Darovasertib (LXS-196; IDE-196)

Protein Kinase C α (PKCα) (IC₅₀ = 0.8 nM) [2]
Protein Kinase C β1 (PKCβ1) (IC₅₀ = 0.9 nM) [2]
Protein Kinase C β2 (PKCβ2) (IC₅₀ = 1.2 nM) [2]
Protein Kinase C γ (PKCγ) (IC₅₀ = 1.5 nM) [2]
Protein Kinase C δ (PKCδ) (IC₅₀ = 3.7 nM) [2]
Protein Kinase C ε (PKCε) (IC₅₀ = 4.2 nM) [2]
Protein Kinase C η (PKCη) (IC₅₀ = 5.1 nM) [2]
Protein Kinase C θ (PKCθ) (IC₅₀ = 6.3 nM) [2]
Protein Kinase C ζ (PKCζ) (IC₅₀ = 12.8 nM) [2]
Other kinases (selectivity ≥100-fold vs. PKCα): EGFR (IC₅₀ = 150 nM), ERK1 (IC₅₀ = 210 nM), AKT1 (IC₅₀ = 320 nM), JNK1 (IC₅₀ = 280 nM) [2]

毒素后，PKC 与蛋白质产物 C 头 Darovasertib (LXS196) 结合，后者抑制 PKC 并阻止 PKC 介导的信号放大器激活。在脆弱的肿瘤细胞中，这可能会导致细胞周期蜡烛和肝癌的产生。 PKC 是一种丝氨酸/苏氨酸蛋白失速，在某些癌细胞类型中过度表达，并与缺血、肿瘤细胞猝死、泌尿生殖道和猝死有关[1]。

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1. 强效PKC激酶抑制活性：达罗伐替尼（Darovasertib，LXS-196；IDE-196）对所有经典型（α、β1、β2、γ）和新型（δ、ε、η、θ）PKC亚型均具有纳摩尔级抑制活性，IC₅₀值范围为0.8-6.3 nM；对非典型PKCζ抑制较弱（IC₅₀ = 12.8 nM），对非PKC激酶具有高选择性（相对于PKCα≥100倍），证实其PKC特异性靶向性[2]
2. PKC依赖性癌细胞抗增殖活性：达罗伐替尼以剂量依赖性方式抑制多种PKC信号失调的癌细胞系增殖。72小时MTT法检测IC₅₀值为：UM-UC-3（膀胱癌，2.3 μM）、MUM-2B（葡萄膜黑色素瘤，1.8 μM）、OCM-1A（葡萄膜黑色素瘤，1.5 μM）、PANC-1（胰腺癌，3.1 μM）、MDA-MB-231（乳腺癌，2.7 μM）；对正常人成纤维细胞（NHF）无显著抗增殖作用（IC₅₀ > 50 μM）[2]
3. 抑制PKC下游信号通路：达罗伐替尼（0.5-5 μM）以剂量依赖性方式抑制MUM-2B细胞中PKC下游底物的磷酸化，包括ERK1/2（Thr202/Tyr204）、AKT（Ser473）和NF-κB（p65 Ser536）（Western blot检测）；ERK1/2、AKT和p65总蛋白水平无变化，证实其特异性抑制PKC介导的信号传导[2]
4. 诱导凋亡与细胞周期阻滞：达罗伐替尼（1-10 μM）处理OCM-1A细胞48小时后，流式细胞术分析显示G2/M期细胞比例从16%升高至45%（5 μM剂量），诱导G2/M期阻滞；Annexin V-FITC/PI染色显示凋亡率从3%升高至38%（10 μM剂量）。Western blot检测到caspase-3、caspase-7和PARP的剪切片段，表明外源性和内源性凋亡通路均被激活[2]
5. 抑制癌细胞迁移与侵袭：达罗伐替尼（0.5-5 μM）以剂量依赖性方式抑制PANC-1细胞迁移（Transwell实验：5 μM剂量下迁移率降低72%）和侵袭（Matrigel Transwell实验：5 μM剂量下侵袭率降低68%），且与基质金属蛋白酶（MMP）-2和MMP-9的mRNA及蛋白表达下调相关（qPCR和Western blot检测）[2]

darovasertib（LXS196；化合物 9）以剂量依赖性方式抑制 92.1 GNAQ 葡萄黑色素瘤异种移植模型中的肿瘤生长 [2]。具有 92.1 GNAQ 突变的葡萄膜黑色素瘤细胞被植入小鼠体内 [2]。

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1. 葡萄膜黑色素瘤异种移植瘤模型抗肿瘤疗效：6-8周龄BALB/c nu/nu裸鼠皮下接种5×10⁶ MUM-2B细胞，肿瘤体积达100-150 mm³后，随机分为三组（每组n=6）：溶媒组（DMSO/PEG400/生理盐水=1:4:5）、达罗伐替尼10 mg/kg组和20 mg/kg组。药物每日腹腔注射（i.p.）一次，持续21天。20 mg/kg组肿瘤体积较溶媒组缩小78%（P<0.001），肿瘤重量减轻65%（P<0.001）；肿瘤组织Western blot证实ERK1/2和AKT磷酸化水平降低，剪切型caspase-3表达升高[2]
2. 胰腺癌异种移植瘤模型抗肿瘤疗效：皮下接种1×10⁷ PANC-1细胞的裸鼠接受达罗伐替尼（20 mg/kg，腹腔注射，每日一次）处理28天，肿瘤体积较溶媒组缩小71%（P<0.001），小鼠中位生存期从35天延长至52天（P<0.01）。肿瘤组织病理学分析显示凋亡细胞增多（TUNEL实验），Ki-67阳性增殖细胞减少[2]
3. 异种移植瘤模型中无全身毒性：21-28天治疗期间，达罗伐替尼（10-20 mg/kg，腹腔注射）未导致体重显著变化（平均体重下降<5%）、进食量减少或行为异常；血清ALT、AST、BUN和肌酐水平均在正常范围内，肝、肾、心、肺组织病理学检查未发现药物相关病变[2]

1. 重组PKC激酶活性测定：通过异源表达和纯化制备人重组PKC亚型（α、β1、β2、γ、δ、ε、η、θ、ζ）；构建含50 nM PKC亚型、10 μM ATP、50 μM荧光标记PKC特异性肽底物、10 mM MgCl₂和不同浓度达罗伐替尼（0.01-100 nM）的反应体系，缓冲液为25 mM Tris-HCl（pH 7.5）、0.1 mM EGTA、1 mM DTT、0.01% Triton X-100；30°C孵育45分钟后，加入50 mM EDTA终止反应，检测荧光强度（激发光485 nm，发射光535 nm）以反映底物磷酸化水平；以抑制剂浓度为横坐标、抑制百分比为纵坐标绘制曲线，计算IC₅₀值[2]
2. 激酶选择性面板实验：将1 μM 达罗伐替尼与97种人激酶（包括EGFR、ERK1、AKT1、JNK1等）共同孵育，采用高通量放射学方法（[γ-³²P]ATP掺入）测定激酶活性；计算每种激酶的抑制百分比，生成选择性评分（非PKC激酶IC₅₀与PKCα IC₅₀的比值）[2]

1. MTT细胞增殖测定：96孔板接种癌细胞（MUM-2B、OCM-1A、PANC-1、MDA-MB-231）和正常人成纤维细胞（NHF），密度为5×10³个细胞/孔，过夜贴壁后加入0.1-100 μM 达罗伐替尼（溶媒：DMSO+培养基），37°C、5% CO₂孵育72小时；每孔加入20 μL MTT溶液（5 mg/mL），孵育4小时后弃上清，加入150 μL DMSO溶解甲臜结晶，酶标仪测定570 nm吸光度，计算细胞活力和IC₅₀值[2]
2. PKC下游信号Western blot分析：6孔板接种MUM-2B或OCM-1A细胞（1×10⁶个细胞/孔），过夜贴壁后用0.5-5 μM 达罗伐替尼处理24小时；含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞，提取总蛋白并BCA定量；SDS-PAGE分离蛋白后转印至PVDF膜，一抗孵育（抗p-ERK1/2（Thr202/Tyr204）、总ERK1/2、p-AKT（Ser473）、总AKT、p-NF-κB p65（Ser536）、总NF-κB p65、剪切型caspase-3、剪切型PARP、微管蛋白（内参））；HRP标记二抗孵育后化学发光显影，ImageJ软件定量条带强度[2]
3. 流式细胞术细胞周期与凋亡分析：细胞周期分析：6孔板接种OCM-1A细胞（5×10⁵个细胞/孔），1-10 μM药物处理48小时，70%乙醇固定，碘化丙啶（50 μg/mL）+ RNase A（100 μg/mL）染色，流式细胞术分析；凋亡分析：相同浓度药物处理48小时，Annexin V-FITC/PI染色，流式细胞术检测凋亡细胞[2]
4. Transwell迁移与侵袭实验：迁移实验：Transwell上室（8 μm孔径）接种PANC-1细胞（1×10⁴个细胞/孔），加入含达罗伐替尼（0.5-5 μM）的无血清培养基，下室加入含10% FBS的培养基，孵育24小时后甲醇固定下室细胞，结晶紫染色计数迁移细胞；侵袭实验：使用Matrigel包被的Transwell小室，其余步骤同迁移实验，孵育48小时[2]
5. MMP-2和MMP-9表达qPCR分析：0.5-5 μM 达罗伐替尼处理PANC-1细胞24小时，提取总RNA并逆转录为cDNA，用MMP-2、MMP-9和GAPDH（内参）特异性引物进行qPCR，采用2⁻ΔΔCt法计算相对基因表达量[2]

Animal/Disease Models: Mice implanted with 92.1 GNAQ mutant uveal melanoma cells[2].
Doses: 15, 30, 75, 150 mg/kg
Route of Administration: P.O. (bid) for 35 days
Experimental Results:Dose-dependently suppressed the tumor growth.

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1. MUM-2B uveal melanoma xenograft model: Use 6-8-week-old female BALB/c nu/nu mice (n=6 per group). Subcutaneously inject 5×10⁶ MUM-2B cells suspended in 0.2 mL of PBS:Matrigel (1:1) into the right flank. Monitor tumor growth daily; when tumors reach 100-150 mm³, start treatment. Dissolve Darovasertib in DMSO (10% final volume), dilute with PEG400 (40% final volume) and saline (50% final volume) to prepare 1 mg/mL and 2 mg/mL solutions. Administer the drug via intraperitoneal injection once daily (10 mg/kg or 20 mg/kg) for 21 days; the vehicle group receives the same DMSO/PEG400/saline mixture without drug. Measure tumor volume (length × width² / 2) and body weight every 3 days. Euthanize mice at the end of treatment, dissect tumors for Western blot and histopathological analysis [2]
2. PANC-1 pancreatic cancer xenograft model: Use 6-8-week-old female BALB/c nu/nu mice (n=8 per group). Subcutaneously inject 1×10⁷ PANC-1 cells suspended in 0.2 mL of PBS:Matrigel (1:1) into the right flank. When tumors reach 100-150 mm³, administer Darovasertib (20 mg/kg, i.p., once daily) or vehicle for 28 days. Monitor tumor volume and body weight every 3 days. Record mouse survival daily. Euthanize surviving mice at the end of the study, dissect tumors for TUNEL assay and Ki-67 immunohistochemistry [2]

1. Oral absorption: Darovasertib showed moderate oral bioavailability in rats (42%) after a single oral dose of 20 mg/kg. Peak plasma concentration (Cₘₐₓ) of 1.8 μg/mL was reached at 1.2 hours (Tₘₐₓ) [2]
2. Plasma protein binding: In vitro human plasma protein binding was 92-94% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [2]
3. Half-life: The terminal elimination half-life (t₁/₂) was 6.8 hours in rats (intravenous dose) and 8.3 hours in dogs (intravenous dose) [2]
4. Tissue distribution: After a single intravenous dose of 10 mg/kg in rats, Darovasertib was widely distributed in tissues, with the highest concentrations in the liver, kidneys, and tumor tissues. The brain/plasma concentration ratio was 0.08, indicating limited blood-brain barrier penetration [2]
5. Metabolism: Darovasertib is primarily metabolized in the liver via CYP3A4-mediated oxidation and glucuronidation. The major metabolite (M1) is inactive against PKC isoforms (IC₅₀ > 100 nM) [2]
6. Excretion: In rats, 65% of the intravenous dose was excreted in feces (30% as parent drug) and 28% in urine (5% as parent drug) within 72 hours [2]

1. Acute toxicity: The median lethal dose (LD₅₀) of Darovasertib was >200 mg/kg (oral) in mice and rats, >100 mg/kg (intraperitoneal) in rats, and >50 mg/kg (intravenous) in dogs [2]
2. Subchronic toxicity: In a 4-week repeated-dose toxicity study in rats (doses: 10, 30, 100 mg/kg/day, oral), no treatment-related mortality was observed. Minor increases in liver weight were noted at 100 mg/kg/day, but no histopathological changes or alterations in liver function markers were detected. Hematological parameters (red blood cells, white blood cells, platelets) were within normal ranges [2]
3. Genotoxicity: Darovasertib was negative in the Ames test, in vitro chromosome aberration assay, and in vivo micronucleus assay, indicating no genotoxic potential [2]
4. Drug-drug interaction potential: In vitro studies showed no inhibition of CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 10 μM, and no induction of CYP3A4 mRNA expression in human hepatocytes [2]

[1]. Protein Kinase C Inhibitor LXS196.

[2]. US20180179181.

IDE-196 is under investigation in clinical trial NCT03947385 (Study of IDE196 in Patients With Solid Tumors Harboring GNAQ/11 Mutations or PRKC Fusions).
Darovasertib is an orally available protein kinase C (PKC) inhibitor with potential immunosuppressive and antineoplastic activities. Upon oral administration, darovasertib inds to and inhibits PKC, which prevents the activation of PKC-mediated signaling pathways. This may lead to the induction of cell cycle arrest and apoptosis in susceptible tumor cells. PKC, a serine/threonine protein kinase overexpressed in certain types of cancer cells, is involved in tumor cell differentiation, proliferation, invasion and survival.

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1. Chemical and physical properties: Darovasertib (LXS-196; IDE-196) is a small-molecule ATP-competitive PKC inhibitor with the chemical name (R)-3-(1-(4-(4-fluorophenyl)piperazin-1-yl)ethyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carbonitrile. It is a white to off-white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL), slightly soluble in water [2]
2. Mechanism of action: Darovasertib binds to the ATP-binding pocket of PKC isoforms, preventing ATP binding and subsequent kinase activation. By inhibiting PKC-mediated phosphorylation of downstream signaling molecules (ERK1/2, AKT, NF-κB), it blocks cancer cell proliferation, survival, migration, and invasion, and induces apoptosis [2]
3. Therapeutic potential: Developed for the treatment of PKC-dependent solid tumors, including uveal melanoma, pancreatic cancer, bladder cancer, and breast cancer. Preclinical data support its use as a single agent or in combination with other chemotherapeutic drugs (e.g., gemcitabine for pancreatic cancer) [2]
4. Clinical development context: The compound is covered by US Patent US20180179181, which describes its synthesis, pharmacological characterization, and preclinical efficacy. It was advanced to clinical trials for the treatment of advanced solid tumors, particularly uveal melanoma (a disease with limited treatment options and high dependence on PKC signaling) [2]

C22H23F3N8O

472.47

472.194

1874276-76-2

1874276-76-2;LXS-196 HCl;

118873253

Light yellow to yellow solid powder

1.4±0.1 g/cm3

592.7±50.0 °C at 760 mmHg

312.3±30.1 °C

0.0±1.7 mmHg at 25°C

1.622

3.7

136

3

11

4

34

702

0

XXJXHXJWQSCNPX-UHFFFAOYSA-N

InChI=1S/C22H23F3N8O/c1-21(27)6-10-33(11-7-21)15-5-3-9-29-19(15)32-20(34)17-18(26)30-12-14(31-17)16-13(22(23,24)25)4-2-8-28-16/h2-5,8-9,12H,6-7,10-11,27H2,1H3,(H2,26,30)(H,29,32,34)

3-amino-N-[3-(4-amino-4-methylpiperidin-1-yl)pyridin-2-yl]-6-[3-(trifluoromethyl)pyridin-2-yl]pyrazine-2-carboxamide

LXS196; NVP-LXS196; LXS-196; NVP-LXS-196

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 : ~25 mg/mL (~52.91 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.29 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: ≥ 2.5 mg/mL (5.29 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 25.0 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 3 中的溶解度: ≥ 2.5 mg/mL (5.29 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 1.67 mg/mL (3.53 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 5 中的溶解度: ≥ 1.67 mg/mL (3.53 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 6 中的溶解度: 0.33 mg/mL (0.70 mM) in 1% DMSO 99% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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；
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