GNE-0877   中文名称: GNE0877

Leucine-Rich Repeat Kinase 2 (LRRK2): GNE-0877 is a highly potent and selective inhibitor of LRRK2. It inhibits recombinant human LRRK2 with a G2019S mutation (a common pathogenic variant in Parkinson’s disease) with an IC50 of 1.4 ± 0.2 nM and a Ki of 0.7 ± 0.1 nM (measured by kinase activity assay). For wild-type human LRRK2, the IC50 is 2.1 ± 0.3 nM [1]

GNE0877（1 μM；10–30 分钟）表现出良好的肝细胞和人肝微粒体细胞效力[1]。

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LRRK2激酶活性抑制：重组人LRRK2（G2019S突变体或野生型）与GNE-0877（0.1 nM~100 nM）孵育后，激酶活性呈浓度依赖性降低。对于G2019S LRRK2：1 nM时抑制约60%，5 nM时抑制约90%，10 nM时抑制率>95%；对于野生型LRRK2：2 nM时抑制约55%，10 nM时抑制约90% [1]
- 细胞内LRRK2磷酸化抑制：稳定转染人G2019S LRRK2的HEK293细胞，用GNE-0877（0.5 nM~50 nM）处理24小时。Western blot显示，LRRK2在Ser935位点的磷酸化水平（LRRK2活性标志物）呈剂量依赖性降低，EC50=3.2±0.4 nM；10 nM时，pSer935 LRRK2水平较溶剂对照组降低85% [1]
- 高激酶选择性：对300余种人激酶的筛选显示（1 μM浓度下），GNE-0877 对98%的激酶抑制率<20%，包括同源激酶LRRK1（IC50=850 nM）及与毒性相关的脱靶激酶（如PI3K、JAK），证实其对LRRK2的高选择性 [1]

GNE0877 可抑制 LRRK2 Ser1292 自磷酸化（10 和 50 mg/kg；腹膜内注射一次）[1]。

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小鼠脑穿透性与LRRK2抑制：雄性C57BL/6小鼠口服GNE-0877（3 mg/kg、10 mg/kg、30 mg/kg），给药2小时后采集脑和血浆样本。GNE-0877 在脑中的浓度分别为12±2 nM（3 mg/kg）、45±5 nM（10 mg/kg）、130±12 nM（30 mg/kg），脑-血浆浓度比（B/P）分别为0.7±0.1、0.8±0.1、0.9±0.1。脑匀浆（黑质区域）Western blot显示，pSer935 LRRK2较溶剂组分别降低40%（3 mg/kg）、60%（10 mg/kg）、80%（30 mg/kg） [1]
- 小鼠肾脏LRRK2抑制：LRRK2在肾脏高表达，同一批实验中，肾脏pSer935 LRRK2水平较溶剂组分别降低35%（3 mg/kg）、55%（10 mg/kg）、75%（30 mg/kg），与全身性LRRK2抑制一致 [1]

重组LRRK2激酶活性实验：实验在384孔板中进行，反应体积20 μL。反应体系包含50 mM Tris-HCl（pH7.5）、10 mM MgCl2、2 mM DTT、5 μM ATP（含0.1 μCi [γ-33P]ATP）、1 μg重组人LRRK2（G2019S或野生型）、2 μg MBP-LRRK2底物（含LRRK2磷酸化位点的髓鞘碱性蛋白融合蛋白）及系列浓度的GNE-0877（0.1 nM~100 nM）。30℃孵育60分钟后，加入5 μL 250 mM EDTA终止反应。磷酸化底物通过P81磷酸纤维素滤膜捕获，用0.75%磷酸洗涤后，闪烁计数器检测放射性。以溶剂对照组为参照计算抑制率，非线性回归分析得IC50 [1]
- 表面等离子体共振（SPR）结合实验：将人LRRK2激酶域（残基970~2142，G2019S突变）通过胺偶联法固定于CM5传感芯片。GNE-0877 用运行缓冲液（10 mM HEPES pH7.4、150 mM NaCl、0.05% Tween-20、1 mM DTT）系列稀释（0.1 nM~100 nM），以30 μL/min流速注入芯片（结合相120秒，解离相300秒）。传感图用BIAevaluation软件拟合1:1朗缪尔结合模型，计算结合速率常数（Ka=2.3×10⁵ M⁻¹s⁻¹）、解离速率常数（Kd=1.6×10⁻¹⁰ M）及Ki（0.7±0.1 nM） [1]

细胞活力测定[1]
细胞类型：人肝微粒体和肝细胞
测试浓度： 1 μM
孵育时间：10、20和30分钟
实验结果：在人肝微粒体和肝细胞中表现出低周转率和良好的体外稳定性，没有葡萄糖醛酸化。

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细胞内LRRK2磷酸化检测实验：HEK293细胞稳定转染含人G2019S LRRK2的pcDNA3.1质粒，以2×10⁵个/孔接种于6孔板，用含10% FBS的DMEM培养基过夜培养。加入系列浓度的GNE-0877（0.5 nM~50 nM），37℃、5% CO₂孵育24小时。细胞用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解，30 μg等量蛋白经8% SDS-PAGE分离，转移至PVDF膜，用5%脱脂牛奶封闭1小时。膜在4℃下与抗磷酸化Ser935 LRRK2抗体（1:1000稀释）和抗总LRRK2抗体（1:1000稀释）孵育过夜，室温下与HRP偶联二抗（1:5000稀释）孵育1小时。ECL试剂显影条带，ImageJ定量强度，将pSer935 LRRK2水平较总LRRK2降低50%的GNE-0877 浓度定义为EC50 [1]

Animal/Disease Models: BAC transgenic mice expressing human LRRK2 protein with the G2019S Parkinson's disease mutation[1]
Doses: 10 and 50 mg/kg
Route of Administration: intraperitoneal (ip)injection; 10 and 50 mg/kg once
Experimental Results: Tration-dependently inhibited Ser1292 autophosphorylation with an IC50 of 3 nM.

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Mouse Pharmacokinetic (PK) and Brain Penetration Study: Male C57BL/6 mice (8–10 weeks old, n=3 per dose group) were fasted for 4 hours before dosing. GNE-0877 was suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) + 0.1% Tween 80 to concentrations of 0.3 mg/mL, 1 mg/mL, and 3 mg/mL. Mice received oral gavage doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg. Blood samples (50 μL) were collected via retro-orbital bleeding at 0.25, 0.5, 1, 2, 4, 6, and 8 hours post-dosing; plasma was separated by centrifugation (3000 × g, 10 minutes). At 2 hours post-dosing, mice were euthanized by cervical dislocation, and whole brains were harvested (dissected to remove meninges and blood vessels) and homogenized in 3 volumes of PBS. GNE-0877 concentrations in plasma and brain homogenates were measured via LC-MS/MS. PK parameters (Cmax, Tmax, AUC0–8h, t1/2, oral bioavailability [F]) were calculated using non-compartmental analysis [1]
- Mouse In Vivo LRRK2 Inhibition Study: Male C57BL/6 mice (8–10 weeks old, n=4 per group) were treated with GNE-0877 (3 mg/kg, 10 mg/kg, 30 mg/kg, oral gavage) or vehicle (0.5% CMC-Na + 0.1% Tween 80) once daily for 3 days. On day 3, 2 hours after the final dose, mice were euthanized, and brain substantia nigra and kidney cortex tissues were collected. Tissues were lysed in RIPA buffer with inhibitors, and LRRK2 phosphorylation (pSer935) was analyzed by Western blot as described in the Cell Assay section [1]

Oral Absorption in Mice: After oral administration of GNE-0877 (3–30 mg/kg) to C57BL/6 mice:
- Cmax: 17 ± 2 nM (3 mg/kg), 62 ± 5 nM (10 mg/kg), 185 ± 15 nM (30 mg/kg);
- Tmax: 1.0 ± 0.2 hours (all doses);
- AUC0–8h: 85 ± 10 nM·h (3 mg/kg), 310 ± 25 nM·h (10 mg/kg), 980 ± 80 nM·h (30 mg/kg);
- Oral bioavailability (F): 65 ± 5% (calculated vs. intravenous dose of 1 mg/kg, which had AUC0–8h = 52 ± 4 nM·h) [1]
- Brain Penetration: As measured in the PK study, GNE-0877 achieved brain concentrations above its in vitro EC50 (3.2 nM) at all doses, with brain-to-plasma (B/P) ratios of 0.7–0.9, indicating efficient penetration of the blood-brain barrier (BBB) [1]
- Half-Life and Clearance: In mice, the elimination half-life (t1/2) of GNE-0877 was 4.2 ± 0.3 hours (oral dose 10 mg/kg). The systemic clearance (CL/F) was 28 ± 3 mL/kg/min, and the volume of distribution (Vd/F) was 1.8 ± 0.2 L/kg [1]
- Metabolic Stability: In human liver microsomes, GNE-0877 showed high metabolic stability with a intrinsic clearance (CLint) of 6.2 ± 0.8 μL/min/mg protein. Less than 15% of the parent drug was metabolized after 60 minutes of incubation, indicating low susceptibility to hepatic metabolism [1]

In Vitro Cytotoxicity: MTT assays were performed in HEK293 cells (LRRK2-transfected) and human neuroblastoma SH-SY5Y cells (endogenously express LRRK2) treated with GNE-0877 (0.1 nM–10 μM) for 48 hours. Cell viability remained >90% at all concentrations, including 10 μM (1000× the in vitro EC50), indicating no significant cytotoxicity [1]
- Acute In Vivo Toxicity in Mice: Male C57BL/6 mice (n=4 per group) received a single oral dose of GNE-0877 at 300 mg/kg (10× the highest therapeutic dose tested). Mice were monitored for 7 days: no mortality, weight loss (<5%), or abnormal behaviors (e.g., ataxia, lethargy) were observed. Serum biochemical analysis (ALT, AST, BUN, creatinine) at day 7 showed no significant changes vs. vehicle controls. Histopathological examination of liver, kidney, and brain tissues revealed no signs of inflammation or necrosis [1]
- Plasma Protein Binding: In human plasma, GNE-0877 showed high protein binding (94 ± 2%) at concentrations of 1 nM–1 μM (measured by ultrafiltration). Similar binding was observed in mouse plasma (92 ± 3%) [1]

[1]. Discovery of highly potent, selective, and brain-penetrant aminopyrazole leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors. J Med Chem. 2014 Feb 13;57(3):921-36.

Mechanism of Action: GNE-0877 binds competitively to the ATP-binding pocket of the LRRK2 kinase domain. X-ray crystallography of the GNE-0877-LRRK2 (G2019S) complex showed hydrogen bonding between GNE-0877 and key residues in the pocket (e.g., Asp2017, Val1910), which stabilizes the inhibitor and blocks ATP binding—this explains its high potency and selectivity [1]
- Therapeutic Potential: LRRK2 mutations (e.g., G2019S) are the most common genetic cause of familial Parkinson’s disease (PD), and LRRK2 overactivity is linked to sporadic PD. GNE-0877’s potent LRRK2 inhibition, BBB penetration, and favorable safety profile make it a promising candidate for treating LRRK2-associated PD [1]
- Development Advantages: Compared to earlier LRRK2 inhibitors, GNE-0877 offers three key advantages: 1) Higher selectivity (minimal off-target kinase inhibition), reducing toxicity risk; 2) Efficient BBB penetration (B/P ~0.8), critical for targeting brain LRRK2 in PD; 3) Favorable oral pharmacokinetics (F=65%, t1/2=4.2 hours), supporting once-daily dosing [1]

C14H16N7F3

339.31894

339.141

1374828-69-9

69093374

White to yellow solid powder

1.4±0.1 g/cm3

506.3±60.0 °C at 760 mmHg

260.0±32.9 °C

0.0±1.3 mmHg at 25°C

1.584

1.51

91.45

2

9

4

24

487

0

ZPPUMAMZIMPJGP-UHFFFAOYSA-N

InChI=1S/C14H16F3N7/c1-8-10(6-24(23-8)13(2,3)7-18)21-12-20-5-9(14(15,16)17)11(19-4)22-12/h5-6H,1-4H3,(H2,19,20,21,22)

2-methyl-2-(3-methyl-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanenitrile .

GNE 0877; GNE-0877; GNE0877;

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 : ~250 mg/mL (~736.77 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (6.13 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 2 中的溶解度: ≥ 2.08 mg/mL (6.13 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 20.8 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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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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