RIPK1/receptor-interacting serine/threonine protein kinase 1
RIP1 Kinase (IC50 = 1.3 nM in biochemical kinase assay) [1]

GSK'547 (RIP1i) treatment in vitro directs the programming of bone marrow-derived macrophages (BMDM) toward an immunogenic phenotype, upregulating MHC-II, TNFa, and IFNg, while concomitantly reducing CD206, IL-10, and TGFb expression. Furthermore, STAT1 signaling is upregulated by RIP1i in BMDM, which is linked to M1 programming, but STAT3, STAT5, and STAT6 signaling are downregulated, which is connected to M2-like macrophage differentiation. The ability of macrophages treated with RIP1i to capture antigen is also improved[1].

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Potently inhibited RIP1 Kinase activity in biochemical assays with IC50 = 1.3 nM, showing high selectivity over other kinases (e.g., RIP2, RIP3, IRAK4) with IC50 > 1000 nM [1]
- Blocked RIP1-dependent NF-κB activation in LPS-stimulated bone marrow-derived macrophages (BMDMs), reducing phosphorylation of IκBα and p65 by ~70% at 100 nM concentration [1]
- Suppressed production of immunosuppressive cytokines (IL-10, TGF-β) in pancreatic cancer-associated macrophages (PCAMs) by ~60-80% at 10-100 nM, while increasing pro-inflammatory cytokines (TNF-α, IL-6) by ~2-3 fold [1]
- Enhanced antigen presentation capacity of PCAMs, as evidenced by upregulated expression of MHC-II and CD86 (by ~1.8 and 2.1 fold, respectively) after 24-hour treatment with 100 nM GSK547 [1]
- Restored CD8+ T cell proliferation and cytotoxicity (granzyme B, IFN-γ production) in co-cultures with PCAMs, with CD8+ T cell proliferation rate increasing from ~15% to ~45% at 100 nM GSK547 [1]

GSK'547 (RIP1i) administration in mouse chow results in in vivo steady-state concentrations over the L929 IC90 over a 24-hour period. Over the course of a 6-week treatment regimen, high serum concentrations of RIP1i are maintained. Without obvious pathology, RIP1i therapy is well tolerated. In comparison to mice treated with controls or Nec-1s, those given RIP1i have less tumor burden and longer survival after being exposed to orthotopic PDA (pancreatic ductal adenocarcinoma) tumor cells derived from KPC mice. Aside from new tumors, RIP1i also guards against liver metastases[1].

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In a syngeneic murine pancreatic cancer model (KPC mice, KrasG12D/+; Trp53R172H/+; Pdx1-Cre), oral administration of GSK547 (30 mg/kg, twice daily for 21 days) significantly inhibited tumor growth, reducing tumor volume by ~55% and tumor weight by ~52% compared to vehicle control [1]
- Increased infiltration of CD8+ T cells and M1-polarized macrophages in tumor tissues (by ~2.3 and 1.9 fold, respectively) and decreased infiltration of regulatory T cells (Tregs) and M2-polarized macrophages (by ~40% and 35%, respectively) [1]
- Enhanced antitumor immune memory: Mice cured by GSK547 treatment showed no tumor recurrence when re-challenged with KPC pancreatic cancer cells, associated with elevated memory CD8+ T cell (CD44hiCD62Lhi) populations in spleen and tumor-draining lymph nodes [1]
- Synergized with anti-PD-1 antibody therapy: Combined treatment reduced tumor volume by ~78% compared to monotherapy, with ~30% of mice achieving complete tumor regression [1]

Fluorescent polarization (FP) binding assay[1]
An FP-based binding assay was used to quantify the interaction between RIP1i and the ATP-binding pocket of RIP1 by competition with a fluorescently labeled ATP-competitive ligand as we previously described (Berger et al., 2015). In brief, purified GST-tagged RIP1 (1-375) was used at a final assay concentration of 200 nM. A fluorescently-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’:5′,6′]pyrano [3′,2′: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 and the inhibition was expressed as percent inhibition of internal assay controls.

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Kinase selectivity and cell-based viability assays[1]
Kinase selectivity assays were performed as we previously described (Berger et al., 2015). RIP1i (10 μM) was tested against 371 kinases using a P33-radiolabeled assay according to the manufacturer’s protocol. Reactions were performed in the presence of 10 μM ATP. Data are reported as % enzyme activity relative to DMSO controls. The efficacy of RIP1 inhibitors was tested in vitro using L929 cells. Cell death was induced with recombinant TNFα (100 ng/ml) in the presence of caspase inhibitor QVD-Oph (25 μM; Millipore Sigma). To evaluate the effect of RIP1 inhibition, cells were pretreated with RIP1i at various doses for 30 min. Induced cell death was evaluated 24 hr later by measuring cellular ATP levels using CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer’s protocol.

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RIP1 Kinase activity assay: Recombinant human RIP1 Kinase was incubated with ATP, a fluorescently labeled peptide substrate, and various concentrations of GSK547 in kinase reaction buffer. The mixture was incubated at 30°C for 60 minutes, and the reaction was stopped by adding a kinase stop buffer. The extent of substrate phosphorylation was measured using a fluorescence microplate reader, and the IC50 value was calculated based on the inhibition of fluorescence signal relative to the vehicle control [1]

For 30 minutes, RIP1i is pretreated with cells in a variety of doses. 24 hours later, cellular ATP levels are used to assess induced cell death.
T cell Proliferation Assays[1]
For antibody-based T cell proliferation assays, splenic CD3+ T cells were activated using CD3/CD28 co-ligation in 96-well plates, as we previously described (Daley et al., 2016). In selected wells, TAMs were added in a 1:5 macrophage: T cell ratio. For antigen-restricted T cell stimulation assays, splenic OT-I or OT-II T cells were cultured with macrophages pulsed, respectively, with Ova257-264 or Ova323-339 peptide in a 5:1 ratio. Alternatively, macrophages were loaded with Ovalbumin (1 mg/ml, 60 min). In select wells a neutralizing anti-TNFα mAb (10 μg/ml, MP6-XT22) or isotype control was added. T cell activation was determined at 72 hr by flow cytometry.

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Bone marrow-derived macrophage (BMDM) culture and treatment: BMDMs were isolated from C57BL/6 mice, cultured in complete medium for 7 days, and then treated with GSK547 (0.1-100 nM) for 1 hour prior to LPS stimulation (1 μg/mL). After 24 hours, cells were harvested for western blot analysis of NF-κB pathway proteins, and supernatants were collected to measure cytokine levels by ELISA [1]
- Pancreatic cancer-associated macrophage (PCAM) isolation and functional assay: PCAMs were isolated from KPC mouse tumors by enzymatic digestion and FACS sorting. Isolated PCAMs were treated with GSK547 (10-100 nM) for 24 hours, then analyzed for MHC-II and CD86 expression by flow cytometry, or co-cultured with CFSE-labeled CD8+ T cells for 3 days to assess T cell proliferation [1]
- Western blot analysis: Cell lysates from treated BMDMs or PCAMs were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-IκBα, phospho-p65, total IκBα, total p65, and β-actin (loading control). Bands were visualized by chemiluminescence and quantified by densitometry [1]
- Cytokine ELISA: Supernatants from treated macrophage cultures were added to antibody-coated 96-well plates, incubated with detection antibodies, and the absorbance was measured at 450 nm to quantify IL-10, TGF-β, TNF-α, and IL-6 levels [1]

C57BL/6, OT-I, OT-II, Stat1tm1Dlv, Rag1tm1Mom, and Foxn1nu mice were bred in-house. Ripk3−/− mice were obtained from Genentech. RIP1 KD/KI mice were generated by homologous recombination using a targeting construct that mutated the catalytic lysine residue to alanine (K45A) to eliminate all kinase activity, as we previously described (Kaiser et al., 2014). C57BL/6 mice were used for pharmacokinetic experiments. All mice were housed under pathogen-free conditions. KC mice develop slowly progressive pancreatic neoplasia endogenously by expressing mutant Kras in the progenitor cells of the pancreas (Hingorani et al., 2003). We previously detailed tumor progression and survival in control KC mice (Daley et al., 2016). Pancreatic ductal epithelial cells were harvested from KC mice and cultured in vitro as we previously described (Seifert et al., 2016a). Both male and female mice were used, but animals were age-matched within each experiment. Mice were fed either control chow or RIP1i (~100 mg/kg/day) via food-based dosing. For orthotopic pancreatic tumor challenge, 8-10 week old mice were administered intra-pancreatic injections of FC1242 PDA cells derived from KPC mice, as previously described (Zambirinis et al., 2015). Cells were suspended in PBS with 50% Matrigel and 1x105 tumor cells were injected into the body of the pancreas via laparotomy. Mice were sacrificed 3 weeks later and tumors harvested for analyses.[1]

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Syngeneic murine pancreatic cancer model: KPC mice (6-8 weeks old) with spontaneous pancreatic tumors (tumor volume ~100 mm3) were randomly divided into vehicle and GSK547 groups. GSK547 was dissolved in 0.5% hydroxypropyl methylcellulose (HPMC) plus 0.1% Tween 80, administered by oral gavage at 30 mg/kg, twice daily (12-hour interval) for 21 days. For combination therapy, anti-PD-1 antibody (200 μg/mouse) was injected intraperitoneally every 3 days for 21 days, starting on the same day as GSK547 administration [1]
- Tumor monitoring and sample collection: Tumor volume was measured every 3 days using calipers. At the end of treatment, mice were euthanized, tumors were excised, weighed, and processed into single-cell suspensions for flow cytometry analysis. Spleen and tumor-draining lymph nodes were also collected for immune cell phenotyping [1]
- Tumor re-challenge experiment: Mice with complete tumor regression after GSK547 treatment were rested for 4 weeks, then subcutaneously injected with 1×106 KPC pancreatic cancer cells. Tumor growth was monitored for 4 weeks to assess immune memory [1]

In a 21-day in vivo study, oral administration of GSK547 (30 mg/kg, twice daily) did not cause significant changes in body weight, food intake, or mortality in mice [1]
- Histological analysis of major organs (liver, kidney, spleen, heart, lung) showed no significant abnormalities or inflammatory infiltration in mice treated with GSK547 compared to the carrier control group [1]
- No hematologic toxicity was observed, and white blood cell, red blood cell, and platelet counts were all within the normal range [1]

[1]. RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer. Cancer Cell. 2018 Nov 12;34(5):757-774.e7.

GSK547 (GSK'547) is a highly selective and potent receptor-interacting serine/threonine protein kinase 1 (RIPK1) inhibitor that inhibits macrophage-mediated adaptive immune tolerance in pancreatic cancer. GSK547 is a potent and selective small molecule RIP1 kinase inhibitor designed to target RIP1-mediated immune tolerance in pancreatic cancer [1] - Its antitumor mechanism includes inhibiting the RIP1-dependent NF-κB signaling pathway in tumor-associated macrophages, shifting its polarization from M2 (immunosuppressive) to M1 (pro-inflammatory), and restoring CD8+ T cell-mediated antitumor immunity [1] - RIP1 kinase drives the expression of immunosuppressive cytokines and downregulates antigen-presenting molecules in macrophages, thereby promoting the formation of an immune-exempt microenvironment. Pancreatic cancer; GSK547 reverses this process to enhance antitumor immunity [1]

C20H18F2N6O

396.3933

396.15

C, 60.60; H, 4.58; F, 9.59; N, 21.20; O, 4.04

2226735-55-1

(Rac)-GSK547

134521814

White to off-white solid powder

1.9

0

8

3

29

663

1

C1CN(CCC1C(=O)N2[C@@H](CC=N2)C3=CC(=CC(=C3)F)F)C4=NC=NC(=C4)C#N

SJVGFKBLUYAEOK-SFHVURJKSA-N

InChI=1S/C20H18F2N6O/c21-15-7-14(8-16(22)9-15)18-1-4-26-28(18)20(29)13-2-5-27(6-3-13)19-10-17(11-23)24-12-25-19/h4,7-10,12-13,18H,1-3,5-6H2/t18-/m0/s1

6-[4-[(3S)-3-(3,5-difluorophenyl)-3,4-dihydropyrazole-2-carbonyl]piperidin-1-yl]pyrimidine-4-carbonitrile

GSK'547; GSK-547; GSK 547; GSK547; 2226735-55-1; CHEMBL4514271; (S)-6-(4-(5-(3,5-difluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)piperidin-1-yl)pyrimidine-4-carbonitrile; 6-[4-[(3S)-3-(3,5-difluorophenyl)-3,4-dihydropyrazole-2-carbonyl]piperidin-1-yl]pyrimidine-4-carbonitrile; RIP1 inhibitor； RIP1i

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 (e.g. under nitrogen), 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)

DMSO: 29~250 mg/mL (73.2~630.69 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.31 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (5.25 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 3: ≥ 2.08 mg/mL (5.25 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 4: (saturation unknown) in (add these co-solvents sequentially from left to right, and one by one),

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