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Purity: ≥98%
PF-06447475 (PF06447475) is a novel, highly potent, selective and brain penetrable inhibitor of Leucine rich repeat kinase 2 (LRRK2) with anti-PD activity. It inhibits LRRK2 with IC50 of 3 nM. LRRK2 has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). PF-06447475 inhibits LRRK2 enzyme and LRRK2 in the whole cell assay with IC50s of 3 and 25 nM, respectively. Cells incubated with PF-06447475 alone (0.5, 1, 3 μM) or in the presence of ROT significantly reduces (S935)-LRRK2 kinase phosphorylation to control. PF-06447475 has the potential for the treatment of PD.
| Targets |
PF-06447475 specifically targets Leucine-rich Repeat Kinase 2 (LRRK2) (a serine-threonine protein kinase); IC50 is approximately 5 nM (determined by the ratio of Ser(P)-935 to total LRRK2 in mouse macrophage Raw264.7 cells). [3]
PF-06447475 is a potent and selective LRRK2 kinase inhibitor, with inhibitory activity verified in recombinant LRRK2 FRET assay and human peripheral blood monocytic cells (PBMCs) ActivX assay. [1] |
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| ln Vitro |
With IC50s of 3 and 25 nM, respectively, PF-06447475 inhibits LRRK2 enzyme and LRRK2 in the whole cell assay[1]. Cells treated with PF-06447475 alone (0.5, 1, 3 μM) or with ROT significantly lower the phosphorylation of (S935)-LRRK2 kinase compared to control. Compared to untreated and control, PF-06447475 significantly preserves the nucleus morphology and ΔΨm of NLCs exposed to ROT. PF-475 considerably reduces ROS production induced by ROT to a level comparable to cells exposed to PF-475 alone[2].
1. In human nerve-like differentiated cells (NLCs) exposed to oxidative stress induced by 50 μM rotenone (a PD model inducer): PF-06447475 at 1 μM completely blocked LRRK2 phosphorylation at serine 935 (p-S935-LRRK2), eliminated reactive oxygen species (ROS) overproduction (~100% increase induced by rotenone), reversed mitochondrial membrane potential (∆Ψₘ) reduction (~21% decrease), and normalized the overexpression of apoptosis-related markers: transcription factors NF-κB (5.6-fold increase), p53 (5.3-fold increase), c-Jun (5.4-fold increase), and proteins caspase-3 (8.0-fold increase)、AIF (6.8-fold increase). It also inhibited nuclear condensation/fragmentation (16% induced by rotenone). [2] 2. In mouse macrophage Raw264.7 cells: PF-06447475 treatment for 24 hours dose-dependently inhibited LRRK2 phosphorylation at Ser935, with a half-maximal inhibitory concentration (IC50) of ~5 nM (calculated by the ratio of Ser(P)-935 to total LRRK2). [3] 3. In vitro kinase activity assay: PF-06447475 exhibited potent inhibitory activity against recombinant full-length GST-tagged LRRK2 protein using LRRKtide as a substrate in a FRET assay (1 mM ATP present). It also showed inhibitory potential in human PBMCs tested by a commercial ActivX assay. [1] |
| ln Vivo |
Microgliosis in G2019S+ rats treated with PF-06447475 was significantly reduced to levels observed in wild-type rats. In confocal sections, the proinflammatory marker MHC-II, which is expressed on myeloid cells but not neurons, also seems to be less prevalent in G2019S+ rats given PF-06447475. In G2019S+ rats, treatment with PF-06447475 dramatically reduces the amount of CD68 cells recruited to the SNpc. PF-06447475 effectively inhibits the increased neuroinflammation linked to the expression of G2019S-LRRK2. TH expression in the dorsal striatum, which is in line with medication that lessens SNpc neurodegeneration[3]. Rats are able to tolerate PF-06447475 well[1].
1. In rat models of α-synuclein-induced neurodegeneration (PD model): [3] - Animal models: 10- to 12-week-old wild-type Sprague-Dawley rats and G2019S-LRRK2 transgenic rats. - Therapeutic effects: Oral administration of PF-06447475 at 30 mg·kg⁻¹ b.i.d. for 4 weeks mitigated dopaminergic neurodegeneration induced by unilateral intracranial injection of 6 × 10⁹ rAAV2 α-synuclein viral particles. It preserved tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) (confirmed by unbiased stereological counting), maintained TH fiber density in the dorsal striatum, and reduced neuroinflammation (decreased fractional area of Iba1 immunoreactivity and recruitment of CD68-positive cells in the SNpc). - Synergistic effect in transgenic rats: G2019S-LRRK2 transgenic rats showed exacerbated neurodegeneration and inflammation after α-synuclein overexpression, which were significantly attenuated by PF-06447475 treatment. 2. Brain penetrance: PF-06447475 penetrated the blood-brain barrier in rats, with unbound brain concentrations (Cbu) equivalent to unbound plasma concentrations (Cpu). [1] 3. Pharmacodynamic effects in rats: Oral administration of PF-06447475 to Sprague-Dawley rats for 14 days (b.i.d.) significantly reduced the ratio of Ser(P)-935-LRRK2 to total LRRK2 in the brain and kidney. [1] |
| Enzyme Assay |
1. LRRK2 kinase activity FRET assay: [3]
Recombinant full-length GST-tagged LRRK2 protein was prepared as the enzyme source, and LRRKtide was used as a surrogate kinase substrate. The assay was performed in the presence of 1 mM ATP. Different concentrations of PF-06447475 were added to the reaction system, and FRET signals were detected to evaluate the inhibitory effect on LRRK2 kinase activity. The half-maximal inhibitory concentration (IC50) was calculated based on the signal changes. 2. Human PBMCs ActivX assay: [1] Human peripheral blood monocytic cells (PBMCs) were treated with PF-06447475 at specified concentrations. A commercial ActivX assay kit was used to detect the binding of the compound to LRRK2 kinase, thereby verifying its inhibitory potential against endogenous LRRK2 in primary cells. [1] |
| Cell Assay |
1. Oxidative stress-induced cell injury and neuroprotection assay (human nerve-like differentiated cells, NLCs): [2]
NLCs were first induced and differentiated from mesenchymal stem cells. The cells were divided into control group, rotenone-treated group (50 μM, 6 hours), and PF-06447475 intervention group (1 μM, co-treated or pre-treated with rotenone). After incubation, ROS levels were detected by fluorescent probes; mitochondrial membrane potential (∆Ψₘ) was measured by flow cytometry or fluorescent staining; nuclear morphology was observed under a microscope to evaluate condensation/fragmentation; Western blot was performed to detect the expression levels of p-S935-LRRK2, total LRRK2, NF-κB, p53, c-Jun, caspase-3, and AIF. 2. LRRK2 phosphorylation inhibition assay (mouse macrophage Raw264.7 cells): [3] Raw264.7 cells were seeded in culture plates and adhered overnight. The cells were treated with serial concentrations of PF-06447475 for 24 hours. Total protein was extracted from the cells, and protein concentration was determined by BCA assay. Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were incubated with primary antibodies against Ser(P)-935-LRRK2 and total LRRK2, followed by HRP-conjugated secondary antibodies. The protein bands were visualized by chemiluminescence, and the ratio of Ser(P)-935-LRRK2 to total LRRK2 was calculated to determine the IC50 value. [3] |
| Animal Protocol |
3 and 30 mg/kg PF-06447475 (p.o. b.i.d.)
G2019S+ rats 1. α-synuclein-induced neurodegeneration rat model experiment: [3] - Animals: 10- to 12-week-old male Sprague-Dawley wild-type rats (G2019S⁻) and G2019S-LRRK2 BAC transgenic rats (G2019S⁺). - Model induction: Unilateral intracranial injection of 6 × 10⁹ rAAV2 α-synuclein viral particles into the substantia nigra (SNpc) of rats. - Drug administration: Starting after viral transduction, PF-06447475 was administered by oral gavage at a dose of 30 mg·kg⁻¹ twice a day (b.i.d.) for 4 weeks; the control group received a control compound via the same route and frequency. - Sample collection: Rats were sacrificed 90 minutes or 2 hours after the last dose. Brains (substantia nigra, striatum), kidneys, lungs, livers, and spleens were harvested. - Detection methods: Western blot to quantify Ser(P)-935-LRRK2 and total LRRK2 in brain and kidney tissues; histological staining (H&E, Masson's trichrome, LAMP2-DAB, CD68-DAB, Oil Red O) of visceral tissues; unbiased stereological counting of TH-positive neurons in the SNpc; quantification of TH fiber density in the dorsal striatum by LI-COR analysis. 2. Rat short-term pharmacodynamic and safety study: [1] - Animals: Groups of six Sprague-Dawley rats (three males and three females). - Drug administration: Oral gavage of PF-06447475 or vehicle control twice a day (b.i.d.) for 14 days. - Sample collection: Rats were sacrificed 90 minutes after the last dose, and whole brains and kidneys were rapidly removed. - Detection method: Protein lysates were prepared from tissues, and 50 μg of total protein per lane was subjected to Western blot to quantify Ser(P)-935-LRRK2 and total LRRK2 expression. [1] |
| ADME/Pharmacokinetics |
1. Passive permeability: assessed in RRCK cells. [1]
2. P-glycoprotein (P-gp) efflux rate: determined in MDR1 cells. [1] 3. Liver microsomal stability: assessed in rat liver microsomes. [1] 4. Brain permeability: in rats, the free brain concentration (Cbu) was comparable to the free plasma concentration (Cpu), indicating good brain permeability. [1] |
| Toxicity/Toxicokinetics |
1. Acute and subchronic toxicity in rats: Oral administration of PF-06447475 at a dose of 30 mg·kg⁻¹ twice daily for 4 weeks (or 14 days) did not cause significant changes in body weight; histological examination of the liver, kidneys, lungs and spleen showed no abnormal pathological changes (e.g., inflammation, tissue damage). [3]
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| References |
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| Additional Infomation |
1. Background: Leucine-rich repeat kinase 2 (LRRK2) is genetically associated with Parkinson's disease (PD). The G2019S missense mutation of LRRK2 enhances its kinase activity, which is involved in oxidative stress, neurodegeneration, and neuroinflammation in PD. [1][2][3]
2. Drug characteristics: PF-06447475 is a highly effective, selective, blood-brain barrier-penetrating LRRK2 kinase inhibitor with in vivo activity. Its selectivity for the kinase community was improved through structure-activity relationship (SAR) optimization and alternative crystallography screening. [1] 3. Mechanism of action: PF-06447475 inhibits LRRK2 kinase activity, blocking phosphorylation of LRRK2 at the Ser935 site, thereby inhibiting oxidative stress-induced neuronal apoptosis and α-synuclein-mediated dopaminergic neurodegeneration and neuroinflammation. [2][3] 4. Therapeutic potential: PF-06447475 has the potential to treat Parkinson's disease (PD) because it can alleviate PD-related pathological changes (neurodegenesis, neuroinflammation) and protect neurons in preclinical models. [1][2][3] |
| Molecular Formula |
C17H15N5O
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| Molecular Weight |
305.33
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| Exact Mass |
305.127
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| CAS # |
1527473-33-1
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| Related CAS # |
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| PubChem CID |
72706840
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| Appearance |
Off-white to pink solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.711
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| LogP |
2
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
23
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| Complexity |
457
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BHTWDJBVZQBRKP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H15N5O/c18-9-12-2-1-3-13(8-12)14-10-19-16-15(14)17(21-11-20-16)22-4-6-23-7-5-22/h1-3,8,10-11H,4-7H2,(H,19,20,21)
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| Chemical Name |
3-(4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (9.83 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 30.0 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. Solubility in Formulation 2: ≥ 3 mg/mL (9.83 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 30.0 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 | 3.2751 mL | 16.3757 mL | 32.7514 mL | |
| 5 mM | 0.6550 mL | 3.2751 mL | 6.5503 mL | |
| 10 mM | 0.3275 mL | 1.6376 mL | 3.2751 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.
 Efficacy and pharmacodynamic properties of the LRRK2 kinase inhibitor PF-06447475.J Biol Chem.2015 Aug 7;290(32):19433-44. |
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 LRRK2 kinase inhibition is well tolerated in rats.J Biol Chem.2015 Aug 7;290(32):19433-44. |
 PF-06447475 administration blocks α-synuclein-induced dopaminergic neurodegeneration.J Biol Chem.2015 Aug 7;290(32):19433-44. |
 Microgliosis associated with G2019S-LRRK2 expression is attenuated by PF-06447475 in G2019S-LRRK2 rats.J Biol Chem.2015 Aug 7;290(32):19433-44. |
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 Reduced CD68 cell recruitment in G2019S-LRRK2 rats treated with PF-06447475.J Biol Chem.2015 Aug 7;290(32):19433-44. |
 G2019S-LRRK2 expression enhances α-synuclein-induced dopaminergic neurodegeneration.J Biol Chem.2015 Aug 7;290(32):19433-44. |
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