Human Respiratory Syncytial Virus (hRSV) Small Hydrophobic (SH) protein ion channel. Pyronin B is a small-molecule inhibitor of this pentameric viroporin. The dissociation constant (Kd) for inhibition was determined to be approximately 6.8 µM.[1]

At a concentration of 10 μM, Pyronin B (0-15 μM) suppresses the channel activity of small hydrophobic (SH) proteins by around 60%, with a Kd value of 6.8 μM [1]. Pyronin B (0-0.25 μM) has a dosage-dependent effect on RSV replication in Vero cells, with a TCID50 (50% tissue culture infectious dose) = 0 at 0.25 μM [1]. When tiny amounts of Ti (IV) are added, Pyronin B (10 μM) can oxidize with bromate and the reaction rate is greatly accelerated, with a distinctive absorption spectra of 555 nm[2].

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Pyronin B was used as a colorimetric indicator in a kinetic spectrophotometric method for the determination of trace titanium(IV). In a solution of 0.02 mol/L sulfuric acid, Pyronin B undergoes a slow oxidation reaction with hydrogen peroxide. This reaction rate is sharply increased (catalyzed) by the addition of trace amounts of Ti(IV). The decrease in absorbance of Pyronin B at its characteristic wavelength of 555 nm over a fixed time interval (1.0–8.0 min) is proportional to the concentration of Ti(IV) catalyst. The reaction was studied in aqueous solution under controlled temperature (40°C). [2]
Pyronin B inhibited the ion channel activity of the hRSV SH protein reconstituted in planar lipid bilayers (black lipid membranes, BLM). At a concentration of 10 µM, Pyronin B led to approximately 60% inhibition of channel activity, reducing the single-channel conductance from 300 ± 70 pS (control) to 120 ± 60 pS.[1]
A dose-response curve was constructed using Pyronin B concentrations of 0, 2.5, 3, 5, 7, 10, and 15 µM. Fitting the data to a standard sigmoidal dose-response curve yielded an effective Kd (dissociation constant) of approximately 6.8 µM.[1]
Pyronin B reduced human respiratory syncytial virus (RSV strain VR-1580) replication in Vero cells in a plaque assay. The 50% tissue culture infective dose (TCID50) of RSV was reduced to zero at a Pyronin B concentration of 0.25 µM.[1]
Binding of Pyronin B to the SH protein was monitored by solution NMR using uniformly 15N-labeled SH protein in dodecylphosphocholine (DPC) micelles. Titration with Pyronin B (up to 4.8 mM) caused significant chemical shift perturbations (CSPs) in the backbone resonances of specific residues, primarily at both ends of the transmembrane (TM) domain (e.g., Ile-6, Ile-21 at the N-terminal end; and Ile-38, Ala-39, Ile-40, Leu-41, Lys-43 at the C-terminal end). This mapped the likely binding site to a conserved surface on the SH pentamer.[1]
Docking studies predicted two possible binding sites for Pyronin B on the SH pentamer surface, located at the N-terminal and C-terminal ends of the TM domain, consistent with the NMR CSP data.[1]
The inhibitory effect of Pyronin B (10 µM) was tested on SH protein mutants. Inhibition was nearly abolished (~10% inhibition) for the A39S mutant located at the proposed C-terminal binding site. Mutants at the N-terminal end (I21F, I21Y, H22F) also showed reduced sensitivity to inhibition (~20% inhibition), suggesting potential cross-talk between the two ends of the TM domain.[1]

Plaque Assay for Antiviral Activity: Vero cells were seeded in 12-well plates and cultured overnight to reach about 90% confluence. Cells were infected with RSV strain VR-1580 at a multiplicity of infection (MOI) of approximately 1. At 2 hours post-infection, the virus inoculum was removed and replaced with fresh Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal bovine serum (FBS) and 1 µL of dimethyl sulfoxide (DMSO), with or without different concentrations of Pyronin B. At 90 hours post-infection, cultured supernatants were collected, and the 50% tissue culture infective doses (TCID50) of RSV were calculated using the Reed-Muench method. The experiment was repeated three times, with each sample titrated twice.[1]
Whole-Cell Patch Clamp (Attempted): HEK293 Phoenix cells were transfected with a plasmid expressing the full-length hRSV SH protein. Single cells expressing a green fluorescent protein (AcGFP) marker were selected for whole-cell patch clamp recordings to attempt to measure SH channel activity in a cellular context. However, no SH protein-dependent current could be detected at pH 7.4 or 5.5. The measured currents were attributed to endogenous potassium channels, and the previously reported low-pH activation of SH protein in patch clamp could not be reproduced in this study.[1]

[1]. Inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment. J Virol. 2014 Oct;88(20):11899-914.

[2]. Study of the Catalytic Effects of Trace Titanium on Oxidation of Pyronin B with Hydrogen Peroxide and Its Application[C]//Advanced Materials Research. Trans Tech Publications Ltd, 2013, 602: 1233-1237.

Pironine B is an organochloride salt with a cation of 6-(diethylamino)-N,N-diethyl-3H-xanthon-3-imine. Depending on the preparation method, pironine B can also exist as a FeCl3 complex. It is a histological dye. It is an organochloride and imine salt. It contains pironine B(1+) ions. In this study, pironine B was used as a chemical reagent (dye/indicator). It was oxidized by hydrogen peroxide in an acidic medium, and the reaction was catalyzed by titanium(IV) ions. [2] The method developed was used to analyze trace amounts of titanium(IV) in samples such as rocks, rather than to evaluate the efficacy of pironine B as a therapeutic agent. [2] The optimal conditions for the catalytic reaction using pironine B were determined: 0.02 mol/L sulfuric acid medium, 1.0 × 10⁻⁵ mol/L pironine B, 0.08 mol/L hydrogen peroxide, and a reaction temperature of 40 °C. The absorbance was monitored at 555 nm. [2]
0.0104 g of piron B was dissolved in water in a 100 mL volumetric flask to prepare a 1.0 × 10⁻⁴ mol/L piron B solution. [2]

C21H27N2O+.CL-

358.90488

358.181

2150-48-3

62669-69-6 (perchlorate)

16524

Brown to black solid powder

174-176ºC

1.199

0

3

5

25

530

0

CXZRDVVUVDYSCQ-UHFFFAOYSA-M

InChI=1S/C21H27N2O.ClH/c1-5-22(6-2)18-11-9-16-13-17-10-12-19(23(7-3)8-4)15-21(17)24-20(16)14-18;/h9-15H,5-8H2,1-4H3;1H/q+1;/p-1

[6-(diethylamino)xanthen-3-ylidene]-diethylazanium;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20.83 mg/mL (~58.04 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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