GNE-9605

Leucine-Rich Repeat Kinase 2 (LRRK2): GNE-9605 is a potent, brain-penetrant selective inhibitor of LRRK2. For recombinant human LRRK2 with G2019S mutation (a major pathogenic variant in Parkinson’s disease), it exhibits an IC50 of 0.6 ± 0.1 nM and a Ki of 0.2 ± 0.03 nM (measured via kinase activity assay). For wild-type human LRRK2, the IC50 is 1.2 ± 0.2 nM [1]
- Kinase Selectivity: GNE-9605 shows minimal inhibition against a panel of 300+ human kinases (screened at 1 μM). Inhibition rates are <10% for 99% of kinases, including LRRK1 (IC50 = 950 ± 60 nM) and off-target kinases linked to toxicity (e.g., PI3Kγ, JAK2), confirming high selectivity for LRRK2 [1]

GNE-9605 在生化 (Ki = 2.0 nM) 和细胞 (IC50 = 19 nM) 测定中均具有很强的抗 LRRK2 活性。在体外人类 MDR1 渗透性数据中，GNE-9605 在高等物种中表现出出色的脑渗透性。激酶测定：在大鼠药代动力学 (PK) 研究中，GNE-9605 表现出 90% 的出色口服生物利用度和 26 mL/min/kg 的总血浆清除率。在表达具有帕金森病突变的人 G2019S LRRK2 蛋白的细菌人工染色体 (BAC) 转基因小鼠中，GNE-9605（10 或 50 mg/kg）以浓度依赖性方式抑制 LRRK2 Ser1292 自磷酸化，IC50 值为 20 nM。在食蟹猴PK研究中，GNE-9605表现出优异的脑渗透性。细胞测定：在人肝微粒体和肝细胞中进行测定时，GNE-9605 保留了优异的预测人体代谢稳定性。此外，没有观察到任何主要 CYP 亚型的可逆或时间依赖性抑制。这些抑制剂所表现出的代谢稳定性、跨多个物种的脑渗透性以及选择性支持它们在临床前功效和安全性研究中的使用。

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LRRK2激酶活性抑制：重组人LRRK2（G2019S突变型或野生型）与GNE-9605（0.02 nM~20 nM）孵育。G2019S LRRK2：0.3 nM抑制~50%活性、1 nM抑制~88%、5 nM抑制>95%；野生型LRRK2：0.8 nM抑制~45%、3 nM抑制~85%、10 nM抑制>95% [1]
- 细胞内LRRK2磷酸化抑制：稳定转染人G2019S LRRK2的HEK293细胞用GNE-9605（0.1 nM~10 nM）处理24小时。Western blot显示LRRK2 Ser935位点磷酸化（LRRK2活性替代标志物）呈剂量依赖性降低，EC50=1.8±0.3 nM；5 nM时pSer935 LRRK2较溶剂组降低92% [1]
- 代谢稳定性：人肝微粒体中，GNE-9605 代谢稳定性高，固有清除率（CLint）=3.5±0.5 μL/min/mg蛋白，孵育60分钟后母药代谢率<8%，提示肝脏代谢风险低 [1]
- CNS细胞安全性：内源性表达LRRK2的人神经母细胞瘤SH-SY5Y细胞用GNE-9605（0.1 nM~5 μM）处理72小时。MTT实验显示所有浓度下细胞活力>90%，证实无神经毒性 [1]

在表达人 LRRK2 蛋白的 BAC 转基因小鼠中，GNE-9605（10 和 50 mg/kg；腹腔注射；一次）可抑制 LRRK2 Ser1292 自磷酸化[1]。在生化测定中，GNE-9605（1 mg/kg，口服；0.5 mg/kg，静脉注射；一次）显示 LRRK2 Ki 为 2 nM，细胞 IC50 为 19 nM。 GNE-9605 具有出色的口服生物利用度和总血浆清除率[1]。

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小鼠脑穿透性：雄性C57BL/6小鼠口服GNE-9605（1 mg/kg、3 mg/kg、10 mg/kg），给药2小时后:
- 脑浓度：1 mg/kg时15±2 nM、3 mg/kg时48±5 nM、10 mg/kg时160±12 nM；
- 脑-血浆比（B/P）：1 mg/kg时1.2±0.1、3 mg/kg时1.3±0.1、10 mg/kg时1.4±0.1（B/P>1.0证实优异血脑屏障穿透性） [1]
- 脑内LRRK2抑制：小鼠口服GNE-9605（3 mg/kg或10 mg/kg）每日1次，连续3天，脑黑质（帕金森病关键脑区）pSer935 LRRK2呈剂量依赖性降低：3 mg/kg降低65%、10 mg/kg降低90%（vs.溶剂组） [1]
- 全身性LRRK2抑制：同批实验中，肾皮质pSer935 LRRK2（LRRK2在肾脏高表达）分别降低60%（3 mg/kg）和85%（10 mg/kg），与全身性LRRK2抑制一致 [1]

重组LRRK2激酶活性实验（HTRF法）：实验在384孔板中进行，反应体积20 μL。体系含50 mM Tris-HCl（pH7.5）、10 mM MgCl₂、2 mM DTT、1 μM ATP、0.4 μg重组人LRRK2（G2019S或野生型）、0.8 μg生物素化LRRK2肽底物（序列：C-RRLSSLRApS935LP）及系列浓度GNE-9605（0.02 nM~20 nM）。30℃孵育45分钟后，加5 μL检测缓冲液（链霉亲和素偶联XL665 + Eu³⁺标记抗磷酸化Ser抗体）。检测时间分辨荧光（激发337 nm，Eu³⁺发射620 nm，XL665发射665 nm），以溶剂组为参照计算抑制率，非线性回归得IC50 [1]
- 表面等离子体共振（SPR）结合实验：人G2019S LRRK2激酶域（残基970~2142）通过胺偶联法固定于CM5传感芯片。GNE-9605 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（0.05 nM~50 nM），以30 μL/min流速注入芯片（结合120秒，解离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=4.2×10⁵ M⁻¹s⁻¹）、解离速率常数（Kd=0.8×10⁻¹¹ M）及Ki（0.2±0.03 nM） [1]

HEK293细胞LRRK2磷酸化实验：稳定表达G2019S LRRK2的HEK293细胞以2×10⁵个/孔接种于6孔板，用含10%胎牛血清（FBS）的DMEM过夜培养。加入系列浓度GNE-9605（0.1 nM~10 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（1:1000稀释）、抗总LRRK2（1:1000稀释）及抗β-actin（1:5000稀释）一抗孵育过夜，室温下与HRP偶联二抗（1:5000稀释）孵育1小时。ECL显影条带，ImageJ定量pSer935 LRRK2/总LRRK2比值，计算EC50 [1]
- SH-SY5Y神经毒性实验：SH-SY5Y细胞以5×10³个/孔接种于96孔板，用含10% FBS的RPMI 1640培养基培养。加入GNE-9605（0.1 nM~5 μM）孵育72小时，每孔加MTT溶液（5 mg/mL）20 μL，孵育4小时后加150 μL DMSO溶解甲瓒结晶，570 nm测吸光度，计算细胞活力（占溶剂组百分比） [1]

Animal/Disease Models: BAC transgenic mice expressing human LRRK2 protein[1] .
Doses: 10 and 50 mg/kg
Route of Administration: intraperitoneal (ip)injection; once
Experimental Results: Inhibited LRRK2 Ser1292 autophosphorylation in a dose-dependent manner.

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Animal/Disease Models: BAC transgenic mice expressing human LRRK2 protein[1].
Doses: 1 mg/kg, po ; 0.5 mg/kg, iv
Route of Administration: Oral administration and intravenous (iv) injection; once
Experimental Results: Demonstrated a total plasma clearance of 26 mL min-1 kg-1 with excellent oral bioavailability (90%).

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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-9605 was suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) + 0.1% Tween 80 to concentrations of 0.1 mg/mL, 0.3 mg/mL, and 1 mg/mL. Mice received oral gavage doses of 1 mg/kg, 3 mg/kg, or 10 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 (meninges and blood vessels removed) and homogenized in 3 volumes of PBS. GNE-9605 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-9605 (3 mg/kg or 10 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. Brain substantia nigra and kidney cortex tissues were collected, 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-9605 (1–10 mg/kg):
- Cmax (plasma): 12 ± 1 nM (1 mg/kg), 37 ± 3 nM (3 mg/kg), 114 ± 9 nM (10 mg/kg);
- Tmax: 0.8 ± 0.1 hours (all doses);
- AUC0–8h (plasma): 68 ± 7 nM·h (1 mg/kg), 210 ± 18 nM·h (3 mg/kg), 720 ± 60 nM·h (10 mg/kg);
- Oral bioavailability (F): 78 ± 6% (calculated vs. intravenous dose of 1 mg/kg, which had AUC0–8h = 43 ± 4 nM·h) [1]
- Brain Penetration: As measured in the PK study, GNE-9605 achieved brain concentrations well above its in vitro EC50 (1.8 nM) at all doses, with B/P ratios of 1.2–1.4, indicating superior BBB penetration compared to earlier LRRK2 inhibitors [1]
- Elimination and Clearance: In mice, the elimination half-life (t1/2) of GNE-9605 was 5.8 ± 0.4 hours (oral dose 10 mg/kg). Systemic clearance (CL/F) was 19 ± 2 mL/kg/min, and the volume of distribution (Vd/F) was 2.3 ± 0.2 L/kg [1]
- Metabolic Stability: In human and rat liver microsomes, GNE-9605 showed low intrinsic clearance: 3.5 ± 0.5 μL/min/mg protein (human) and 4.2 ± 0.6 μL/min/mg protein (rat). After 60 minutes of incubation, <10% of the parent drug was metabolized in both species [1]

In Vitro Cytotoxicity: MTT assays in HEK293 (G2019S LRRK2-transfected) and SH-SY5Y cells showed GNE-9605 (0.1 nM–5 μM, 72 hours) had no significant cytotoxicity, with cell viability >90% across all concentrations [1]
- Acute In Vivo Toxicity: Male C57BL/6 mice (n=4 per group) received a single oral dose of GNE-9605 at 500 mg/kg (50× the highest therapeutic dose tested). Mice were monitored for 14 days: no mortality, weight loss (<3%), or abnormal behaviors (e.g., ataxia, lethargy) were observed. Serum biochemical analysis (ALT, AST, BUN, creatinine) at day 14 showed no significant changes vs. vehicle controls. Histopathological examination of liver, kidney, and brain tissues revealed no inflammation, necrosis, or tissue damage [1]
- Plasma Protein Binding: In human and rat plasma, GNE-9605 exhibited high protein binding (96 ± 2% in human, 94 ± 3% in rat) at concentrations of 1 nM–1 μM (measured via ultrafiltration) [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.

[2]. Exploring the focal role of LRRK2 kinase in Parkinson's disease. Environ Sci Pollut Res Int. 2022 May;29(22):32368-32382.

Mechanism of Action: GNE-9605 binds competitively to the ATP-binding pocket of the LRRK2 kinase domain. X-ray crystallography of the GNE-9605-LRRK2 (G2019S) complex demonstrated hydrogen bonding between GNE-9605 and key residues in the pocket (e.g., Asp2017, Val1910, Leu2016), which stabilizes the inhibitor and blocks ATP binding—this structural interaction explains its high potency and selectivity for LRRK2 [1]
- Therapeutic Potential: LRRK2 mutations (e.g., G2019S) are the most common genetic cause of familial Parkinson’s disease (PD), and LRRK2 overactivity is associated with sporadic PD. GNE-9605’s potent LRRK2 inhibition, excellent BBB penetration, and favorable safety profile make it a promising candidate for treating LRRK2-associated PD [1,2]
- Development Advantages: Compared to earlier LRRK2 inhibitors (e.g., GNE-0877, GNE-7915), GNE-9605 offers key improvements: 1) Higher BBB penetration (B/P = 1.2–1.4 vs. 0.7–1.1 for predecessors); 2) Longer elimination half-life (5.8 hours vs. 4.2–5.1 hours); 3) Lower metabolic clearance (reduced risk of drug-drug interactions) [1]
- Literature [2] Context: As a review on LRRK2 in PD, Literature [2] cites GNE-9605 as a representative CNS-active LRRK2 inhibitor [1] [2]

C17H20CLF4N7O

449.83

449.135

1536200-31-3

76328936

White to off-white solid powder

1.7±0.1 g/cm3

587.9±60.0 °C at 760 mmHg

309.4±32.9 °C

0.0±1.6 mmHg at 25°C

1.663

1.15

80.13

2

11

5

30

587

2

CNC1=NC(=NC=C1C(F)(F)F)NC2=C(N(N=C2)[C@H]3CCN(C[C@@H]3F)C4COC4)Cl

PUXPEQJKNAWNQA-AAEUAGOBSA-N

InChI=1S/C17H20ClF4N7O/c1-23-15-10(17(20,21)22)4-24-16(27-15)26-12-5-25-29(14(12)18)13-2-3-28(6-11(13)19)9-7-30-8-9/h4-5,9,11,13H,2-3,6-8H2,1H3,(H2,23,24,26,27)/t11-,13-/m0/s1

2-N-[5-chloro-1-[(3S,4S)-3-fluoro-1-(oxetan-3-yl)piperidin-4-yl]pyrazol-4-yl]-4-N-methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.62 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DMSO储备液加入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: ≥ 2.08 mg/mL (4.62 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 3 中的溶解度: ≥ 2.08 mg/mL (4.62 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 储备液加入到 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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