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Purity: ≥98%
BL-918, a racemic mixture, is a novel and potent small molecule activator of UNC-51-like kinase 1 (ULK1), inducing cytoprotective autophagy for Parkinson’s disease treatment. UNC-51-like kinase 1 (ULK1), the yeast Atg1 ortholog, is the sole serine-threonine kinase and initiating enzyme in autophagy, which may be regarded as a target in Parkinson's disease (PD). 33i (BL-918) as a potent activator of ULK1 by structure-based drug design. Subsequently, some key amino acid residues (Arg18, Lys50, Asn86, and Tyr89) were found to be crucial to the binding pocket between ULK1 and 33i by site-directed mutagenesis. Moreover, it was found that 33i induced autophagy via the ULK complex in SH-SY5Y cells. Intriguingly, this activator displayed a cytoprotective effect on MPP+-treated SH-SY5Y cells, as well as protected against MPTP-induced motor dysfunction and loss of dopaminergic neurons by targeting ULK1-modulated autophagy in mouse models of PD. Together, these results demonstrate the therapeutic potential to target ULK1, and 33i, the novel activator of ULK1, may serve as a candidate drug for future PD treatment.
| Targets |
UNC-51-like kinase 1 (ULK1) activator (EC50 = 24.14 nM)[1]
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|---|---|
| ln Vitro |
BL-918 (compound 33i) exhibits a high binding affinity for ULK1 (KD=0.719 μM) [1]. In neuronal-like SH-SY5Y cells, BL-918 (5 μM) induces autophagy over a 24-hour period [1]. -918 (0.5-50 μM; 24 hours) partially reverses MPP+-induced cell death, as determined by enhancing cell viability [1]. BL-918 (5 μM; duration 6-36 hours) time-drivenly increases the expression levels and phosphorylation status of LC3-II and Beclin-1, while reducing the levels of the autophagy substrate SQSTM1/p62. BL-918 increases the phosphorylation of ULK1 at Ser317 and Ser555, and decreases the phosphorylation of ULK1 at Ser757 [1]. Cell Autophagy Assay[1]
BL-918 activated ULK1 kinase activity with EC50 of 24.14 nM in ADP-Glo kinase assay.[1] It induced cytoprotective autophagy in SH-SY5Y and PC-12 cells, as evidenced by increased LC3-II levels, decreased SQSTM1/p62, and elevated autophagosome formation.[1] BL-918 partially reversed MPP+-induced cell death in SH-SY5Y cells, enhancing cell viability by 42% at 0.5 µM and 67% at 5 µM.[1] Knockdown of ULK1 reversed the cytoprotective effect of BL-918.[1] The compound did not activate AMPK or eEF2K in kinase assays, indicating selectivity for ULK1.[1] |
| ln Vivo |
Compound 33i, also known as BL-918 (compound 20, 40, or 80 mg/kg/day; border gavage; started two days prior to the first saline/MPTP injection and continued for five days following the last saline/MPTP injection), dramatically lowers dopamine (DA), homovanillic acid (HVA), and 3,4-diphenyl acetamide (DOPAC) levels [1].
BL-918 improved motor dysfunction in MPTP-induced Parkinson’s disease mouse models (pole test and swimming test) at doses of 40 and 80 mg/kg.[1] It attenuated the loss of dopamine, DOPAC, and HVA in the striatum of MPTP-treated mice.[1] BL-918 reduced the loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and striatum.[1] It induced cytoprotective autophagy and reduced apoptosis in vivo, as shown by increased p-ULK1, LC3-II, and decreased SQSTM1/p62 and caspase-3 activation.[1] |
| Enzyme Assay |
ULK1 kinase activity was measured using ADP-Glo Kinase Assay with recombinant human ULK1. The compound, substrate (MBP), ATP, and kinase enzyme were incubated in kinase buffer (50 µM DTT, 20 mM MgCl₂, 40 mM Tris pH 7.5, 0.1 mg/mL BSA). After 60 min incubation, ADP-Glo reagent was added, followed by kinase detection reagent. Luminescence was recorded and EC50 values were calculated using nonlinear regression.[1]
SPR analysis was performed to determine binding affinity of BL-918 to wild-type and mutant ULK1. The compound was injected at different concentrations and dissociation constants (KD) were evaluated.[1] |
| Cell Assay |
Cell Autophagy Assay[1]
Cell Types: SH-SY5Y cells Tested Concentrations: 5 μM Incubation Duration: For 24 hrs (hours) Experimental Results: Induced autophagy. Cell viability assay [1] Cell Types: SH-SY5Y Cell Tested Concentrations: 0.5, 5, 50 μM Incubation Duration: 24 hrs (hours) Experimental Results: MPP+-induced cell death can be partially reversed, as determined by enhanced cell viability. Western Blot Analysis[1] Cell Types: SH-SY5Y Cell Tested Concentrations: 5 μM Incubation Duration: 6, 12, 24, 36 hrs (hours) Experimental Results: Time-dependent increase in the expression of LC3-II (key marker of autophagy), Beclin level-1 and its phosphorylation status, thereby reducing the levels of the selective autophagy substrate SQSTM1/p62. SH-SY5Y and PC-12 cells were cultured in DMEM or RPMI-1640 medium with 10% FBS. Cells were treated with BL-918 and autophagy markers (LC3, SQSTM1/p62, Beclin-1) were analyzed by western blot. Autophagic flux was assessed using bafilomycin A1. MDC staining and GFP-LC3 transfection were used to visualize autophagosomes. Cell viability was measured by MTT assay after MPP+ treatment.[1] ULK1 knockdown was performed using siRNA transfection in SH-SY5Y cells to assess the role of ULK1 in BL-918-induced autophagy.[1] |
| Animal Protocol |
Animal/Disease Models: Male C57BL/6 mice (eight weeks old) weighing between 20 and 25 g [1]
Doses: 20, 40 or 80 mg/kg Route of Administration: po (oral gavage); daily; first time Start 2 days before injecting saline/MPTP and continue for 5 days after the last injection of saline/MPTP. Experimental Results: Reduce the loss of DA and its metabolites. Male C57BL/6 mice (8 weeks old) were intraperitoneally injected with MPTP-HCl (30 mg/kg) daily for 5 days. BL-918 was administered orally at 20, 40, or 80 mg/kg/day starting 2 days before MPTP injection and continued for 5 days after the last MPTP dose. Behavioral tests (pole test, swimming test) were performed after treatment. Brain tissues (striatum, substantia nigra) were collected for biochemical and immunohistochemical analysis.[1] |
| Toxicity/Toxicokinetics |
No significant toxicity was observed in mice treated with BL-918 (at doses up to 80 mg/kg). No significant abnormalities were observed in body weight, blood parameters (white blood cell count, hemoglobin, platelet count, alanine aminotransferase, aspartate aminotransferase), or histological examination of the heart, thymus, spleen, kidneys, colon, and ileum. [1]
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| References | |
| Additional Infomation |
BL-918 is a small molecule ULK1 activator based on structure-based drug design and high-throughput screening. It binds to key amino acid residues (Arg18, Lys50, Asn86, and Tyr89) in ULK1 to activate the ULK complex (ULK1-mAtg13-FIP200-Atg101). It induces protective autophagy in cells and has shown therapeutic potential in a Parkinson's disease model without significant toxicity. [1]
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| Molecular Formula |
C23H15F8N3OS
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|---|---|
| Molecular Weight |
533.4369
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| Exact Mass |
533.08
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| CAS # |
2101517-69-3
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| Related CAS # |
(Rac)-BL-918;2435589-07-2
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| PubChem CID |
146014432
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.592
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| LogP |
6.85
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
36
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| Complexity |
737
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1=CC=C(C=C1)[C@H](C(=O)NC2=C(C=C(C=C2)F)F)NC(=S)NC3=CC(=CC(=C3)C(F)(F)F)C(F)(F)F
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| InChi Key |
BDBWQANRZRCMMD-LJQANCHMSA-N
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| InChi Code |
InChI=1S/C23H15F8N3OS/c24-15-6-7-18(17(25)11-15)33-20(35)19(12-4-2-1-3-5-12)34-21(36)32-16-9-13(22(26,27)28)8-14(10-16)23(29,30)31/h1-11,19H,(H,33,35)(H2,32,34,36)/t19-/m1/s1
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| Chemical Name |
(2R)-2-[[3,5-bis(trifluoromethyl)phenyl]carbamothioylamino]-N-(2,4-difluorophenyl)-2-phenylacetamide
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| Synonyms |
BL-918; BL 918; BL918
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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 (~468.66 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.90 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 | 1.8746 mL | 9.3731 mL | 18.7463 mL | |
| 5 mM | 0.3749 mL | 1.8746 mL | 3.7493 mL | |
| 10 mM | 0.1875 mL | 0.9373 mL | 1.8746 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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