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| Targets |
BMS-986176 (also named LX-9211): Adaptor Protein-2 Associated Kinase 1 (AAK1) (Ki = 0.8 nM; IC50 for AAK1 enzymatic activity = 1.8 nM) [2]
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| ln Vitro |
The serine/threonine kinase Arkl/Prkl family includes adapter-associated kinase 1 (AAK1). There are two spliced versions of AAK1 mRNA: short and long. In the brain and heart, the long form predominates and is highly expressed. In cultured cells, AAK1 colocalizes with endocytic structures and is abundant in synaptosome preparations. AAK1 controls receptor-mediated endocytosis and clathrin-coated endocytosis, two processes crucial to synaptic vesicle recycling [1].
1. BMS-986176 exhibited potent and selective inhibition of AAK1 enzymatic activity, with an IC50 of 1.8 nM and Ki of 0.8 nM; it showed >1000-fold selectivity over other kinases in a panel of 403 kinases, including closely related BIKE (AP2-associated kinase 2, IC50 = 1600 nM) [2] 2. In human embryonic kidney (HEK293) cells overexpressing AAK1, BMS-986176 inhibited AAK1-mediated phosphorylation of AP2M1 (μ2 subunit of AP2) with an IC50 of 19 nM [2] 3. In dorsal root ganglion (DRG) neurons, BMS-986176 dose-dependently inhibited nerve growth factor (NGF)-induced AAK1 activity and downstream signaling pathways related to neuropathic pain [2] |
| ln Vivo |
1. In a mouse spared nerve injury (SNI) model of neuropathic pain, oral administration of BMS-986176 (3, 10, 30 mg/kg) produced dose-dependent reversal of mechanical allodynia and thermal hyperalgesia, with significant efficacy observed at doses ≥10 mg/kg; the analgesic effect persisted for at least 6 hours post-dosing [2]
2. In a rat chronic constriction injury (CCI) model of neuropathic pain, BMS-986176 (10, 30 mg/kg, p.o.) also showed dose-dependent analgesic activity, reversing mechanical allodynia by 50–70% at 30 mg/kg [2] 3. BMS-986176 demonstrated excellent central nervous system (CNS) penetration, with a brain/plasma ratio of 2.8 in mice and 1.2 in rats after oral administration [2] 4. In rodent models, BMS-986176 did not cause motor impairment or sedation at analgesic doses, as assessed by rotarod and open-field tests [2] |
| Enzyme Assay |
1. For AAK1 enzymatic activity assay: Recombinant human AAK1 protein was incubated with a peptide substrate (derived from AP2M1) and ATP in the presence of various concentrations of BMS-986176; the reaction was initiated by adding ATP and incubated at room temperature for a set period. Phosphorylation of the substrate was detected using a time-resolved fluorescence resonance energy transfer (TR-FRET) method, and the fluorescence signal was measured to calculate the inhibition rate and IC50/Ki values [2]
2. For kinase selectivity assay: BMS-986176 was tested against a panel of 403 human kinases at a concentration of 1 μM; the activity of each kinase was measured using radiometric or luminescent assays, and the selectivity score was calculated based on the percentage of kinases inhibited by <50% [2] |
| Cell Assay |
1. For AP2M1 phosphorylation assay in HEK293 cells: HEK293 cells stably expressing FLAG-tagged AAK1 were seeded in multi-well plates and cultured overnight. The cells were pretreated with different concentrations of BMS-986176 for 1 hour, then stimulated with pervanadate (a phosphatase inhibitor) to enhance AAK1 activity. Cells were lysed, and the level of phosphorylated AP2M1 (p-AP2M1) was detected by Western blot analysis; the band intensity was quantified to determine the IC50 of BMS-986176 for inhibiting AP2M1 phosphorylation [2]
2. For DRG neuron assay: Primary DRG neurons were isolated from neonatal rats and cultured in neurobasal medium. Neurons were treated with BMS-986176 at various doses for 2 hours, then stimulated with NGF (50 ng/mL) for 15 minutes. The phosphorylation of AAK1 and downstream signaling molecules (e.g., ERK1/2, AKT) was analyzed by Western blot, and the expression of pain-related genes was quantified by quantitative real-time PCR (qPCR) [2] |
| Animal Protocol |
1. For mouse SNI neuropathic pain model: Male C57BL/6 mice (8–10 weeks old) were subjected to SNI surgery (ligation and transection of the tibial and common peroneal nerves, leaving the sural nerve intact). Seven days post-surgery, mice with established mechanical allodynia (assessed by von Frey filaments) were randomized into treatment groups. BMS-986176 was formulated in a vehicle consisting of 0.5% methylcellulose and 0.1% Tween 80 in water, and administered orally by gavage at doses of 3, 10, 30 mg/kg once daily for 7 days. Mechanical allodynia was measured using von Frey filaments (0.02–2 g) at 1, 3, 6, and 24 hours post-dosing, and thermal hyperalgesia was assessed using a hot plate test (52 ± 0.5 °C) [2]
2. For rat CCI neuropathic pain model: Male Sprague-Dawley rats (200–250 g) underwent CCI surgery (four loose ligatures around the sciatic nerve). Ten days post-surgery, rats with confirmed mechanical allodynia received oral gavage of BMS-986176 (10, 30 mg/kg) or vehicle once daily for 5 days. Mechanical allodynia was evaluated using von Frey filaments (2–15 g) at 2 and 4 hours post-dosing each day [2] 3. For CNS penetration study: Mice and rats were administered a single oral dose of BMS-986176 (10 mg/kg). At predetermined time points (0.5, 1, 2, 4, 6 hours), animals were euthanized, blood was collected to obtain plasma, and brains were dissected and homogenized. The concentration of BMS-986176 in plasma and brain homogenates was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the brain/plasma ratio was calculated [2] 4. For pharmacokinetic (PK) study: Male CD-1 mice, Sprague-Dawley rats, and Beagle dogs were used. BMS-986176 was administered via oral gavage (10 mg/kg) or intravenous (IV) injection (2 mg/kg). Blood samples were collected at multiple time points (0–24 hours) and analyzed by LC-MS/MS to determine plasma drug concentrations. PK parameters (Cmax, Tmax, AUC, t1/2, F) were calculated using non-compartmental analysis [2] |
| ADME/Pharmacokinetics |
1. Absorption: BMS-986176 showed good oral bioavailability: 72% in mice (10 mg/kg orally), 68% in rats (10 mg/kg orally), and 45% in dogs (10 mg/kg orally) [2] 2. Distribution: It has excellent central nervous system penetration, with brain/plasma ratios of 2.8 (mice) and 1.2 (rats) after oral administration; the volume of distribution (Vdss) was 3.2 L/kg in rats and 5.8 L/kg in dogs [2] 3. Metabolism: BMS-986176 is mainly metabolized by cytochrome P450 (CYP) enzymes, of which CYP3A4 is the major isoenzyme responsible for its biotransformation; minor metabolites include hydroxylated and demethylated derivatives [2] 4. Elimination: After oral administration, the terminal half-life (t1/2) in mice, rats and dogs was 4.2 hours, 6.8 hours and 12.5 hours, respectively; renal excretion accounted for approximately 15% of the administered dose and fecal excretion accounted for approximately 75% of the administered dose [2]. 5. Pharmacokinetic parameters (rat, 10 mg/kg orally): Cmax = 1250 ng/mL, Tmax = 1 hour, AUC0-∞ = 8900 ng·h/mL [2].
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| Toxicity/Toxicokinetics |
1. Plasma protein binding: BMS-986176 showed high plasma protein binding in humans (96%), mice (94%), rats (95%) and dogs (97%) [2]
2. In vitro drug interactions: BMS-986176 did not inhibit major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM, nor did it induce CYP3A4 activity in human hepatocytes [2] 3. Acute toxicity: In single-dose toxicity studies in mice and rats, BMS-986176 was well tolerated at oral doses up to 300 mg/kg, with no deaths or serious adverse reactions (e.g., weight loss, behavioral changes) observed [2] 4. Repeat-dose toxicity: In a 14-day repeated-dose study in rats (oral doses of 10, 30, and 100 mg once daily), BMS-986176 did not cause significant histopathological changes in major organs (liver, kidneys, brain, heart); the No Adverse Effect Level (NOAEL) was 100 mg/kg/day [2]. 5. Neurotoxicity: BMS-986176 did not cause motor impairment in the rotarod test (mice, oral administration 30 mg/kg) or cognitive impairment in the new object recognition test (rats, oral administration 30 mg/kg) [2]. |
| References | |
| Additional Infomation |
1. BMS-986176 (LX-9211) is a highly selective, centrally penetrating, orally effective AAK1 inhibitor discovered through structure-based drug design and bipyridine-based pharmacochemistry optimization.[2] 2. This drug is undergoing a Phase I/II clinical trial (NCT04119276) for the treatment of chronic neuropathic pain, including diabetic peripheral neuropathy (DPN) and postherpetic neuralgia (PHN).[2] 3. BMS-986176 reduces neuronal sensitization and neuropathic pain signaling by inhibiting AAK1 and blocking the endocytosis of pain-related receptors (e.g., TrkA, TRPV1) in sensory neurons.[2] 4. This patent [1] discloses biaryl kinase inhibitors as potential therapeutic agents, but does not specifically mention BMS-986176 or its therapeutic indications.[1]
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| Molecular Formula |
C19H23F4N3O
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|---|---|
| Molecular Weight |
385.4066
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| Exact Mass |
385.177
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| Elemental Analysis |
C, 59.21; H, 6.02; F, 19.72; N, 10.90; O, 4.15
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| CAS # |
1815613-42-3
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| Related CAS # |
1815613-42-3;
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| PubChem CID |
118419773
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
470.8±45.0 °C at 760 mmHg
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| Flash Point |
238.5±28.7 °C
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| Vapour Pressure |
0.0±1.2 mmHg at 25°C
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| Index of Refraction |
1.501
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| LogP |
3.11
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
27
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| Complexity |
455
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| Defined Atom Stereocenter Count |
1
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| SMILES |
FC([H])(C1=C(C([H])=C([H])C(C2C([H])=C([H])N=C(C([H])(F)F)C=2[H])=N1)OC([H])([H])[C@](C([H])([H])[H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])[H])F
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| InChi Key |
RKAHOQATMSONTM-IBGZPJMESA-N
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| InChi Code |
InChI=1S/C19H23F4N3O/c1-11(2)9-19(3,24)10-27-15-5-4-13(26-16(15)18(22)23)12-6-7-25-14(8-12)17(20)21/h4-8,11,17-18H,9-10,24H2,1-3H3/t19-/m0/s1
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| Chemical Name |
(2S)-1-[2-(difluoromethyl)-6-[2-(difluoromethyl)pyridin-4-yl]pyridin-3-yl]oxy-2,4-dimethylpentan-2-amine
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| Synonyms |
AAK1-IN-1LX-9211LX9211 LX 9211 BMS 986176BMS-986176 BMS986176
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~220 mg/mL (~570.84 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 5.5 mg/mL (14.27 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 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: ≥ 5 mg/mL (12.97 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 50.0 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. View More
Solubility in Formulation 3: ≥ 5 mg/mL (12.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5946 mL | 12.9732 mL | 25.9464 mL | |
| 5 mM | 0.5189 mL | 2.5946 mL | 5.1893 mL | |
| 10 mM | 0.2595 mL | 1.2973 mL | 2.5946 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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