MDCK cells treated with ML303 (20 μM) for 6 hours exhibit increased levels of IFN-β mRNA [1].

MDCK cells treated with ML303 (20 μM) for 6 hours exhibit increased levels of IFN-β mRNA [1].

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ML303 (compound 32) reduced viral titer of influenza A/PR/8/34 (H1N1) in MDCK cells with an IC90 of 155 nM in a TCID50 assay. At 10 µM, it achieved a 128-fold reduction in viral titer.
In a cytopathic effect (CPE) protection assay against seasonal influenza A/California/7/2009 (H1N1), ML303 exhibited an EC50 of 0.7 µM and an EC90 of >50 µM. Its CC50 in MDCK cells was 25.3 µM, resulting in a selectivity index (SI50) of 38.
ML303 reversed the NS1-induced slow-growth phenotype in a yeast-based assay, restoring yeast growth at 50 µM concentration, indicating functional NS1 antagonism without acute toxicity in yeast.
In A/PR/8/34-infected MDCK cells, ML303 (20 µM) significantly restored IFN-β mRNA levels as measured by RT-PCR, comparable to the positive control poly(I:C). This restoration was specific to infected cells, as the compound did not induce IFN-β mRNA in uninfected cells.
In a luciferase reporter assay in 293 cells, ML303 (20 µM) restored poly(I:C)-induced IFN-β promoter activity that was suppressed by co-transfected NS1, confirming its ability to block NS1 function. The compound had no effect on luciferase activity in the absence of NS1 expression.
Western blot analysis confirmed that ML303 did not affect the steady-state level of NS1 protein in infected cells, indicating it acts functionally rather than by degrading NS1. [1]

Western Blot Analysis[1]
Cell Types: MDCK cells were infected with A/PR/8/34 at MOI 2 for 6 hrs (hours).
Tested Concentrations: 20μM.
Incubation Duration: 6 hrs (hours).
Experimental Results: IFN-β mRNA levels were restored in MDCK cells.

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Viral Replication Inhibition: MDCK cells were infected with influenza A/PR/8/34 at a multiplicity of infection (MOI) of 0.1. Compounds were added and incubated for 48 hours. Supernatants were collected, and viral titer was determined using hemagglutination assay and standard TCID50 analysis. Fold reduction was calculated relative to a DMSO control. [1]
Cytotoxicity Assay: Cytotoxicity of compounds was tested in uninfected MDCK cells using a cell viability assay based on ATP content (e.g., CellTiter-Glo). The CC50 was determined. [1]
CPE Protection Assay: MDCK cells were infected with various influenza strains. Compounds were added, and after an incubation period, cell viability was measured to determine the compound's ability to protect cells from virus-induced death. EC50, EC90, and CC50 values were derived from dose-response curves. [1]
Yeast Growth Restoration Assay: A yeast strain expressing influenza NS1 protein exhibits a slow-growth phenotype. Yeast was grown in the presence of compound or DMSO. Growth (OD600nm) was monitored at 22, 42, and 60 hours. Reversal of the slow-growth phenotype indicates NS1 antagonism. [1]
IFN-β mRNA Restoration (RT-PCR): MDCK cells were infected with A/PR/8/34 at an MOI of 2 and treated with compound for 6 hours. Total RNA was harvested, and IFN-β mRNA levels were analyzed by RT-PCR, with β-actin serving as a loading control. [1]
IFN-β Luciferase Reporter Assay: 293 cells were co-transfected with a firefly luciferase reporter plasmid under the control of the human IFN-β promoter and an NS1 expression plasmid. After 16 hours, cells were treated with poly(I:C) to induce the promoter and with the test compound. Luciferase activity was measured 24 hours later to assess the compound's ability to counteract NS1-mediated suppression. [1]
Western Blot for NS1 Protein: Infected and compound-treated MDCK cells were lysed. Proteins were separated by SDS-PAGE, transferred to a membrane, and probed with antibodies against NS1 and α-tubulin (loading control) to assess NS1 protein levels. [1]

Pharmacokinetics Study: Male C57BL/6 mice (N=3 per compound) were administered a single intraperitoneal (IP) dose of ML303 at 30 mg/kg. The formulation/solvent used for dosing is not specified in the provided text. Blood (for plasma), liver, and lung tissues were collected at various time points post-dose. Concentrations of ML303 in these matrices were determined to calculate pharmacokinetic parameters. [1]

In vitro, ADME:ML303 exhibited good stability in mouse liver microsomes (MLM), with residual amounts of the parent compound at 66% and 45% after 15 and 30 minutes of incubation, respectively. Its water solubility was 3.1 µM. In Caco-2 monolayer cell permeability assays, its apparent permeability from apex to lateral basal surface (Papp, AB) was 0.10 × 10⁻⁶ cm/s, and its apparent permeability from lateral basal surface to apex (Papp, BA) was 0.22 × 10⁻⁶ cm/s, with an efflux ratio of 2.29. This low permeability is attributed to its low solubility. [1]
Pharmacokinetics in vivo (mice, intraperitoneal injection): After a single intraperitoneal injection of 30 mg/kg ML303, the Cmax in plasma was 2457 ng/mL, in liver it was 49,600 ng/mL, and in lung it was 18,567 ng/mL, with a time to peak concentration (Tmax) of 0.25 hours.
The terminal half-life (t₁/₂) was approximately 4.6 hours in plasma, approximately 4.7 hours in liver, and approximately 4.5 hours in lung.
The area under the concentration-time curve (AUC) in plasma was 53,894 hng/mL. The exposure in liver (AUC ratio approximately 10.5) and lung (AUC ratio approximately 7.0) was significantly higher than in plasma.
The concentration of this compound in the lung (the main site of influenza virus infection) remained above 1 µM for more than 12 hours. [1]

In MDCK cell viability assays, ML303 showed no cytotoxicity at concentrations up to 100 µM, which gives it a high selectivity index (SI50 > 100). In yeast growth recovery assays, the compound showed no acute toxicity at a concentration of 50 µM. In single-dose pharmacokinetic studies in mice (30 mg/kg, intraperitoneal injection), no adverse reactions or signs of toxicity were observed. [1]

[1]. Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists. Bioorg Med Chem Lett. 2019 May 1;29(9):1113-1119.

ML303 belongs to a class of novel pyrazolopyridine small molecules and was identified as an influenza virus NS1 antagonist by quantitative high-throughput screening (qHTS) in a yeast system. Its mechanism of action is believed to be to inhibit the function of the influenza A virus NS1 protein, thereby reversing the NS1-mediated suppression of the host's innate immune response, especially the induction of IFN-β. This compound is positioned as a tool for studying NS1 function and is expected to verify the effectiveness of NS1 as a therapeutic target for influenza in future in vivo proof-of-concept studies. [1]

C21H16F3N3O2

399.36585521698

399.119

1638211-04-7

136141525

White to off-white solid powder

5

1

7

3

29

556

0

FC(C1C=CC(=CC=1)N1C2C(C(C)=N1)=C(C=CN=2)C1C=CC(=C(C=1)OC)O)(F)F

YBRHGNCGFMRGSV-UHFFFAOYSA-N

InChI=1S/C21H16F3N3O2/c1-12-19-16(13-3-8-17(28)18(11-13)29-2)9-10-25-20(19)27(26-12)15-6-4-14(5-7-15)21(22,23)24/h3-11,28H,1-2H3

2-methoxy-4-[3-methyl-1-[4-(trifluoromethyl)phenyl]pyrazolo[3,4-b]pyridin-4-yl]phenol

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)