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| Targets |
Pronethalol is described as a β-adrenergic receptor antagonist.[2]
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| ln Vitro |
In ReNcell VM cells, pronethalol (2, 10, 20 μM) suppresses EGFP expression in a time- and dose-dependent manner. After two days of treatment, Sox2 expression is reduced to less than 10% by pronethalol (10 μM; two days) [2].
In a high-throughput screen using ReNcell VM human neural progenitor cells stably expressing EGFP under the control of the Sox2 promoter, treatment with increasing doses of pronethalol repressed EGFP expression in a dose- and time-dependent manner. Specifically, 10 µM pronethalol reduced Sox2 expression to less than 10% after 2 days of treatment.[2] In human brain microvascular endothelial cells (HBMECs) depleted of matrix Gla protein (MGP CRISPR cells, modeling MGP deficiency), treatment with 10 µM pronethalol for 48 hours abolished the induction of Sox2, its downstream target JMJD5, the mesenchymal marker N-cadherin, and the lumen-associated genes Par3 and Rasip1, as shown by immunoblotting.[2] The inhibitory effect of pronethalol on Sox2 expression was shown not to involve β-adrenergic receptors. Depletion of individual β1-, β2-, or β3-adrenergic receptors using siRNA in ReNcell VM cells or MGP CRISPR cells did not affect the ability of pronethalol to suppress Sox2 expression.[2] Several other β-adrenergic receptor antagonists (e.g., propranolol, metoprolol, nadolol, sotalol) also inhibited Sox2 expression in ReNcell VM and MGP CRISPR cells, while the β-agonist isoproterenol had no effect. Treatment of MGP CRISPR cells with propranolol or nadolol (10 µM) decreased the expression of JMJD5, N-cadherin, and Par3.[2] |
| ln Vivo |
In Mgp-/-mice, pronethalol (0.15 mg/g; daily; for 14 days) improves cerebral arteriovenous malformations (AVMs) and stabilizes endothelial differentiation and lumen formation [2].
Treatment of matrix Gla protein-deficient mice (\(Mgp^{−/−}\) mice, a model of cerebral arteriovenous malformations (AVMs)) with pronethalol hydrochloride (0.15 mg/g daily for 14 days) improved cerebral AVMs. Injection of GFP-labeled microspheres showed increased retention in the brain tissues of treated \(Mgp^{−/−}\) mice, indicating improved cerebral capillary network and reduced arteriovenous shunting.[2] Micro-computed tomography (µCT) imaging revealed a significant improvement in the cerebral vasculature of pronethalol-treated \(Mgp^{−/−}\) mice, with a decrease in the frequency of vessels with lumen radii between 20 to 50 µm (characteristic of AVMs).[2] Isolation of cerebral endothelial cells from treated \(Mgp^{−/−}\) mice showed decreased expression of Sox2, JMJD5, N-cadherin, and Par3.[2] Pronethalol treatment had no effect on pulmonary and renal AVMs in \(Mgp^{−/−}\) mice, as determined by fluorescent microsphere injection, indicating its effect is specific to cerebral AVMs in this model.[2] Pronethalol treatment did not affect BMP activity in \(Mgp^{−/−}\) cerebral endothelial cells, as assessed by immunoblotting for phosphorylated SMAD1/5/8 (pSMAD1/5/8).[2] |
| Cell Assay |
High-throughput screening: A robotic high-throughput system was used to screen chemical compounds for their ability to suppress Sox2 expression. ReNcell VM human neural progenitor cells, which stably express EGFP under the control of the Sox2 promoter (via lentiviral integration), were used. Cells were seeded in 384-well plates coated with laminin. Chemical compounds (including pronethalol) were added to the wells using a pinning system. Plates were incubated for at least 3 days. GFP expression was then imaged and quantified using fluorescence microscopy and image analysis software to identify compounds that repressed the Sox2 promoter-driven EGFP signal.[2]
Immunoblotting (Western Blot): Cells (e.g., MGP CRISPR HBMECs or ReNcell VM cells) were treated with pronethalol or other compounds for specified durations (e.g., 48 hours). Cells were then lysed, and equal amounts of protein were subjected to SDS-PAGE, followed by transfer to membranes. Membranes were probed with primary antibodies against target proteins (e.g., Sox2, JMJD5, N-cadherin, Par3, Rasip1, β-actin) and then with appropriate secondary antibodies. Protein bands were visualized using chemiluminescence and quantified.[2] Real-time PCR (qPCR): To measure mRNA expression levels of genes of interest (e.g., Sox2, JMJD5, N-cadherin, Par3, β-adrenergic receptors), cells were treated with pronethalol or transfected with siRNA. Total RNA was extracted, reverse-transcribed into cDNA, and analyzed by real-time PCR using specific primers and probes. Gene expression levels were normalized to a housekeeping gene (e.g., GAPDH).[2] siRNA Transfection: To deplete specific proteins (e.g., β1-, β2-, β3-adrenergic receptors, Sox2, JMJD5), cells were transfected with predesigned siRNA oligonucleotides using optimized transfection protocols. Transfection efficiency was validated by measuring mRNA and protein levels via real-time PCR and immunoblotting, respectively, showing 90% to 95% decrease compared to control scrambled siRNA.[2] |
| Animal Protocol |
In vivo efficacy study in AVM mouse model: Matrix Gla protein-deficient mice (\(Mgp^{−/−}\) mice) on a C57BL/6J background were used as a model for cerebral arteriovenous malformations (AVMs). Pronethalol hydrochloride was administered daily at a dose of 0.15 mg/g body weight for a duration of 14 days. The specific formulation, route of administration (e.g., oral, intraperitoneal), and vehicle are not detailed in the provided text.[2]
Assessment of cerebral AVMs: After the treatment period, cerebral AVMs were assessed by two main methods: 1) Fluorescent microsphere injection: Mice were injected with 15 µm GFP-labeled fluorescent microspheres into the left ventricle. Tissues were then examined under UV light; retention of microspheres in brain tissue indicates a functional capillary network, whereas washout suggests arteriovenous shunting. 2) Micro-computed tomography (µCT) imaging: Mice were perfused with a radiopaque compound (MICROFIL). The brains were then scanned using a high-resolution volumetric µCT scanner. Images were analyzed to determine the frequency distribution of vessel lumen radii, with a focus on the abnormal 20-50 µm range characteristic of AVMs.[2] Tissue and cell analysis: After in vivo treatment, mice were euthanized. Cerebral endothelial cells were isolated from the brains for subsequent RNA and protein analysis (e.g., real-time PCR, immunoblotting) to evaluate the expression of relevant markers (Sox2, JMJD5, N-cadherin, Par3).[2] |
| References |
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| Additional Infomation |
Propranol belongs to the naphthalene family.
Propranol was discovered through high-throughput screening of more than 3,000 compounds and is a Sox2 expression inhibitor. Sox2 is a transcription factor whose overexpression in brain endothelial cells drives endothelial-mesenchymal transitions (EndMTs), leading to luminal disruption and the development of arteriovenous malformations (AVMs). [2] The main mechanism of action of propranol in this process is to inhibit the expression of Sox2, thereby normalizing endothelial cell differentiation, reducing EndMTs (manifested as a reduction in N-cadherin), and correcting the expression of lumen-related genes (Par3, Rasip1), thereby improving lumen formation and alleviating AVMs. This effect is independent of its known β-adrenergic receptor antagonism. [2] This study suggests that propranolol and other β-blockers (e.g., propranolol, nadolol) may exert some clinical efficacy by inhibiting Sox2, highlighting the potential new mechanisms of these drugs in the treatment of vascular malformations. [2] Studies have shown that propranolol specifically improves cerebral arteriovenous malformations in (Mgp−/−) mouse models, but has no effect on pulmonary or renal arteriovenous malformations, suggesting that its therapeutic effect is organ-specific or mechanism-specific. [2] |
| Molecular Formula |
C15H19NO
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|---|---|
| Molecular Weight |
229.31746
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| Exact Mass |
229.147
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| CAS # |
54-80-8
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| Related CAS # |
Pronethalol hydrochloride;51-02-5
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| PubChem CID |
4930
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| Appearance |
White to off-white solid powder
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| Density |
1.074g/cm3
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| Boiling Point |
322.4ºC at 760mmHg
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| Flash Point |
84.3ºC
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| LogP |
3.262
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
17
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| Complexity |
229
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
HRSANNODOVBCST-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H19NO/c1-11(2)16-10-15(17)14-8-7-12-5-3-4-6-13(12)9-14/h3-9,11,15-17H,10H2,1-2H3
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| Chemical Name |
1-naphthalen-2-yl-2-(propan-2-ylamino)ethanol
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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 : ~50 mg/mL (~218.04 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (10.90 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 25.0 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: ≥ 2.5 mg/mL (10.90 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (10.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.3607 mL | 21.8036 mL | 43.6072 mL | |
| 5 mM | 0.8721 mL | 4.3607 mL | 8.7214 mL | |
| 10 mM | 0.4361 mL | 2.1804 mL | 4.3607 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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