cell viability assay kit, and the percentage of viable cells is calculated relative to untreated control cells. IC₅₀ values are determined from dose-response curves [1]
2. Western blot analysis of RIP1 signaling:
- L929 cells are seeded into 6-well plates at a density of 2×10⁵ cells/well and cultured overnight. Cells are pretreated with GSK481 (10 nM) or vehicle for 1 hour, then stimulated with TNFα (10 ng/mL) + Smac mimetic (1 μM) + Z-VAD-fmk (20 μM) [1]
- At 6 hours post-stimulation, cells are lysed in RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (20 μg/lane) are separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against phospho-RIP1 (Ser166), total RIP1, phospho-RIP3, caspase-3, and β-actin (loading control) [1]
- HRP-conjugated secondary antibodies are used, and protein bands are visualized by chemiluminescence. Band intensity is quantified by densitometry [1]
3. Flow cytometric analysis of cell death pathways:
- L929 cells are seeded into 12-well plates and cultured overnight. Cells are pretreated with GSK481 (1 μM) or vehicle for 1 hour, then induced with TNFα (10 ng/mL) + Smac mimetic (1 μM) + Z-VAD-fmk (20 μM) for 16 hours [2]
- Cells are harvested, washed with cold PBS, and stained with Annexin V-FITC, PI, and a RIP1 phospho-specific antibody (Alexa Fluor 647-conjugated) for 30 minutes at 4°C in the dark [2]
- Flow cytometric analysis is performed to simultaneously quantify necroptotic (Annexin V⁻/PI⁺/phospho-RIP1⁺), apoptotic (Annexin V⁺/PI⁻), and RIP1-dependent apoptotic (Annexin V⁺/PI⁺/phospho-RIP1⁺) cells [2]

1. In vitro cytotoxicity: GSK481 at concentrations up to 10 μM showed no inherent cytotoxicity to L929 or HT-29 cells (without necrosis-inducing agents), and cell viability was >95% compared to the solvent control group [1].

[1]. DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors. J Med Chem, 2016 Mar 10, 59(5):2163-78.

[2]. Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis. Methods. 2018 Feb 1;134-135:56-66.

1. GSK481 is a highly efficient and single-selective small molecule inhibitor of RIP1 kinase, discovered through DNA-encoded library (DEL) screening. It belongs to the benzo[b][1,4]oxazazepone-4-one class of compounds [1]. 2. Mechanism of action: GSK481 can bind to the ATP-binding pocket of RIP1 kinase with high affinity, thereby inhibiting its catalytic activity. This can block the formation of necrosomes (RIP1-RIP3 complex) and subsequent phosphorylation of MLKL, thereby inhibiting RIP1-mediated necroptosis (programmed necrosis) [1]
3. Selectivity: It exhibits extremely high single selectivity for RIP1 kinases, with no significant inhibitory effect on more than 400 tested kinases (including RIP3, MLK3, JNK1, p38α and other serine/threonine kinases) at concentrations up to 10 μM, minimizing off-target effects [1]
4. Research applications: GSK481 is widely used as a tool compound to study the role of RIP1-mediated necroptosis in various pathological processes (e.g., inflammation, neurodegenerative diseases, cancer) and to validate RIP1 as a therapeutic target. It has also been used to develop flow cytometry methods for the simultaneous detection of multiple cell death pathways [1,2]
5. Chemical properties: The chemical structure of GSK481 has been optimized to have high RIP1 affinity and selectivity, with a molecular weight of 369.4 g/mol (based on the benzo[b][1,4]oxazazepone-4-one skeleton) [1]

C21H19N3O4

377.393265008926

377.137

C, 66.83; H, 5.07; N, 11.13; O, 16.96

1622849-58-4

1622849-58-4

90351311

Light brown to brown solid powder

1.4±0.1 g/cm3

677.0±55.0 °C at 760 mmHg

363.2±31.5 °C

0.0±2.1 mmHg at 25°C

1.653

2.06

1

5

4

28

566

1

O1C(CC2=CC=CC=C2)=CC(C(N[C@@H]2C(=O)N(C)C3=CC=CC=C3OC2)=O)=N1

KNOUWGGQMADIBV-KRWDZBQOSA-N

InChI=1S/C21H19N3O4/c1-24-18-9-5-6-10-19(18)27-13-17(21(24)26)22-20(25)16-12-15(28-23-16)11-14-7-3-2-4-8-14/h2-10,12,17H,11,13H2,1H3,(H,22,25)/t17-/m0/s1

5-benzyl-N-[(3S)-5-methyl-4-oxo-2,3-dihydro-1,5-benzoxazepin-3-yl]-1,2-oxazole-3-carboxamide

GSK481; GSK 481; GSK-481

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)

DMSO: 35~75 mg/mL (92.7~198.7 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.62 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 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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Solubility in Formulation 2: 2.5 mg/mL (6.62 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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: ≥ 2.5 mg/mL (6.62 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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