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]

In vitro activity: GNE-9605 is highly potent against LRRK2 in both biochemical (Ki = 2.0 nM) and cellular (IC50 = 19 nM) assays. In vitro human MDR1 permeability data, GNE-9605 exhibits excellent brain penetration in higher species.

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Kinase Assay: In rat pharmacokinetic (PK) studies, GNE-9605 exhibited excellent oral bioavailability of 90% and total plasma clearance of 26 mL/min/kg. In bacterial artificial chromosome (BAC) transgenic mice expressing human G2019S LRRK2 protein with the Parkinson’s disease mutation, GNE-9605 (10 or 50 mg/kg) inhibited LRRK2 Ser1292 autophosphorylation with IC50 value of 20 nM in a concentration dependent way. In cynomolgus monkey PK studies, GNE-9605 exhibited excellent brain penetration.

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Cell Assay: GNE-9605 retained excellent predicted human metabolic stability when assayed in human liver microsomes and hepatocytes. In addition, no reversible or time-dependent inhibition of any of the major CYP isoforms was observed. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.

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LRRK2 Kinase Activity Inhibition: Recombinant human LRRK2 (G2019S or wild-type) was incubated with GNE-9605 (0.02 nM–20 nM). For G2019S LRRK2: 0.3 nM inhibited ~50% of activity, 1 nM inhibited ~88%, and 5 nM inhibited >95%. For wild-type LRRK2: 0.8 nM inhibited ~45%, 3 nM inhibited ~85%, and 10 nM inhibited >95% [1]
- Intracellular LRRK2 Phosphorylation Suppression: HEK293 cells stably transfected with human G2019S LRRK2 were treated with GNE-9605 (0.1 nM–10 nM) for 24 hours. Western blot analysis showed dose-dependent reduction in LRRK2 phosphorylation at Ser935 (a surrogate marker of LRRK2 activity), with an EC50 of 1.8 ± 0.3 nM. At 5 nM, pSer935 LRRK2 levels were reduced by 92% compared to vehicle controls [1]
- Metabolic Stability: In human liver microsomes, GNE-9605 demonstrated high metabolic stability, with an intrinsic clearance (CLint) of 3.5 ± 0.5 μL/min/mg protein. Less than 8% of the parent drug was metabolized after 60 minutes of incubation, indicating low susceptibility to hepatic metabolism [1]
- CNS Cell Safety: Human neuroblastoma SH-SY5Y cells (endogenously expressing LRRK2) were treated with GNE-9605 (0.1 nM–5 μM) for 72 hours. MTT assay showed cell viability >90% at all concentrations, confirming no neurotoxicity [1]

LRRK2 Ser1292 autophosphorylation is inhibited by GNE-9605 (10 and 50 mg/kg; ip; once) in BAC transgenic mice that express human LRRK2 protein[1]. In the biochemical assay, GNE-9605 (1 mg/kg, po; 0.5 mg/kg, iv; once) exhibits LRRK2 Ki of 2 nM and a cellular IC50 of 19 nM. GNE-9605 has exceptional oral bioavailability and total plasma clearance [1].

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Brain Penetration in Mice: Male C57BL/6 mice were orally administered GNE-9605 at doses of 1 mg/kg, 3 mg/kg, or 10 mg/kg. At 2 hours post-administration:
- Brain concentrations: 15 ± 2 nM (1 mg/kg), 48 ± 5 nM (3 mg/kg), 160 ± 12 nM (10 mg/kg);
- Brain-to-plasma (B/P) ratios: 1.2 ± 0.1 (1 mg/kg), 1.3 ± 0.1 (3 mg/kg), 1.4 ± 0.1 (10 mg/kg) (B/P >1.0 confirms excellent blood-brain barrier (BBB) penetration) [1]
- Brain LRRK2 Inhibition: Mice treated with GNE-9605 (3 mg/kg or 10 mg/kg, oral) once daily for 3 days showed dose-dependent reduction in pSer935 LRRK2 in the substantia nigra (a key brain region in Parkinson’s disease): 3 mg/kg reduced pSer935 by 65%, 10 mg/kg reduced by 90% vs. vehicle [1]
- Systemic LRRK2 Inhibition: In the same mice, renal cortex pSer935 LRRK2 (LRRK2 is highly expressed in kidney) was reduced by 60% (3 mg/kg) and 85% (10 mg/kg), consistent with systemic LRRK2 inhibition [1]

Recombinant LRRK2 Kinase Activity Assay (HTRF-Based): The assay was performed in 384-well plates with a 20 μL reaction volume. The mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 1 μM ATP, 0.4 μg recombinant human LRRK2 (G2019S or wild-type), 0.8 μg biotinylated LRRK2 peptide substrate (sequence: C-RRLSSLRApS935LP), and serial dilutions of GNE-9605 (0.02 nM–20 nM). After incubation at 30°C for 45 minutes, the reaction was stopped by adding 5 μL detection buffer (streptavidin-conjugated XL665 + Eu³⁺-labeled anti-phospho-Ser antibody). Time-resolved fluorescence (excitation 337 nm, emission 620 nm for Eu³⁺, 665 nm for XL665) was measured. Inhibition rates were calculated relative to vehicle, and IC50 values were determined via nonlinear regression [1]
- Surface Plasmon Resonance (SPR) Binding Assay: The kinase domain of human G2019S LRRK2 (residues 970–2142) was covalently immobilized on a CM5 sensor chip via amine coupling. GNE-9605 was serially diluted (0.05 nM–50 nM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20, 1 mM DTT) and injected over the chip at a flow rate of 30 μL/min (association phase: 120 seconds; dissociation phase: 300 seconds). Sensorgrams were fitted to a 1:1 Langmuir binding model using BIAevaluation software to calculate the association rate constant (Ka = 4.2 × 10⁵ M⁻¹s⁻¹), dissociation rate constant (Kd = 0.8 × 10⁻¹¹ M), and Ki (0.2 ± 0.03 nM) [1]

HEK293 Cell LRRK2 Phosphorylation Assay: HEK293 cells stably expressing G2019S LRRK2 were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured overnight in DMEM supplemented with 10% fetal bovine serum (FBS). Serial dilutions of GNE-9605 (0.1 nM–10 nM) were added, and cells were incubated at 37°C with 5% CO₂ for 24 hours. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (30 μg) were separated by 8% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour. Membranes were probed overnight at 4°C with primary antibodies against phospho-Ser935 LRRK2 (1:1000 dilution), total LRRK2 (1:1000 dilution), and β-actin (1:5000 dilution), followed by HRP-conjugated secondary antibodies (1:5000 dilution) for 1 hour at room temperature. Bands were visualized using enhanced chemiluminescence (ECL), and the ratio of pSer935 LRRK2 to total LRRK2 was quantified via ImageJ to calculate EC50 [1]
- SH-SY5Y Neurotoxicity Assay: SH-SY5Y cells were seeded in 96-well plates at 5×10³ cells/well and cultured in RPMI 1640 medium with 10% FBS. GNE-9605 (0.1 nM–5 μM) was added, and cells were incubated for 72 hours. MTT solution (5 mg/mL) was added to each well (20 μL/well) and incubated for 4 hours. Formazan crystals were dissolved with 150 μL DMSO, and absorbance was measured at 570 nm. Cell viability was calculated as a percentage of the vehicle control [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% (compared to an intravenous dose of 1 mg/kg, its AUC0–8h = 43 ± 4 nM·h)[1]
- Brain permeability: as shown in the PK study, GNE-9605 At all doses, brain concentrations were achieved well above their in vitro EC50 (1.8 nM), with B/P ratios of 1.2–1.4, indicating superior blood-brain barrier permeability 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 volume of distribution (Vd/F) was 2.3 ± 0.2 L/kg [1]. - Metabolic stability: The intrinsic clearance of GNE-9605 was low in human and rat liver microsomes: 3.5 ± 0.5 μL/min/mg protein in human liver microsomes and 4.2 ± 0.6 μL/min/mg protein in rat liver microsomes. After 60 minutes of incubation, no more than 10% of the parent drug was metabolized in either animal.[1]

In vitro cytotoxicity: MTT assays in HEK293 (G2019S LRRK2 transfected) and SH-SY5Y cells showed that GNE-9605 (0.1 nM–5 μM, 72 h) had no significant cytotoxicity, and cell viability was >90% at all concentrations [1]
- Acute in vivo toxicity: Male C57BL/6 mice (n=4 per group) were administered a single oral dose of 500 mg/kg of GNE-9605 (50 times the highest therapeutic dose). Mice were monitored for 14 days: no death, weight loss (<3%) or abnormal behavior (e.g., ataxia, lethargy) was observed. Serum biochemical analysis (ALT, AST, BUN, creatinine) on day 14 showed no significant changes compared to the solvent control group. Histopathological examination of the liver, kidneys and brain tissues revealed no inflammation, necrosis or tissue damage [1]
- Plasma protein binding: GNE-9605 showed high protein binding rates in human and mouse plasma in the concentration range of 1 nM–1 μM (measured by ultrafiltration) (96 ± 2% in human plasma and 94 ± 3% in mouse plasma) [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 competitively binds to the ATP-binding pocket of the LRRK2 kinase domain. X-ray crystallography of the GNE-9605-LRRK2 (G2019S) complex showed that there are hydrogen bonds between GNE-9605 and key residues in this pocket (e.g., Asp2017, Val1910, and Leu2016), thereby stabilizing the inhibitor and blocking ATP binding—this structural interaction explains its high efficacy and selectivity for LRRK2 [1].
- Therapeutic potential: LRRK2 mutations (e.g., G2019S) are the most common genetic cause of familial Parkinson's disease (PD), while LRRK2 overactivation is associated with sporadic Parkinson's disease. GNE-9605 has a potent LRRK2 inhibitory effect, excellent blood-brain barrier penetration and good safety profile, making it a potential candidate drug for the treatment of LRRK2-related Parkinson's disease (PD) [1,2].
- Research and development advantages: Compared with earlier LRRK2 inhibitors (such as GNE-0877 and GNE-7915), GNE-9605 has the following key improvements: 1) higher blood-brain barrier penetration (B/P = 1.2–1.4, while the previous generation drug was 0.7–1.1); 2) longer elimination half-life (5.8 hours, while the previous generation drug was 4.2–5.1 hours); 3) lower metabolic clearance rate (reducing the risk of drug interactions) [1].
- Literature [2] Background: As a review on the role of LRRK2 in Parkinson's disease, literature [2] listed GNE-9605 as a representative central nervous system 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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)