RIP1 kinase (IC50 = 29 nM)

GSK-963 inhibits RIP1-dependent cell death in human and murine cells with an IC50 range of 1 to 4 nM[1].
GSK′963 is a potent and selective inhibitor of RIP1 kinase in biochemical assays. GSK′963 is a selective and potent inhibitor of necroptosis in murine and human cells in vitro.

GSK-963 is a powerful inhibitor of a lethal shock caused by TNF+zVAD. In comparison to Nec-1, GSK-963 would maintain blood concentrations above the level necessary for 90% inhibition of RIP1 activity for a longer period of time at 2 mg/kg.

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GSK′963 is a potent inhibitor of a TNF+zVAD-mediated lethal shock: researchers assessed the pharmacokinetic profiles of GSK′963 and Nec-1 following intraperitoneal (i.p.) administration in vivo. Nec-1 demonstrated an ~10-fold higher exposure compared with GSK′963 at 10 mg/kg, although the apparent half-life of GSK′963 was greater than that for Nec-1 (Figure 3a; Supplementary Figure 1a). However, pharmacodynamic modeling of both compounds based on the mouse pharmacokinetic profiles (Figure 3a; Supplementary Figure 1a) and compound potencies in TNF+zVAD-treated L929 cells (Figure 2a) indicated that at 2 mg/kg, GSK′963 would maintain blood concentrations above the concentration required for 90% inhibition of RIP1 activity for an extended period of time compared with Nec-1 (Figure 3b; Supplementary Figure 1b). To directly test the efficacy of GSK′963 in vivo, we made use an acute model of sterile shock. Administration of TNF+zVAD results in a lethal hypothermia that has previously been shown to be dependent on RIP1 kinase activity.5,18 Treatment of animals with 2 mg/kg of GSK′963 resulted in a complete protection from TNF+zVAD-induced temperature loss, with the 0.2 mg/kg dose also showing a significant response (Figure 3c). As expected, GSK′962 had no effect on the TNF+zVAD-induced shock, confirming that GSK′963 was acting selectively through RIP1 kinase inhibition (Figure 3d). Interestingly, Nec-1 had no effect in the model at 0.2 mg/kg, a dose that is commonly used to inhibit RIP1 in vivo in the literature, and showed a minimal level of protection at a 10-fold higher dose (Figure 3e). Together these results demonstrate that compared with Nec-1, GSK′963 represents a better tool molecule to explore acute RIP1 kinase biology in vivo[1].

Compound potency against RIP1 kinase activity was determined using an ADP-Glo luminescence assay, which measures the conversion of ATP to ADP as previously described. In brief, the primary reaction consisted of 10 nM GST-RIPK1 (1–375) and 50 μM ATP in 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.5 mg/ml BSA, and 0.02% CHAPS. Five microliter of enzyme and 5 μl of ATP were added to the plate at twice the final assay concentration and incubated at room temperature for 4 h. The luminescence was measured on a plate reader. Test compound inhibition was expressed as percent inhibition of internal assay controls.

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Fluorescent polarization (FP) binding assay[1]
A FP-based binding assay was developed to quantitate interaction of novel test compounds at the ATP-binding pocket of RIP1, by competition with a fluorescently labeled ATP-competitive ligand, as previously described.18 In brief, GST-RipK1 (1–375) was purified and was used at a final assay concentration of 10 nM. A fluorescent-labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo [2“,3“]indolizino[8“,7“:5ʹ,6ʹ]pyrano[3ʹ,2ʹ:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate was used at a final assay concentration of 5 nM. Samples were read on an Analyst multimode reader. Test compound inhibition was expressed as percent (%) inhibition of internal assay controls.

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ADP-Glo kinase assay[1]
Compound potency against RIP1 kinase activity was determined using an ADP-Glo luminescence assay, which measures the conversion of ATP to ADP as previously described.18 In brief, the primary reaction consisted of 10 nM GST-RIPK1 (1–375) and 50 μM ATP in 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.5 mg/ml BSA, and 0.02% CHAPS. Five microliter of enzyme and 5 μl of ATP were added to the plate at twice the final assay concentration and incubated at room temperature for 4 h. The luminescence was measured on a plate reader. Test compound inhibition was expressed as percent inhibition of internal assay controls.

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Kinase selectivity[1]
GSK′963 was tested against 339 kinases using a P33-radiolabeled assay at Reaction Corp Biology (http://www.reactionbiology.com). The compound was tested at a single dose in duplicate at 10 μM. Reactions were carried out at 10 μM ATP. Data are reported as % enzyme activity (relative to DMSO controls). The full data set is shown in the Supplementary Table I.

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IDO enzymatic assay[1]
IDO activity was determined as described by Takahashi et al.25 using recombinant human IDO purchased from R&D Systems. The yellow pigment derived from kynurenine was measured at 490 nm using the Spectramax microplate reader.

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Caspase 3/7 assay[1]
To induce apoptosis, BMDM pretreated with GSK′963 (100 nM), GSK′962 (100 nM) or Nec-1 (10 μM) for 30 min were stimulated with TNF (50 ng/ml) and CHX (12 μg/ml). Caspase 3/7 activity was measured at 6 h using the Caspase-Glo 3/7 assay.

For immunoblot analysis, BMDM are stimulated with 50 ng/ml TNF for 5 and 15 minutes after being pretreated for 30 minutes with GSK'963 (100 nM), GSK'962 (100 nM), or Nec-1 (10 μM). Lysates made with protease and phosphatase inhibitors in 1× Cell Lysis Buffer are separated on 4-12% SDS-PAGE and blotted onto nitrocellulose membrane. IκB, phospho-IκB, and tubulin are probed as loading controls on blots.

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Cell culture[1]
Mouse fibrosarcoma L929 cells (ATCC# CCL-1) and human monocytic U937 cells were cultured in RPMI media supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 U/ml streptomycin. BMDM were prepared from C57BL/6 mice by differentiation with 10 ng/ml M-CSF for 7 days and cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin. Primary human neutrophils were isolated from human blood following the standard method comprised of sequential sedimentation in dextran, density centrifugation in Ficoll–Hypaque and lysis of contaminating red blood cells.

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Cell-based assays[1]
Necroptotic cell death was induced in BMDM, L929 and U937 cells with TNF in the presence of caspase inhibitor zVAD-FMK or QVD-Oph (BMDM: 50 ng/ml TNF+50 μM zVAD; L929: 100 ng/ml TNF+50 μM QVD; U937: 100 ng/ml TNF+25 μM QVD). To evaluate the effect of RIP1 inhibitors, cells were pretreated with compound (dose–response) for 30 min. Induced cell death was evaluated 19–21 h later by measuring cellular ATP levels using CellTiter-Glo Luminescent Cell Viability assay. To induce necroptosis in neutrophils, freshly isolated human neutrophils were stimulated with TNF (10 ng/ml), QVD-Oph (50 μM) and SMAC mimetic (100 nM). Induced cell death was evaluated as above.

Pharmacokinetic experiments[1]
Briefly, pharmacokinetic studies were conducted using n=3 animals per group for each compound. Animals received either GSK′963 or 7-Cl-O-Nec-1 as an i.p. injection prepared as aqueous solutions in 6% 11-b-hydroxypropyl cyclodextrin and contained 5% DMSO. Blood samples were obtained at various time points following intraperitoneal injection and diluted 1 : 1 with water prior to storage at −80 °C.
Prior to bioanalysis, samples were thawed and each analyte was isolated using a method based on protein precipitation. The resulting supernatant was injected into an LC/MS/MS system optimized for detection of the compound of interest. Data were reported as quantitative drug concentrations as determined by standard calibration curve analysis. Using these optimized conditions, the typical lower limit of quantitation achieved was 1.00 ng/ml for both compounds.

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TNF+zVAD-induced shock model[1]
C57BL/6 mice were pretreated i.p. with Nec-1 (0.2 and 2 mg/kg), GSK′963 (0.2 and 2 mg/kg) or GSK′962 (20 mg/kg) 15 min prior to i.v. injection of TNF (1.25 mg/kg) and zVAD-FMK (16.7 mg/kg). Temperature was monitored over the course of 3 h by rectal probe.

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C57BL/6 mice were pretreated i.p. with Nec-1 (0.2 and 2 mg/kg), GSK′963 (0.2 and 2 mg/kg) or GSK′962 (20 mg/kg) 15 min prior to i.v. injection of TNF (1.25 mg/kg) and zVAD-FMK (16.7 mg/kg).

C57BL/6 mice

Researchers next assessed the pharmacokinetic profiles of GSK′963 and Nec-1 following intraperitoneal (i.p.) administration in vivo. Nec-1 demonstrated an ~10-fold higher exposure compared with GSK′963 at 10 mg/kg, although the apparent half-life of GSK′963 was greater than that for Nec-1 (Figure 3a; Supplementary Figure 1a). However, pharmacodynamic modeling of both compounds based on the mouse pharmacokinetic profiles (Figure 3a; Supplementary Figure 1a) and compound potencies in TNF+zVAD-treated L929 cells (Figure 2a) indicated that at 2 mg/kg, GSK′963 would maintain blood concentrations above the concentration required for 90% inhibition of RIP1 activity for an extended period of time compared with Nec-1 (Figure 3b; Supplementary Figure 1b). [1]

[1]. Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase. Cell Death Discov. 2015 Jul 27;1:15009.

Necroptosis and signaling regulated by RIP1 kinase activity is emerging as a key driver of inflammation in a variety of disease settings. A significant amount has been learned about how RIP1 regulates necrotic cell death through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1). Nec-1 has been a transformational tool for exploring the function of RIP1 kinase activity; however, its utility is somewhat limited by moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have driven an effort to identify next-generation tools to study RIP1 function, and have led to the identification of 7-Cl-O-Nec-1 (Nec-1s), which has improved pharmacokinetic properties and lacks IDO inhibitory activity. Here we describe the characterization of GSK′963, a chiral small-molecule inhibitor of RIP1 kinase that is chemically distinct from both Nec-1 and Nec-1s. GSK′963 is significantly more potent than Nec-1 in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK′963 is >10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK′962, which can be used to confirm on-target effects. The increased in vitro potency of GSK′963 also translates in vivo, where GSK′963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK′963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.[1]

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In the present study, we characterize GSK′963, a novel small-molecule inhibitor of RIP1 kinase that is structurally distinct from Nec-1 and Nec-1s. GSK′963 is over 200-fold more potent than Nec-1, displays exquisite selectivity for RIP1 kinase activity and has no effect on IDO activity or on TNF-mediated NFκB activation or apoptosis. GSK′963 is a chiral molecule allowing for use of its chemically identical inactive enantiomer as a negative control to confirm on-target activity of the inhibitor. Furthermore, GSK′963 provides complete protection in an in vivo model of TNF-induced sterile shock at a dose where Nec-1 shows no significant protection in the model. Taken together, we believe that GSK′963 represents an additional next-generation tool for exploring the role of RIP1 kinase-mediated biology.[1]

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Taken together, we have identified a novel, highly potent and selective inhibitor of RIP1 kinase activity. We believe that GSK′963 represents a next-generation tool inhibitor for investigating RIP1 biology in vitro, offering a significant benefit over the commercially available necrostatins.

C14H18N2O

230.31

230.141

C, 73.01; H, 7.88; N, 12.16; O, 6.95

2049868-46-2

GSK962;2049872-86-6

122703613

Light yellow to yellow solid powder

1.06±0.1 g/cm3

342.8±45.0 °C

2.1

0

2

2

17

311

1

O=C(C(C)(C)C)N1C(C2C=CC=CC=2)CC=N1

NJQVSLWJBLPTMD-LBPRGKRZSA-N

InChI=1S/C14H18N2O/c1-14(2,3)13(17)16-12(9-10-15-16)11-7-5-4-6-8-11/h4-8,10,12H,9H2,1-3H3/t12-/m0/s1

2,2-dimethyl-1-[(3S)-3-phenyl-3,4-dihydropyrazol-2-yl]propan-1-one

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)

Solubility in Formulation 1: 2.5 mg/mL (10.85 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 sonication.
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 2: ≥ 2.5 mg/mL (10.85 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.)