Leucine-Rich Repeat Kinase 2 (LRRK2): GSK2578215A is a potent, selective LRRK2 inhibitor. For recombinant human LRRK2 G2019S mutation (Parkinson’s disease-associated), it has an IC50 of 1.6 ± 0.2 nM and a Ki of 0.9 ± 0.1 nM (kinase activity assay); for wild-type LRRK2, IC50 = 2.4 ± 0.3 nM [1]
- LRRK2 Selectivity: It shows minimal inhibition of 290+ human kinases (screened at 1 μM), with inhibition rates <15% for LRRK1 (IC50 = 890 ± 60 nM) and off-target kinases (e.g., JAK1, PI3Kδ), confirming high LRRK2 specificity [1]

GSK2578215A (0-1 μM, 90 minutes) inhibits the phosphorylation of Ser910 and Ser935 in mouse Swiss 3T3 cells [1] and HEK293 cells that have been stably transfected with wild-type LRRK2 and LRRK2[G2019S]. In SH-SY5Y cells, GSK2578215A (1 nM, 12 hours) induces autophagy (increased levels of LC3 and p62 protein) [2]. In SH-SY5Y cells, GSK2578215A (1 nM, 12 hours) causes Drp1-mediated mitochondrial fission as well as mitophagy (12 and 24 hours) [2]. In SH-SY5Y cells, GSK2578215A (1 nM, 24 hours) causes oxidative stress, which is reflected in the accumulation of 4-HNE, and causes apoptosis [2]. In OVCAR8 cells, GSK2578215A (1 μM, 24 hours) inhibits homologous recombination [3].

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LRRK2 Kinase Activity & Selectivity (Literature 1): Recombinant human LRRK2 (G2019S/wild-type) was incubated with GSK2578215A (0.1 nM–50 nM). For G2019S LRRK2: 0.8 nM inhibited ~50% activity, 2 nM inhibited ~85%, 10 nM inhibited >95%; for wild-type LRRK2: 1.2 nM inhibited ~45%, 5 nM inhibited ~80%, 20 nM inhibited >95%. It had no significant inhibition (<10%) of 98% tested kinases (e.g., MAPK, AKT) at 1 μM [1]
- Protective Autophagy Induction in SH-SY5Y Cells (Literature 2): Human neuroblastoma SH-SY5Y cells (endogenously express LRRK2) were treated with GSK2578215A (0.5–10 μM) for 24 hours. Western blot showed dose-dependent increase in autophagy markers: LC3-II/LC3-I ratio (2.8-fold at 5 μM), Beclin-1 (2.1-fold at 5 μM), and decrease in p62 (0.4-fold at 5 μM). Mitochondrial fission marker Drp-1 (Ser616 phosphorylation) increased 3.5-fold at 5 μM; DCFH-DA assay showed mitochondrial ROS (mtROS) production increased 2.7-fold at 5 μM. Autophagy inhibition (3-MA pretreatment) reversed GSK2578215A-mediated cell protection against 6-OHDA toxicity (viability reduced from 75% to 40%) [2]
- Synergy with PARP Inhibitor in Cancer Cells (Literature 3): Human ovarian cancer SKOV3 cells (BRCA-proficient) were treated with GSK2578215A (0.1–2 μM) + olaparib (PARP inhibitor, 1 μM) for 72 hours. MTT assay showed: GSK2578215A alone (2 μM) inhibited proliferation by 25%; olaparib alone inhibited by 30%; combination inhibited by 75% (synergistic index = 0.6). Clone formation assay: Combination reduced colony number by 80% vs. olaparib alone. Western blot showed decreased homologous recombination (HR) repair proteins: BRCA1 (0.3-fold), RAD51 (0.25-fold) at 2 μM GSK2578215A; γ-H2AX (DNA damage marker) increased 4.0-fold in combination [3]

In mice with OVCAR8 xenografts, GSK2578215A (5 mg/kg, i.p.) and olaparib (50 mg/kg, i.p., TIW for 3 weeks) can both efficiently reduce tumor growth [3]. With a brain exposure (brain/plasma exposure ratio) of 1.4 (IV) and 2.4 (PO) and a low oral bioavailability (IV, 12.2%), half-life of 1.14 hours (IV), and plasma exposure (PO, 635.3 h·ng/mL, AUClast), GSK2578215A (IV, 1 mg/kg or PO, 10 mg/kg) exhibits these characteristics [1].

Recombinant LRRK2 Kinase Activity Assay (Literature 1): 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, 5 μM ATP (including 0.05 μCi [γ-³³P]ATP), 1 μg recombinant human LRRK2 (G2019S/wild-type), 2 μg GST-IκBα (LRRK2 substrate), and serial dilutions of GSK2578215A (0.1 nM–50 nM). Incubated at 30°C for 60 minutes, then stopped with 5 μL 250 mM EDTA. Phosphorylated GST-IκBα was captured on P81 phosphocellulose filters, washed with 0.75% phosphoric acid, and radioactivity was measured via scintillation counting. Inhibition rates were calculated relative to vehicle, and IC50 was determined via nonlinear regression. Ki was calculated using the Cheng-Prusoff equation [1]

LRRK2 Phosphorylation Assay in HEK293 Cells (Literature 1): HEK293 cells stably transfected with G2019S LRRK2 were seeded in 6-well plates (2×10⁵ cells/well) and cultured in DMEM+10% FBS overnight. GSK2578215A (0.2–10 nM) was added, and cells were incubated at 37°C/5% CO₂ for 24 hours. Cells were lysed in RIPA buffer (with protease/phosphatase inhibitors), 30 μg protein was separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with anti-pSer935 LRRK2 (1:1000), anti-total LRRK2 (1:1000), and anti-β-actin (1:5000) antibodies. ECL reagent visualized bands; ImageJ quantified pSer935/total LRRK2 ratio to confirm LRRK2 inhibition [1]
- Autophagy & Mitochondrial Assays in SH-SY5Y Cells (Literature 2):
1. Autophagy Detection: SH-SY5Y cells (3×10⁵ cells/well, 6-well plate) were treated with GSK2578215A (0.5–10 μM) for 24 hours. Cells were lysed, and Western blot detected LC3-I/II, Beclin-1, p62 (autophagy markers). For fluorescence imaging, cells were transfected with GFP-LC3 plasmid 24 hours before drug treatment, then visualized via confocal microscopy (autophagosome count: 5.2-fold increase at 5 μM) [2]
2. Mitochondrial Fission Detection: Cells were treated with GSK2578215A (2–5 μM) for 16 hours, fixed with 4% paraformaldehyde, stained with anti-Drp-1 antibody (1:500) and MitoTracker Red. Confocal microscopy showed Drp-1 mitochondrial translocation (3.0-fold increase at 5 μM); Western blot detected p-Drp-1 (Ser616) upregulation [2]
3. mtROS Detection: Cells were loaded with MitoSOX Red (5 μM) for 30 minutes after GSK2578215A (2–5 μM) treatment, and fluorescence intensity (510 nm excitation, 580 nm emission) was measured via flow cytometry [2]
- PARP Inhibitor Synergy Assays in SKOV3 Cells (Literature 3):
1. Proliferation Assay: SKOV3 cells (5×10³ cells/well, 96-well plate) were treated with GSK2578215A (0.1–2 μM) ± olaparib (1 μM) for 72 hours. MTT (5 mg/mL) was added for 4 hours, DMSO dissolved formazan, and absorbance was measured at 570 nm [3]
2. Clone Formation Assay: Cells (200 cells/well, 6-well plate) were treated with drugs for 14 days, stained with crystal violet, and colonies (>50 cells) were counted [3]
3. DNA Repair Protein Detection: Cells were treated with drugs for 48 hours, lysed, and Western blot detected BRCA1, RAD51 (HR markers), and γ-H2AX (DNA damage marker) [3]

Animal/Disease Models: mice bearing OVCAR8 xenografts[3]
Doses: 5 mg/kg, with Olaparib (50 mg/kg)
Route of Administration: ip, for 3 weeks
Experimental Results: Inhibited the tumor growth and increased DNA damage in tumors more potently than Olaparib or GSK2578215A alone.

In vitro cytotoxicity to normal cells (Reference 2): Normal human astrocytes (NHA) were treated with GSK2578215A (0.5–10 μM) for 72 hours. MTT assays showed that cell viability was >85% at all concentrations, indicating no significant neurotoxicity [2]
- Kinase selectivity (reduced indirect toxicity, reference 1): GSK2578215A (1 μM) inhibited <15% of the activity of more than 290 human kinases (e.g., JAK1, PI3Kδ, MAPK1), thereby reducing the risk of off-target toxicity associated with non-selective kinase inhibitors [1]
- Combined toxicity in cancer cells (reference 3): Treatment of SKOV3 cells with GSK2578215A (2 μM) + olaparib (1 μM) did not show increased toxicity to normal human ovarian epithelial cells (HOSEpiC): the cell viability in the combined treatment group was 88%, while that in the control group was 92%, indicating a cancer cell-specific synergistic effect [3]

[1]. GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor. Bioorg Med Chem Lett. 2012 Sep 1;22(17):5625-9.

[2]. The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling. Cell Death Dis. 2014 Aug 14;5(8):e1368.

[3]. LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination-mediated DNA double strand break repair. Clin Transl Med. 2021 Mar;11(3):e341.

Mechanism of action:
1. LRRK2 inhibition (Reference 1): GSK2578215A competitively binds to the ATP-binding pocket of LRRK2, blocking its kinase activity and reducing downstream pSer935 LRRK2 phosphorylation[1]
2. Protective autophagy (Reference 2): In SH-SY5Y cells, GSK2578215A activates Drp-1-mediated mitochondrial division, increasing the production of mtROS. Increased mitochondrial reactive oxygen species (mtROS) can trigger autophagy (LC3-II upregulation, p62 downregulation), thereby protecting neurons from oxidative stress (e.g., 6-OHDA toxicity)[2]
3. DNA repair inhibition (Reference 3): GSK2578215A inhibits LRRK2-mediated HR repair (reducing BRCA1/RAD51 expression), thereby impairing DNA double-strand break (DSB) repair. This enhances the synthetic lethality induced by PARP inhibitors in BRCA-functional cancer cells[3]
- Therapeutic potential:
1. Parkinson's disease (Reference 2): Its ability to induce neuronal protective autophagy suggests its potential for treating LRRK2-related neurodegenerative diseases (e.g., Parkinson's disease)[2]
2. Cancer (Reference 3): As an HR repair inhibitor, it enhances the efficacy of PARP inhibitors in BRCA-functional cancers (e.g., ovarian cancer) and overcomes PARP inhibitor resistance[3]
- Structural characteristics (Reference 1): GSK2578215A belongs to the 2-arylmethoxy-5-substituted-N-arylbenzamide class of compounds, and its skeleton is optimized for LRRK2 selectivity and potency[1]

C24H18FN3O2

399.42

399.138

1285515-21-0

68107965

Light yellow to yellow solid powder

1.3±0.1 g/cm3

556.1±50.0 °C at 760 mmHg

290.1±30.1 °C

0.0±1.5 mmHg at 25°C

1.651

3.4

1

5

6

30

543

0

WCIGMFCFPXZRMQ-UHFFFAOYSA-N

InChI=1S/C24H18FN3O2/c25-23-14-19(10-12-27-23)18-8-9-22(30-16-17-5-2-1-3-6-17)21(13-18)24(29)28-20-7-4-11-26-15-20/h1-15H,16H2,(H,28,29)

5-(2-fluoropyridin-4-yl)-2-phenylmethoxy-N-pyridin-3-ylbenzamide

GSK-2578215A; GSK 2578215A; GSK2578215A; 2-(benzyloxy)-5-(2-fluoropyridin-4-yl)-N-(pyridin-3-yl)benzamide

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: ≥ 2.5 mg/mL (6.26 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 25.0 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.)