GSK547

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

GSK'547 (RIP1i) 体外治疗可引导骨髓源性巨噬细胞 (BMDM) 向免疫原性表型编程，上调 MHC-II、TNFa 和 IFNg，同时减少 CD206、IL-10 和 TGFb 表达。此外，BMDM 中 STAT1 信号传导被 RIP1i 上调，这与 M1 编程有关，但 STAT3、STAT5 和 STAT6 信号传导被下调，这与 M2 样巨噬细胞分化有关。经过RIP1i处理的巨噬细胞捕获抗原的能力也得到提高[1]。

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在生化实验中高效抑制RIP1激酶活性，IC50 = 1.3 nM，对其他激酶（如RIP2、RIP3、IRAK4）具有高选择性，IC50 > 1000 nM [1]
- 在LPS刺激的骨髓来源巨噬细胞（BMDMs）中阻断RIP1依赖的NF-κB激活，100 nM浓度下使IκBα和p65的磷酸化水平降低约70% [1]
- 在10-100 nM浓度下，抑制胰腺癌相关巨噬细胞（PCAMs）中免疫抑制细胞因子（IL-10、TGF-β）的产生约60-80%，同时使促炎细胞因子（TNF-α、IL-6）增加约2-3倍 [1]
- 增强PCAMs的抗原呈递能力，100 nM GSK547处理24小时后，MHC-II和CD86的表达分别上调约1.8倍和2.1倍 [1]
- 在与PCAMs的共培养体系中，恢复CD8+ T细胞的增殖能力和细胞毒性（颗粒酶B、IFN-γ产生），100 nM GSK547处理后CD8+ T细胞增殖率从约15%提升至约45% [1]

在小鼠饲料中施用 GSK'547 (RIP1i) 会在 24 小时内产生超过 L929 IC90 的体内稳态浓度。在 6 周的治疗方案过程中，RIP1i 的血清浓度得以维持。由于没有明显的病理改变，RIP1i 疗法的耐受性良好。与接受对照或 Nec-1 治疗的小鼠相比，接受 RIP1i 治疗的小鼠在暴露于来自 KPC 小鼠的原位 PDA（胰腺导管腺癌）肿瘤细胞后，肿瘤负荷更小，存活时间更长。除了新肿瘤之外，RIP1i 还可以预防肝转移[1]。

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在同源小鼠胰腺癌模型（KPC小鼠，KrasG12D/+; Trp53R172H/+; Pdx1-Cre）中，口服给予GSK547（30 mg/kg，每日两次，持续21天），显著抑制肿瘤生长，与溶媒对照组相比，肿瘤体积减少约55%，肿瘤重量减少约52% [1]
- 增加肿瘤组织中CD8+ T细胞和M1型极化巨噬细胞的浸润（分别约2.3倍和1.9倍），减少调节性T细胞（Tregs）和M2型极化巨噬细胞的浸润（分别约40%和35%）[1]
- 增强抗肿瘤免疫记忆：经GSK547治疗治愈的小鼠，再次接种KPC胰腺癌细胞后无肿瘤复发，脾和肿瘤引流淋巴结中记忆型CD8+ T细胞（CD44hiCD62Lhi）群体升高 [1]
- 与抗PD-1抗体治疗协同：联合治疗相比单药治疗，肿瘤体积减少约78%，约30%的小鼠实现肿瘤完全消退 [1]

荧光极化（FP）结合试验[1]
我们使用了一种基于fp的结合试验，通过与荧光标记的atp竞争配体竞争，量化RIP1i与RIP1的atp结合口袋之间的相互作用（Berger等，2015）。简而言之，纯化的gst标记RIP1（1-375）在最终检测浓度为200 nM时使用。荧光标记配体（14-（2-{[3-{2-[4-（氰乙基）苯基]氨基}-6-[（5-环丙基- 1h -吡唑-3-基）氨基]-4-嘧啶基]氨基}-2-氧乙基）-16,16,18,18-四甲基-6,7,7a,8a,9,10,16,18-八羟基苯并[2 ',3 ']吲哚齐诺[8 ',7 ':5 ',6 ']吡喃[3 ',2 ':3,4]吡喃[1,2- A]吲哚-5- -2-磺酸盐），最终测定浓度为5 nM。样品在Analyst多模式读取器上读取，抑制率表示为内部分析对照的抑制率百分比。

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激酶选择性和细胞活力测定[1]
激酶选择性测定按照我们之前的描述进行（Berger等，2015）。根据制造商的方案，使用p33放射性标记法检测RIP1i （10 μM）对371种激酶的抑制作用。反应在10 μM ATP存在下进行。数据报告为相对于DMSO对照的酶活性%。体外利用L929细胞检测RIP1抑制剂的疗效。用重组TNFα (100 ng/ml)在caspase抑制剂QVD-Oph (25 μM；微孔σ)。为了评估RIP1抑制的效果，用不同剂量的RIP1i预处理细胞30分钟。24小时后，根据制造商的方案，使用CellTiter-Glo发光细胞活力测定法测量细胞ATP水平，以评估诱导的细胞死亡。

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RIP1激酶活性测定：将重组人RIP1激酶与ATP、荧光标记的肽底物及不同浓度的GSK547在激酶反应缓冲液中孵育。30°C反应60分钟后，加入激酶终止缓冲液终止反应。通过荧光酶标仪检测底物磷酸化程度，以溶媒对照组为基准计算抑制率并确定IC50值 [1]

用不同剂量的细胞预处理RIP1i 30分钟。24小时后，用细胞ATP水平评估诱导的细胞死亡。
T细胞增殖试验[1]
对于基于抗体的T细胞增殖试验，如我们之前所述，在96孔板中使用CD3/CD28共连接激活脾脏CD3+ T细胞（Daley等，2016）。在选定的孔中，以1:5的巨噬细胞：T细胞比例加入tam。对于抗原限制性T细胞刺激试验，分别用Ova257-264或Ova323-339肽以5:1的比例脉冲巨噬细胞培养脾脏OT-I或OT-II T细胞。或者，巨噬细胞加载卵白蛋白（1mg /ml， 60分钟）。在选定的孔中加入中和性抗tnf α单抗（10 μg/ml, MP6-XT22）或同型对照。72hr时用流式细胞术检测T细胞活化情况。

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骨髓来源巨噬细胞（BMDM）培养与处理：从C57BL/6小鼠中分离BMDMs，在完全培养基中培养7天，随后用GSK547（0.1-100 nM）预处理1小时，再用LPS（1 μg/mL）刺激。24小时后收集细胞用于NF-κB通路蛋白的western blot分析，收集上清液通过ELISA检测细胞因子水平 [1]
- 胰腺癌相关巨噬细胞（PCAM）分离与功能实验：通过酶消化和流式细胞分选从KPC小鼠肿瘤中分离PCAMs。分离的PCAMs用GSK547（10-100 nM）处理24小时，随后通过流式细胞术分析MHC-II和CD86的表达，或与CFSE标记的CD8+ T细胞共培养3天以评估T细胞增殖 [1]
- Western blot分析：将处理后的BMDMs或PCAMs裂解，提取蛋白经SDS-PAGE分离、转印至PVDF膜，与抗磷酸化IκBα、磷酸化p65、总IκBα、总p65及β-肌动蛋白（内参）抗体孵育，化学发光法显影条带并通过光密度法定量 [1]
- 细胞因子ELISA检测：将处理后的巨噬细胞培养上清液加入抗体包被的96孔板，与检测抗体孵育后，在450 nm处测量吸光度，定量IL-10、TGF-β、TNF-α和IL-6的水平 [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 the 21-day in vivo study, oral administration of GSK547 (30 mg/kg, twice daily) did not cause significant changes in mouse body weight, food intake, or mortality [1]
- Histological analysis of major organs (liver, kidney, spleen, heart, lung) showed no obvious abnormalities or inflammatory infiltration in GSK547-treated mice compared to vehicle control [1]
- No hematological toxicity was observed, with white blood cell, red blood cell, and platelet counts remaining within normal ranges [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 inhibitor of receptor-interacting serine/threonine protein kinase 1 (RIPK1), inhibits macrophage-mediated adaptive immune tolerance in pancreatic cancer.

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GSK547 is a potent and selective small-molecule inhibitor of RIP1 Kinase, designed to target RIP1-mediated immune tolerance in pancreatic cancer [1]
- Its antitumor mechanism involves inhibiting RIP1-dependent NF-κB signaling in tumor-associated macrophages, shifting their 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 presentation molecules in macrophages, contributing to the immune-privileged microenvironment of 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

85.5

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.31 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.25 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (5.25 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: (饱和度未知) in (这些助溶剂从左到右依次添加，逐一添加),

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。