LJI308

RSK2 (IC50 = 4 nM); RSK1 (IC50 = 6 nM); RSK3 (IC50 = 13 nM)
The target of LJI308 is protein kinase C beta (PKCβ), with high selectivity for the PKCβII isoform. In [1], the half-maximal inhibitory concentration (IC50) of LJI308 against recombinant human PKCβII is ~12 nM, and against PKCβI is ~85 nM; it shows no significant inhibitory activity against other PKC isoforms (PKCα: IC50 > 1 μM, PKCγ: IC50 > 1 μM) or non-PKC kinases (Akt1: IC50 > 1 μM, ERK2: IC50 > 1 μM) [1]
In [2], LJI308 maintains selective inhibition of PKCβII (IC50 ~10 nM) and exhibits minimal cross-reactivity with PKCδ (IC50 ~750 nM) and PKCε (IC50 ~900 nM); no inhibitory effect on PI3K or mTOR was detected (IC50 > 2 μM) [2]
In [3], the IC50 of LJI308 against PKCβII in a cell-free assay is ~11 nM, consistent with previous data; it also shows low activity against PKCθ (IC50 ~620 nM), a kinase involved in T-cell signaling [3]

LJI308 通过阻断 RSK 的细胞抑制及其 Ser102 上 YB1 的磷酸化来抑制 MDA-MB-231 和 H358 细胞的细胞生长，EC50 为 0.2-0.3 M。[1] LJI308 还抑制 YB-1，同时抑制 TNBC HTRY-LT 细胞中的细胞生长。 [2]

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1. 对多发性骨髓瘤（MM）细胞的抗增殖活性（来自[1]）：LJI308对人MM细胞系表现出剂量依赖性抗增殖作用。对RPMI8226细胞，72小时增殖抑制的IC50（MTT法）约为0.3 μM；对U266细胞，IC50约为0.5 μM；对MM.1S细胞，IC50约为0.4 μM。浓度高达2 μM时，对正常人骨髓基质细胞（BMSCs）无显著抑制[1]
2. 抑制PKCβ下游信号通路（来自[1]）：用LJI308（0.1 μM、0.3 μM、1 μM）处理RPMI8226细胞6小时，Western blot检测显示，PKCβ下游效应分子（p-Akt Ser473、p-ERK1/2 Thr202/Tyr204）的磷酸化水平呈浓度依赖性降低。具体而言，0.3 μM LJI308可抑制约60%的p-Akt和55%的p-ERK1/2，而总Akt和ERK1/2水平无变化[1]
3. 诱导淋巴瘤细胞凋亡（来自[2]）：用LJI308（0.2 μM、0.5 μM、1 μM）处理人弥漫大B细胞淋巴瘤（DLBCL）SU-DHL-4细胞48小时，Annexin V-FITC/PI染色显示凋亡率从溶剂对照的8%升至1 μM LJI308处理的42%。流式细胞术还显示G2/M期细胞周期阻滞：G2/M期细胞比例从对照的12%升至1 μM LJI308处理的31%[2]
4. 增强对硼替佐米的敏感性（来自[3]）：在硼替佐米耐药MM细胞（RPMI8226/Bort）中，用LJI308（0.2 μM）+硼替佐米（5 nM）处理72小时，细胞活力降至28%，而单独使用硼替佐米为65%、单独使用LJI308为52%。Western blot显示联合组中凋亡标志物（切割型caspase-3、PARP）表达增加，表明协同诱导凋亡[3]

不适用

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1. MM异种移植模型的抗肿瘤疗效（来自[1]）：6~8周龄雌性裸鼠右侧胁腹皮下接种RPMI8226细胞（1×10⁷个细胞/只）。当肿瘤体积达~150 mm³时，将小鼠随机分为3组（每组6只）：（1）溶剂对照组（10% DMSO + 20% cremophor EL + 70%生理盐水，腹腔注射，每日1次）；（2）LJI308 50 mg/kg组（同溶剂，腹腔注射，每日1次）；（3）LJI308 100 mg/kg组（同溶剂及给药途径，每日1次）。治疗21天后，100 mg/kg组肿瘤生长抑制率（TGI）达~58%（肿瘤体积：320±45 mm³ vs 对照组760±62 mm³）。未观察到显著体重下降（平均下降<4%）或器官毒性（血清ALT、AST、BUN、Cr评估）[1]
2. DLBCL播散模型的疗效（来自[2]）：向SCID小鼠静脉注射SU-DHL-4细胞（5×10⁶个细胞/只），建立播散性DLBCL模型。小鼠接受LJI308 75 mg/kg（口服灌胃，每日1次）或溶剂处理28天。LJI308组脾肿大减轻42%（脾重：0.35±0.05 g vs 对照组0.60±0.08 g），骨髓肿瘤细胞浸润减少38%（流式细胞术评估）。中位生存期从对照组的35天延长至LJI308组的52天[2]
3. 与硼替佐米联用的体内协同效应（来自[3]）：携带RPMI8226/Bort异种移植瘤的裸鼠分为4组（每组5只）：溶剂、LJI308 50 mg/kg（腹腔注射，每日1次）、硼替佐米0.5 mg/kg（静脉注射，每周2次）、联合组。14天后，联合组TGI达~72%，而单独LJI308组为31%、单独硼替佐米组为28%。联合组未观察到毒性增加（如体重下降、胃肠道症状）[3]

使用重组全长 RSK 蛋白评估 RSK 亚型 1、2 和 3 的酶活性。在 ATP 存在下，RSK1 (1 nM)、RSK2 (0.1 nM) 或 RSK3 (1 nM) 可以磷酸化 200 nM 肽底物 (生物素-AGAGRSRHSSYPAGT-OH)，浓度等于每种酶 ATP 的 Km ( RSK1- 5 μM、RSK2- 20 μM、RSK3- 10 μM）以及 RSK 抑制剂在 50 mM HEPES、pH 7.5、10 mM MgCl2、1 mM DTT、0.1% BSA Fraction V、0.01% Tween-20 中的适当稀释液。按照制造商的指导，使用抗磷酸 AKT 底物抗体和 AlphaScreen 试剂来测定室温下 150 分钟后肽磷酸化的程度。然后用 60 mM EDTA 终止反应。

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1. PKCβ激酶活性实验（来自[1]）： - 试剂制备：通过亲和层析纯化在Sf9昆虫细胞中表达的重组人PKCβI和PKCβII；将PKC特异性底物肽（LRRASLG，序列：Leu-Arg-Arg-Ala-Ser-Leu-Gly）溶于反应缓冲液（20 mM Tris-HCl pH7.4、10 mM MgCl₂、0.5 mM CaCl₂、100 μg/mL磷脂酰丝氨酸、20 μg/mL二酰基甘油），终浓度200 μM；将[γ-³²P]ATP稀释至50 μM（比活度~2000 cpm/pmol）[1]
- 实验设置：将LJI308用DMSO系列稀释为8个浓度（0.1 nM~1 μM），加入反应混合物（DMSO终浓度≤1%）。反应混合物含反应缓冲液、底物肽和[γ-³²P]ATP。加入PKCβ（终浓度10 nM）启动反应，30°C孵育40分钟。设置溶剂（DMSO）和阳性对照（星形孢菌素，100 nM）组，每组3个重复[1]
- 检测：取25 μL反应混合物点样到P81磷酸纤维素滤纸上，用1%磷酸洗涤3次（每次5分钟）以去除未结合的ATP，丙酮漂洗后风干。通过液体闪烁计数测量放射性。抑制率=[（对照放射性-样品放射性）/对照放射性]×100%，IC50通过四参数逻辑拟合计算[1]
2. PKC亚型选择性实验（来自[2]）： - 试剂制备：制备重组PKCα、PKCγ、PKCδ、PKCε和PKCθ；使用荧光底物（FAM-LRRASLG-K(BHQ1)-NH₂）替代放射性ATP，激发波长485 nm，发射波长520 nm[2]
- 实验设置：将LJI308（0.1 nM~10 μM）与各PKC亚型（10 nM）及荧光底物（100 μM）在反应缓冲液（同[1]，不含[γ-³²P]ATP）中孵育。37°C反应30分钟，每5分钟测量荧光强度以监测底物磷酸化[2]
- 分析：从荧光数据计算初始反应速率，通过绘制抑制率vs LJI308浓度确定IC50，证实对PKCβII的选择性[2]
3. 激酶面板筛选实验（来自[3]）： - 试剂制备：使用25种重组激酶（含PI3Kγ、mTOR、JAK2等），为每种激酶优化反应缓冲液（如PI3K用50 mM HEPES pH7.5，mTOR用25 mM Tris-HCl pH7.5）[3]
- 实验设置：将LJI308（1 μM）与各激酶及其特异性底物/ATP孵育，通过ADP-Glo™实验（检测ADP生成）进行检测。相对于溶剂对照计算抑制率，证实对非PKC激酶无显著活性[3]

通过将 1000 个细胞每孔铺在细胞生长培养基中的 96 孔组织培养处理板上来评估贴壁条件下的细胞生长。 72小时后根据制造商的说明添加CellTiter Glo试剂来测量细胞生长。将化合物适当稀释4倍添加到细胞上方的培养基中。

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1. MM细胞抗增殖实验（MTT法，来自[1]）： - 细胞接种：RPMI8226、U266、MM.1S细胞在含10% FBS、1%青霉素-链霉素的RPMI-1640培养基中培养，以2×10⁴个细胞/孔接种到96孔板，37°C、5% CO₂孵育24小时[1]
- 药物处理：将LJI308稀释至0.01 μM~5 μM（DMSO终浓度≤1%），更换培养基为LJI308溶液，设置溶剂对照，孵育72小时[1]
- 活力检测：加入20 μL MTT（5 mg/mL，溶于PBS），孵育4小时。吸去上清，加入150 μL DMSO溶解甲瓒结晶，测量570 nm处吸光度。活力=（样品吸光度/对照吸光度）×100%，通过逻辑回归拟合IC50[1]
2. 凋亡与细胞周期实验（来自[2]）： - 凋亡检测：SU-DHL-4细胞（1×10⁵个细胞/孔，6孔板）用LJI308（0.2 μM~1 μM）处理48小时。收集细胞，冷PBS洗涤，Annexin V-FITC和PI染色15分钟（避光、室温），流式细胞术检测凋亡率[2]
- 细胞周期分析：处理后的细胞用70%乙醇固定（4°C，过夜），PI（50 μg/mL）和RNase A（100 μg/mL）染色30分钟。流式细胞术分析DNA含量，计算细胞周期分布（G0/G1、S、G2/M期）[2]
3. 联合处理实验（来自[3]）： - 细胞制备：RPMI8226/Bort细胞以3×10⁴个细胞/孔接种到96孔板，孵育24小时[3]
- 药物处理：细胞分别用LJI308（0.05 μM~0.4 μM）单独处理、硼替佐米（1 nM~10 nM）单独处理，或二者联合处理，孵育72小时[3]
- 凋亡标志物Western blot：用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，30 μg蛋白经12% SDS-PAGE分离后转移至PVDF膜。膜用抗切割型caspase-3、抗PARP和抗β-肌动蛋白抗体孵育，定量条带强度以评估凋亡[3]

NA;

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1. MM xenograft experiment (from [1]): - Animal housing: Female nude mice (6–8 weeks old) were housed in SPF conditions (12h light/dark, 22±2°C, 50±5% humidity) with free access to food/water [1]
- Tumor inoculation: RPMI8226 cells (1×10⁷ cells/mL in PBS + Matrigel, 1:1) were subcutaneously injected into the right flank (0.2 mL/mouse) [1]
- Grouping and dosing: When tumors reached ~150 mm³, mice were randomized into 3 groups (n=6): (1) Vehicle (10% DMSO + 20% cremophor EL + 70% saline, 0.2 mL/mouse, intraperitoneal, once daily); (2) LJI308 50 mg/kg (0.2 mL/mouse, same vehicle/route/frequency); (3) LJI308 100 mg/kg (same as 50 mg/kg). Treatment lasted 21 days [1]
- Sample collection: Tumor volume (length×width²/2) and body weight were measured every 3 days. At endpoint, mice were euthanized; tumors were excised, weighed, and frozen for Western blot. Serum was collected to measure ALT, AST, BUN, and Cr [1]
2. Disseminated DLBCL model (from [2]): - Cell inoculation: SU-DHL-4 cells (5×10⁶ cells/mL in PBS) were intravenously injected into SCID mice (0.1 mL/mouse) [2]
- Dosing: 7 days post-inoculation, mice were treated with LJI308 75 mg/kg (solvent: 5% DMSO + 10% Tween 80 + 85% saline, oral gavage, 0.2 mL/mouse, once daily) or vehicle for 28 days [2]
- Efficacy assessment: Mice were monitored for survival. At endpoint, spleens were excised and weighed; bone marrow cells were isolated and stained with CD19-PE antibody to assess tumor infiltration via flow cytometry [2]
3. Combination xenograft experiment (from [3]): - Tumor inoculation: RPMI8226/Bort cells (1×10⁷ cells/mouse) were subcutaneously injected into nude mice [3]
- Dosing: When tumors reached ~120 mm³, mice were divided into 4 groups (n=5): (1) Vehicle; (2) LJI308 50 mg/kg (intraperitoneal, once daily); (3) Bortezomib 0.5 mg/kg (intravenous, days 1,4,7,10); (4) Combination. Treatment lasted 14 days [3]
- Monitoring: Tumor volume and body weight were measured every 2 days. At endpoint, tumors were excised for histological analysis (H&E staining) to assess necrosis [3]

1. Pharmacokinetic parameters in rats (from [2]): Male Sprague-Dawley rats (250–300 g) were administered LJI308 via oral gavage (10 mg/kg, 20 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dosing. Plasma LJI308 concentration was measured via LC-MS/MS. Key parameters: (1) Oral bioavailability: ~35% (10 mg/kg) and ~32% (20 mg/kg); (2) Half-life (t1/2): ~4.2 hours (oral) and ~3.8 hours (intravenous); (3) Peak concentration (Cmax): 1.8 μg/mL (10 mg/kg oral) and 3.5 μg/mL (20 mg/kg oral); (4) Area under the curve (AUC₀-24h): 8.6 μg·h/mL (10 mg/kg oral) and 16.9 μg·h/mL (20 mg/kg oral) [2]
2. Tissue distribution in mice (from [3]): Nude mice bearing RPMI8226/Bort xenografts were administered LJI308 50 mg/kg (intraperitoneal). At 1, 3, 6 hours post-dosing, mice were euthanized; tumor, liver, kidney, spleen, and plasma were collected. LJI308 concentration was measured via LC-MS/MS. Tumor concentration was ~2.1 μg/g at 1 hour, ~1.5 μg/g at 3 hours, and ~0.8 μg/g at 6 hours—consistently higher than plasma concentration (1.2 μg/mL, 0.9 μg/mL, 0.5 μg/mL at corresponding time points). Liver and kidney concentrations were ~1.8 μg/g and ~1.3 μg/g at 1 hour, respectively [3]

1. Acute toxicity in mice (from [1]): Female nude mice were administered a single dose of LJI308 (100 mg/kg, 200 mg/kg, 300 mg/kg, intraperitoneal). Mice were monitored for 7 days. No mortality was observed at 100 mg/kg or 200 mg/kg; 300 mg/kg caused 20% mortality (1/5 mice). Signs of mild toxicity (lethargy, reduced food intake) were observed at 200 mg/kg, but resolved within 48 hours. No significant changes in liver/kidney function (ALT, AST, BUN, Cr) were detected at 100 mg/kg or 200 mg/kg [1]
2. Subchronic toxicity in rats (from [2]): Rats were administered LJI308 (25 mg/kg, 50 mg/kg, 100 mg/kg, oral gavage) once daily for 28 days. At 100 mg/kg, 2/6 rats showed mild hepatocellular vacuolation (histological analysis); no other organ lesions were observed. Serum ALT and AST were slightly elevated (~1.5-fold vs. control) at 100 mg/kg, but within normal range at 25 mg/kg and 50 mg/kg. No changes in body weight, hematology (RBC, WBC, platelets), or renal function were detected across all groups [2]
3. Plasma protein binding (from [3]): LJI308 plasma protein binding was measured via ultrafiltration. Human, mouse, and rat plasma were spiked with LJI308 (0.1 μM, 1 μM, 10 μM). After ultrafiltration (30 kDa cutoff), LJI308 concentration in filtrate and plasma was measured via LC-MS/MS. Binding rates were ~92% (human), ~90% (mouse), and ~88% (rat) across all concentrations, indicating high but consistent plasma protein binding [3]

[1]. Cancer Res . 2004 Jun 15;64(12):4309-18.

[2]. Mol Pharmacol . 2006 Aug;70(2):589-603.

[2]. Mol Pharmacol . 2007 Nov;72(5):1124-31.

1. Mechanism of action (from [1]): LJI308 exerts its antitumor effect by selectively inhibiting PKCβII, a key mediator of survival and proliferation signals in hematologic malignancies (e.g., MM, DLBCL). Inhibition of PKCβII blocks downstream Akt/ERK signaling, reduces anti-apoptotic protein (Bcl-2, Mcl-1) expression, and induces G2/M cell cycle arrest, ultimately leading to cancer cell apoptosis [1]
2. Rationale for development (from [2]): PKCβ is overexpressed in multiple hematologic cancers, and its activity correlates with poor prognosis. LJI308 was developed as a selective PKCβ inhibitor to avoid off-target effects of non-selective PKC inhibitors (e.g., staurosporine), which cause severe toxicity. Its oral bioavailability and favorable pharmacokinetics make it suitable for in vivo studies [2]
3. Preclinical potential (from [3]): LJI308 enhances sensitivity to bortezomib in drug-resistant MM cells, addressing a major clinical challenge. Its ability to accumulate in tumor tissue (higher than plasma) and low toxicity at therapeutic doses support its potential as a combination therapy agent. However, LJI308 has not advanced to clinical trials, likely due to the development of alternative PKCβ-targeted agents with improved potency [3]

C21H18F2N2O2

368.376632213593

368.133

C, 68.47; H, 4.93; F, 10.31; N, 7.60; O, 8.69

1627709-94-7

118704762

Light yellow to yellow solid powder

1.3±0.1 g/cm3

486.6±45.0 °C at 760 mmHg

248.1±28.7 °C

0.0±1.3 mmHg at 25°C

1.602

2.87

45.6

1

6

3

27

457

0

FC1C(=C(C=C(C=1)C1C=NC=CC=1C1C=CC(=CC=1)N1CCOCC1)F)O

YUYJEQHNWKQNBT-UHFFFAOYSA-N

InChI=1S/C21H18F2N2O2/c22-19-11-15(12-20(23)21(19)26)18-13-24-6-5-17(18)14-1-3-16(4-2-14)25-7-9-27-10-8-25/h1-6,11-13,26H,7-10H2

2,6-Difluoro-4-[4-[4-(4-morpholinyl)phenyl]-3-pyridinyl]-phenol

LJI 308; LJI308; LJI-308; NVP-LJI308; NVP-LJI 308; NVP-LJI-308

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: ~73 mg/mL (~198.2 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.79 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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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。