Endoplasmic reticulum (ER) α-glucosidases (inhibitor) [1]

In BGK-21 cells, castanospermine (0.01-1000 μM, 48 hours) suppresses the formation of DEN infectious virus in a dose-dependent manner and decreases the glycosylated structural protein DEN prM's electrophoretic mobility [1].

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Castanospermine inhibited dengue virus type 2 (DENV-2 strain 16681) production in BHK-21 cells in a dose-dependent manner with an IC50 of 1 µM. In Huh-7 human hepatoma cells, the IC50 was higher at 85.7 µM. [1]
Castanospermine (50 µM) strongly inhibited virus secretion and cell-to-cell spread of low-passage isolates representing all four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, DENV-4) in BHK-21 cells. [1]
Castanospermine had little to no significant inhibitory effect on West Nile virus (WNV) infection in BHK-21 cells, even at high doses (500 µM). It showed an intermediate inhibitory effect on Yellow Fever virus (YFV), with 50 µM and 500 µM reducing YFV antigen-positive cells by 57% and 93%, respectively. [1]
Castanospermine (500 µM) reduced the infectivity of pseudoinfectious virus-like particles (VLPs) incorporating DENV-1 envelope proteins by >95%, but had little effect on WNV VLPs. [1]
Treatment with Castanospermine (100-500 µM) significantly decreased both the amount of DENV viral RNA and infectious virus in the supernatant of infected BHK-21 cells. By 48 hours, it reduced DENV RNA levels by ~20-fold and infectious virus titers by ~3000-fold, increasing the viral RNA-to-PFU ratio by ~150-fold. An antigen-capture ELISA confirmed that Castanospermine reduced both the secretion and, to a greater extent, the infectivity of DENV particles. [1]
Western blot analysis showed that Castanospermine treatment slowed the electrophoretic mobility of the DENV prM protein, an effect reversed by endoglycosidase H treatment, indicating impaired N-linked glycan processing. [1]
In BHK-21 cells stably replicating DENV-2 or WNV subgenomic replicons (lacking structural genes), treatment with Castanospermine (15-500 µM) for 48 hours reduced marker gene (luciferase) expression only modestly (up to 20-40%), suggesting minimal direct effect on viral RNA replication/translation. [1]

Mice can become more susceptible to DEN-2 virus infection when given castanospermine (10, 50, or 250 mg/kg) intraperitoneally once every ten days [1]. Mice's susceptibility to DEN-2 virus infection can be boosted by intraperitoneal injection of castanospermine (10–500 mg/kg) [1].

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In A/J mice infected intracranially with a lethal dose (10^5 PFU) of mouse-adapted DENV-2, daily intraperitoneal administration of Castanospermine for 10 consecutive days significantly improved survival. Survival rates were 25% (10 mg/kg/day), 90% (50 mg/kg/day), and 85% (250 mg/kg/day), compared to 0% in vehicle-treated controls. [1]
In C57BL/6 mice infected via footpad with WNV (10^2 PFU), daily treatment with Castanospermine (5 mg/day, approximately 250 mg/kg/day) for 10 days had no significant effect on mortality, showing neither protective nor adverse effects. [1]

RT-PCR[1]
Cell Types: BHK-21
Tested Concentrations: 100, 500 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: A modest reduction of up to 20 to 40% in marker gene expression or proliferation of WNV or DEN replicons, respectively. The effect on secretion of infectious WNV or WNV RNA-containing viral particles was minor, but the amount of DEN viral RNA and infectious virus was diminished.

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Plaque Assay for Virus Production: BHK-21 or Huh-7 cells were infected with DENV-2 at various MOIs. After infection, the medium was replaced with growth medium containing a range of Castanospermine concentrations. Supernatants were harvested at 24 or 72 hours, and infectious virus titers were determined by plaque assay on BHK-21 cells. [1]
Flow Cytometry for Infection Spread: BHK-21 cells were infected with DENV isolates (MOI 0.01) or other flaviviruses and cultured in the presence or absence of Castanospermine (e.g., 50 µM). After 48 hours, cells were analyzed by flow cytometry using virus-specific antibodies to determine the percentage of infected cells. [1]
Plaque Reduction Assay: BHK-21 cells were infected with a fixed number of plaque-forming units (PFU) of DENV isolates. Cells were then overlaid with medium containing agarose and Castanospermine (e.g., 500 µM). Plaques were counted after several days of incubation. [1]
Western Blot Analysis: BHK-21 cells infected with DENV (MOI 0.1) were treated with or without Castanospermine. Cells were harvested, lysed, and proteins were separated by SDS-PAGE. Some samples were treated with endoglycosidase H prior to electrophoresis. Blots were probed with an anti-prM antibody. [1]
Viral Particle ELISA (Antigen-Capture): Supernatants from DENV-infected, Castanospermine-treated cells were clarified, concentrated by ultracentrifugation, and added to microtiter plates coated with an anti-prM monoclonal antibody. Captured viral particles were detected using a biotinylated anti-E protein antibody followed by streptavidin-HRP. [1]
Quantitative RT-PCR for Viral RNA: Supernatants from infected, treated cells were harvested. Viral RNA was extracted and quantified using virus-specific primers and fluorogenic RT-PCR (TaqMan). [1]
VLP Infectivity Assay: A stable BHK cell line expressing a WNV subgenomic replicon encoding luciferase was transfected with plasmids expressing DENV or WNV structural proteins (C, prM, E) to produce pseudoinfectious VLPs. VLP production occurred in the presence or absence of Castanospermine (500 µM). VLPs were purified from supernatants and used to infect fresh BHK-21 cells. Infectivity was measured by luciferase activity 40 hours later. [1]
Subgenomic Replicon Assay: BHK-21 cells stably replicating WNV or DENV subgenomic replicons (encoding luciferase and non-structural genes) were seeded and treated with increasing concentrations of Castanospermine for 48 hours. Cells were lysed, and luciferase activity was measured to assess replicon propagation/translation. [1]

Animal/Disease Models: A/J mouse model [1]
Doses: 10, 50, 250 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: The secretion and virus infectivity of DEN-infected mice were diminished, and the survival rate of the virus was increased.

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Animal/Disease Models: Acute pancreatitis (AP) rat model [2]
Doses: 10, 50, 100, 200, 500 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: The extent and severity of pancreatic damage were Dramatically diminished. Reduces serum interleukin production and NF-kB activation. Increased levels of TNF-a, ICAM-1 and VCAM-1.

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DENV-2 Mouse Protection Model: Four-week-old A/J mice were inoculated intracranially with 10^5 PFU of a mouse-adapted DENV-2 strain. Mice were treated intraperitoneally once daily for 10 consecutive days with saline vehicle or Castanospermine at doses of 0.2 mg (10 mg/kg/day), 1 mg (50 mg/kg/day), or 5 mg (250 mg/kg/day). Survival and morbidity were monitored. [1]
WNV Mouse Model: Seven-week-old C57BL/6 mice were inoculated via footpad with 10^2 PFU of WNV. Mice were treated intraperitoneally once daily for 10 days with saline vehicle or Castanospermine at a dose of 5 mg per day (~250 mg/kg/day). Survival was monitored. [1]

In mice, daily administration of caspamine at doses exceeding 250 mg/kg (e.g., 25 mg or 1.25 g/kg) resulted in adverse reactions such as diarrhea and weight loss. [1]

[1]. Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo. J Virol. 2005 Jul;79(14):8698-706.

[2]. Effects of Castanospermine on Inflammatory Response in a Rat Model of Experimental Severe Acute Pancreatitis. Arch Med Res. 2016 Aug;47(6):436-445.

[3]. Elbein AD. Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing. FASEB J. 1991 Dec;5(15):3055-63.

[4]. Combined donor leucocyte administration and immunosuppressive drug treatment for survival of rat heart allografts. Transpl Immunol. 2004 Nov;13(3):177-84.

Kasparmine is a tetrahydroxyindolizidine alkaloid composed of octahydroindolizidine with four hydroxyl substituents (1S, 6S, 7R, 8R, 8aR-diastereomers) at positions 1, 6, 7, and 8, respectively. It has multiple functions, including as a metabolite, an anti-HIV-1 drug, an anti-inflammatory drug, and an EC 3.2.1 (glucosidase) inhibitor.
Kasparmine has been reported to be found in Alexa canaracunensis, Alexa grandiflora, and other organisms with relevant data.

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Kasparmine is a water-soluble natural alkaloid extracted from the seeds of Castanospermum australe. [1]
It acts as an inhibitor of endoplasmic reticulum (ER) α-glucosidase I and/or II, preventing the modification of N-linked glycan chains on viral glycoproteins. This interference is thought to impair the proper folding, oligomerization, and secretion of viral particles in susceptible viruses such as dengue virus. [1]
This study showed that kaspramine has potent and specific antiviral activity against all four serotypes of dengue virus, both in vitro and in vivo, but is ineffective against the closely related West Nile virus. [1]
The authors believe that kaspramine is worth further development and is expected to become a potential treatment for dengue virus infection. [1]

C8H15NO4

189.209

189.1

79831-76-8

141117-12-6 (HCl);79831-76-8;

54445

White to off-white solid powder

1.5±0.1 g/cm3

421.9±45.0 °C at 760 mmHg

213-217 °C(lit.)

267.6±27.4 °C

0.0±2.3 mmHg at 25°C

1.647

-1.61

4

5

0

13

201

5

C1CN2C[C@@H]([C@H]([C@@H]([C@H]2[C@H]1O)O)O)O

JDVVGAQPNNXQDW-TVNFTVLESA-N

InChI=1S/C8H15NO4/c10-4-1-2-9-3-5(11)7(12)8(13)6(4)9/h4-8,10-13H,1-3H2/t4-,5-,6+,7+,8+/m0/s1

(1S,6S,7R,8R,8aR)-1,2,3,5,6,7,8,8a-octahydroindolizine-1,6,7,8-tetrol

Castanospermine

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)

H2O : ~100 mg/mL (~528.51 mM)
DMSO : ~100 mg/mL (~528.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (13.21 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 (13.21 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 (13.21 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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Solubility in Formulation 4: 120 mg/mL (634.22 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)