ATP1A1 (alpha-1 subunit of Na+,K+-ATPase) – Rostafuroxin is a synthetic digitoxigenin derivative that competitively inhibits ouabain binding to the extracellular domain of ATP1A1, thereby blocking its signaling function [3]

Ouabain binding and signaling are competitively inhibited by rostafuroxin (PST 2238). Rostafuroxin reverses ouabain-induced Src-dependent phosphorylation and activates Na+,K+-ATPase, counteracting the molecular and functional effects of ouabain [3][4]. Within 24 hours of treatment, the viability of A549 and HSAEC cells was lowered by less than 20% by rostafuroxin (0.125–128 μM). In HSAEC (IC50=1.8 μM) and A549 cells (IC50=14.8 μM), prostafuroxin suppresses RSV-GFP expression [3]. Rostafuroxin lacks cardiotonic activity in isolated guinea pig atria, replaces [3H]Ouabain at the dog kidney Na+,K+-ATPase receptor (IC50=1.5 nM), and is consistent with hormone (estrogen, progesterone, androgen, mineralocorticoid) and general (R1, R2, a1, a2, A1, A2, M1, M2, H1, H2, 5-HT1, 5-HT2, Ca2+ channels, TXA2/PGH2, PAF, GABAA, GABAB, DA-NE-5-HT uptake, glutamate, glycine, benzodiazepines) receptors [1].

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Rostafuroxin (20 μM) significantly reduced RSV infection in human airway epithelial A549 cells, as measured by a 89% reduction in viral GFP expression and a 2.0 log10 reduction in progeny virus yield at 24 hours post-infection (p.i.). This inhibitory effect was specific to RSV and not observed with vesicular stomatitis virus (VSV). Pretreatment with rostafuroxin also blocked RSV-triggered phosphorylation of EGFR at Tyr845, a key downstream event in the ATP1A1-Src-EGFR signaling pathway required for RSV entry. In primary human small airway epithelial cells (HSAEC), rostafuroxin (3.1 μM) showed even stronger inhibition of RSV-GFP infection compared to A549 cells, with an IC50 value 8.2-fold lower than in A549 cells. Rostafuroxin treatment did not induce clustering or removal of ATP1A1 from the plasma membrane, nor did it affect EGFR expression or localization, unlike ouabain. Furthermore, rostafuroxin significantly reduced RSV-induced macropinocytosis (dextran uptake into vesicles >1.0 μm³) in A549 cells [3].

Rostafuroxin (PST 2238; gavage; 1 mg/kg/day; 3 weeks) improves acetylcholine-induced relaxation and lowers SBP [4].

Cytotoxicity Assay: Cell viability was assessed using an ATP-based assay (CellTiter-Glo). A549 cells were treated with serially diluted rostafuroxin for 24 hours. After lysis, ATP concentration was determined by measuring luciferase activity, which correlates with viable cell number. Viability was reported relative to mock-treated cells. Concentrations with less than 20% reduction in viability were considered non-cytotoxic for subsequent experiments (e.g., 20 μM in A549 cells) [3].
Viral Infection and Inhibition Assay: A549 or HSAEC cells were pre-treated with rostafuroxin (e.g., 20 μM for A549, 3.1 μM for HSAEC) for 16 hours. Cells were then inoculated with recombinant RSV expressing GFP (RSV-GFP) at a specified multiplicity of infection (MOI, e.g., 1 plaque-forming unit (PFU)/cell) in the continued presence of the drug. Viral infection was quantified 17-24 hours p.i. by either: 1) Measuring total GFP fluorescence intensity per well using an ELISA plate reader, or 2) By flow cytometry analysis of GFP median fluorescence intensity (MFI) in single, live cells. Results were expressed relative to mock-treated, infected controls [3].
Time-of-Addition Assay: To determine the stage of infection inhibited, A549 cells were infected with RSV-GFP (MOI=3 PFU/cell). Rostafuroxin (20 μM) was added at different time points post-infection (e.g., 0, 2, 4, 6, 8, 10 hours). Cells were harvested at 24 hours p.i., and GFP expression was analyzed by flow cytometry. Maximum inhibition occurred when the drug was added at the time of infection (0 hours), suggesting it blocks an early step like viral entry [3].
Macropinocytosis Assay (Dextran Uptake): To measure RSV-induced macropinocytosis, A549 cells were serum-starved for 16 hours, optionally pre-treated with rostafuroxin. Cells were then infected with wild-type RSV (MOI=5 PFU/cell) in medium containing AF568-conjugated dextran (10,000 MW) and incubated for 5 hours. Cells were fixed, nuclei stained with DAPI, and imaged by confocal microscopy. Z-stack images were analyzed using imaging software. Dextran-positive vesicles with a volume >1.0 μm³ were identified and their total fluorescence intensity was quantified, normalized to the number of nuclei per field, and reported relative to control infected cells [3].
EGFR Phosphorylation Analysis: Serum-starved A549 cells, pre-treated with rostafuroxin or transfected with siRNA, were infected with wild-type RSV (MOI=5 PFU/cell) for 5 hours. Cells were lysed, and protein concentration was determined. Lysates (containing 150 μg total protein) were incubated on an EGFR phosphorylation antibody array membrane. After washing, bound phosphorylated EGFR (specifically at Tyr845) was detected using a biotinylated pan-EGFR antibody, followed by horseradish peroxidase-conjugated streptavidin and chemiluminescence detection on X-ray film. Spot intensities were quantified, normalized to internal controls and total EGFR signal, and reported relative to control samples [3].

Animal/Disease Models: Male 7weeks old Wistar rats [4]
Doses: 1 mg/kg
Route of Administration: gavage; daily; for 3 weeks
Experimental Results: diminished systolic blood pressure, improved acetylcholine through enhanced nitric oxide synthesis and bioavailability Induced relaxation reduces superoxide anion production by NAD(P)H oxidase and cyclooxygenase-2, and reduces phosphorylation of the cytoplasmic tyrosine kinase Src.

[1]. 17 beta-(3-furyl)-5 beta-androstane-3 beta, 14 beta, 17 alpha-triol (PST 2238). A very potent antihypertensive agent with a novel mechanism of action. J Med Chem. 1997 May 23;40(11):1561-4.

[2]. PST 2238: A new antihypertensive compound that modulates Na,K-ATPase in genetic hypertension. J Pharmacol Exp Ther. 1999 Mar;288(3):1074-83.

[3]. The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells. PLoS Pathog. 2019 Aug 5;15(8):e1007963.

[4]. Rostafuroxin ameliorates endothelial dysfunction and oxidative stress in resistance arteries from deoxycorticosterone acetate-salt hypertensive rats: the role of Na+,K+-ATPase/cSRC pathway. J Hypertens. 20.

Rosifuxine has been used in clinical trials for the treatment of essential hypertension. Rosifuxine (PST2238) was identified as a potential candidate drug against respiratory syncytial virus (RSV) in this study. Its mechanism of action is believed to be competitive inhibition of the RSV-triggered ATP1A1 signaling pathway, which is essential for downstream Src kinase activation, EGFR transactivation (Tyr845 phosphorylation), and induction of macropinocytosis (the main pathway for RSV entry into respiratory epithelial cells). Unlike ouabain (an ATP1A1 agonist), rosifuxine itself does not induce the ATP1A1 signaling pathway or activate the pump via clathrin-mediated endocytosis. This study speculates that rosifuxine may also inhibit other viruses sensitive to ouabain [3].

C23H34O4

374.51366

374.245

156722-18-8

153976

White to off-white solid powder

1.2±0.1 g/cm3

451.3±45.0 °C at 760 mmHg

226.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.591

3.56

3

4

1

27

609

8

C[C@]12CC[C@@H](C[C@H]1CC[C@@H]3[C@@H]2CC[C@]4([C@@]3(CC[C@@]4(C5=COC=C5)O)O)C)O

AEAPORIZZWBIEX-DTBDINHYSA-N

InChI=1S/C23H34O4/c1-20-8-5-17(24)13-15(20)3-4-19-18(20)6-9-21(2)22(25,10-11-23(19,21)26)16-7-12-27-14-16/h7,12,14-15,17-19,24-26H,3-6,8-11,13H2,1-2H3/t15-,17+,18+,19-,20+,21-,22+,23+/m1/s1

(3S,5R,8R,9S,10S,13S,14S,17S)-17-(furan-3-yl)-10,13-dimethylhexadecahydro-14H-cyclopenta[a]phenanthrene-3,14,17-triol

PST2238; PST 2238; PST-2238; Rostafuroxin

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)

DMSO : ≥ 50 mg/mL (~133.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.68 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.68 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.68 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.)