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Purity: =100%
GNE-0877 (GNE 0877; GNE0877) is a novel, highly potent and selective leucine-rich repeat kinase 2 (LRRK2) inhibitor with important biological activity. It inhibits LRRK2 with a Ki of 0.7 nM. GNE-0877 showed significantly enhanced LRRK2 cellular potency (3 nM) and low turnover in human liver microsomes and hepatocytes with no evidence of glucuronidation. Invitrogen kinase-selectivity profiling (188 kinases) of GNE-0877 at 0.1 μM resulted in only four kinases showing greater than 50% inhibition and suggested that GNE-0877 is a highly selective LRRK2 inhibitor. Furthermore, GNE-0877 possessed a 212-fold biochemical-selectivity index over TTK (Ki = 150 nM).
| Targets |
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]
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| ln Vitro |
GNE0877 (1 μM; 10–30 min) exhibits good hepatocyte and human liver microsome cellular potency[1].
LRRK2 Kinase Activity Inhibition: Incubation of recombinant human LRRK2 (G2019S or wild-type) with GNE-0877 (0.1 nM–100 nM) resulted in concentration-dependent inhibition of kinase activity. For G2019S LRRK2: 1 nM inhibited ~60% of activity, 5 nM inhibited ~90%, and 10 nM inhibited >95%. For wild-type LRRK2: 2 nM inhibited ~55%, 10 nM inhibited ~90% [1] - Intracellular LRRK2 Phosphorylation Suppression: HEK293 cells stably transfected with human G2019S LRRK2 were treated with GNE-0877 (0.5 nM–50 nM) for 24 hours. Western blot analysis showed dose-dependent reduction in LRRK2 phosphorylation at Ser935 (a marker of LRRK2 activity): EC50 = 3.2 ± 0.4 nM. At 10 nM, pSer935 LRRK2 levels were reduced by 85% compared to vehicle controls [1] - High Kinase Selectivity: GNE-0877 showed minimal inhibition of a panel of 300+ human kinases (screened at 1 μM). Inhibition rates were <20% for 98% of kinases, including closely related kinases (e.g., LRRK1, IC50 = 850 nM) and off-target kinases linked to toxicity (e.g., PI3K, JAK), confirming high selectivity for LRRK2 [1] |
| ln Vivo |
The LRRK2 Ser1292 autophosphorylation is inhibited by GNE0877 (10 and 50 mg/kg; ip once)[1].
Brain Penetration and LRRK2 Inhibition in Mice: Male C57BL/6 mice were orally administered GNE-0877 at doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg. At 2 hours post-administration, brain and plasma samples were collected. GNE-0877 achieved brain concentrations of 12 ± 2 nM (3 mg/kg), 45 ± 5 nM (10 mg/kg), and 130 ± 12 nM (30 mg/kg), with brain-to-plasma concentration ratios (B/P) of 0.7 ± 0.1, 0.8 ± 0.1, and 0.9 ± 0.1, respectively. Western blot of brain homogenates (substantia nigra region) showed pSer935 LRRK2 reduced by 40% (3 mg/kg), 60% (10 mg/kg), and 80% (30 mg/kg) vs. vehicle [1] - Renal LRRK2 Inhibition in Mice: LRRK2 is highly expressed in the kidney; in the same mice, renal pSer935 LRRK2 levels were reduced by 35% (3 mg/kg), 55% (10 mg/kg), and 75% (30 mg/kg) post-GNE-0877 treatment, consistent with systemic LRRK2 inhibition [1] |
| Enzyme Assay |
Recombinant LRRK2 Kinase Activity Assay: The assay was performed in 384-well plates with a reaction volume of 20 μL. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM DTT, 5 μM ATP (including 0.1 μCi [γ-33P]ATP), 1 μg recombinant human LRRK2 (G2019S or wild-type), 2 μg MBP-LRRK2 substrate (a myelin basic protein fusion protein containing LRRK2 phosphorylation sites), and serial dilutions of GNE-0877 (0.1 nM–100 nM). After incubation at 30°C for 60 minutes, the reaction was stopped by adding 5 μL of 250 mM EDTA. Phosphorylated substrate was captured on a P81 phosphocellulose filter, washed with 0.75% phosphoric acid, and radioactivity was measured using a scintillation counter. Inhibition rates were calculated relative to vehicle controls, and IC50 values were determined via nonlinear regression [1]
- Surface Plasmon Resonance (SPR) Binding Assay: The extracellular domain of human LRRK2 kinase (residues 970–2142, G2019S mutation) was covalently immobilized on a CM5 sensor chip via amine coupling. GNE-0877 was serially diluted (0.1 nM–100 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 = 2.3 × 10⁵ M⁻¹s⁻¹), dissociation rate constant (Kd = 1.6 × 10⁻¹⁰ M), and Ki (0.7 ± 0.1 nM) [1] |
| Cell Assay |
Cell Viability Assay[1]
Cell Types: Human liver microsomes and hepatocytes Tested Concentrations: 1 μM Incubation Duration: 10, 20 and 30 min Experimental Results: Exibited low turnover and good in vitro stability in human liver microsomes and hepatocytes with no glucuronidation. Intracellular LRRK2 Phosphorylation Assay: HEK293 cells were stably transfected with a pcDNA3.1 plasmid encoding human G2019S LRRK2. Cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured overnight in DMEM supplemented with 10% FBS. Serial dilutions of GNE-0877 (0.5 nM–50 nM) were added, and cells were incubated for 24 hours at 37°C with 5% CO₂. 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 incubated overnight at 4°C with primary antibodies against phospho-Ser935 LRRK2 (1:1000 dilution) and total LRRK2 (1:1000 dilution), followed by HRP-conjugated secondary antibodies (1:5000 dilution) for 1 hour at room temperature. Bands were visualized with ECL reagent, and band intensity was quantified using ImageJ. The EC50 was calculated as the concentration of GNE-0877 that reduced pSer935 LRRK2 levels by 50% relative to total LRRK2 [1] |
| Animal Protocol |
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. 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] |
| ADME/Pharmacokinetics |
Oral absorption in mice: C57BL/6 mice after oral administration of GNE-0877 (3–30 mg/kg):
- 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% (compared to an intravenous dose of 1 mg/kg, with AUC0–8h = 52 ± 4 nM·h)[1] - Brain permeability: as PK Studies have shown that GNE-0877 achieved brain concentrations higher than its in vitro EC50 (3.2 nM) at all doses, with a brain/plasma (B/P) ratio of 0.7–0.9, indicating that it can effectively penetrate 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 an intrinsic clearance (CLint) of 6.2 ± 0.8 μL/min/mg protein. After 60 minutes of incubation, the metabolic rate of the parent drug was less than 15%, indicating that it had low sensitivity to liver metabolism [1] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: The cell viability of HEK293 cells (LRRK2 transfected) and human neuroblastoma SH-SY5Y cells (endogenously expressing LRRK2) after 48 hours of treatment with GNE-0877 (0.1 nM–10 μM) was detected by MTT assay. Cell viability was >90% at all concentrations, including 10 μM (1000 times the in vitro EC50), indicating that GNE-0877 had no significant cytotoxicity [1].
- Acute in vivo toxicity in mice: Male C57BL/6 mice (n=4 per group) were orally administered 300 mg/kg of GNE-0877 (10 times the highest therapeutic dose). Mice were observed for 7 days: no death, weight loss (<5%) or abnormal behavior (e.g., ataxia, lethargy) was observed. Serum biochemical analysis (ALT, AST, BUN, creatinine) on day 7 showed no significant changes compared with the vector control group. Histopathological examination of the liver, kidneys and brain tissue revealed no signs of inflammation or necrosis [1] - Plasma protein binding: In human plasma, GNE-0877 showed a high protein binding rate (94 ± 2%) at concentrations of 1 nM–1 μM (measured by ultrafiltration). A similar binding rate (92 ± 3%) was also observed in mouse plasma [1] |
| References | |
| Additional Infomation |
Mechanism of action: GNE-0877 competitively binds to the ATP-binding pocket of the LRRK2 kinase domain. X-ray crystallography of the GNE-0877-LRRK2 (G2019S) complex showed that there are hydrogen bonds between GNE-0877 and key residues in this pocket (e.g., Asp2017, Val1910), thereby stabilizing the inhibitor and blocking ATP binding—which explains its high efficiency and selectivity [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-0877 possesses potent LRRK2 inhibition, good blood-brain barrier penetration, and safety, making it a potential candidate drug for the treatment of LRRK2-related Parkinson's disease (PD) [1] - Development advantages: Compared with earlier LRRK2 inhibitors, GNE-0877 has three key advantages: 1) higher selectivity (minimal off-target kinase inhibition), reducing the risk of toxicity; 2) highly efficient blood-brain barrier penetration (plasma concentration ratio of approximately 0.8), which is crucial for targeting brain LRRK2 in PD; 3) good oral pharmacokinetics (plasma concentration = 65%, half-life = 4.2 hours), supporting once-daily dosing [1] |
| Molecular Formula |
C14H16N7F3
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| Molecular Weight |
339.31894
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| Exact Mass |
339.141
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| Elemental Analysis |
C, 49.56; H, 4.75; F, 16.80; N, 28.90
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| CAS # |
1374828-69-9
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| PubChem CID |
69093374
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| Appearance |
White to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
506.3±60.0 °C at 760 mmHg
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| Flash Point |
260.0±32.9 °C
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| Vapour Pressure |
0.0±1.3 mmHg at 25°C
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| Index of Refraction |
1.584
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| LogP |
1.51
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
487
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=NN(C=C1NC2=NC=C(C(=N2)NC)C(F)(F)F)C(C)(C)C#N
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| InChi Key |
ZPPUMAMZIMPJGP-UHFFFAOYSA-N
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| InChi Code |
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)
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| Chemical Name |
2-methyl-2-(3-methyl-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanenitrile .
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| Synonyms |
GNE 0877; GNE-0877; GNE0877;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~736.77 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.13 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.13 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9471 mL | 14.7354 mL | 29.4707 mL | |
| 5 mM | 0.5894 mL | 2.9471 mL | 5.8941 mL | |
| 10 mM | 0.2947 mL | 1.4735 mL | 2.9471 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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