Peptidylarginine Deiminase 4 (PAD4): GSK199 is a highly specific PAD4 inhibitor. It inhibits PAD4 enzymatic activity, reducing protein citrullination. IC50 = 0.60 μM against HCoV-OC43 replication in MRC-5 cells. [2]
Selectivity: GSK199 is specific for PAD4 over other PAD isoforms (PAD1, PAD2, PAD3, PAD6). The study shows that PAD4, but not PAD2, is upregulated upon HCoV-OC43 infection, and that PAD4-specific inhibition with GSK199, but not PAD2-specific inhibition with AFM30a, significantly reduces viral replication. [2]

Antiviral Activity Against HCoV-OC43: In human lung fibroblast MRC-5 cells infected with HCoV-OC43 (MOI 0.1), treatment with GSK199 inhibited viral replication in a dose-dependent manner. IC50 = 0.60 μM. Complete blockade of viral replication was achieved at 20 μM. Viral genome copy numbers in both cell lysates and supernatants were drastically reduced. [2]
Viral Protein Expression: Western blot analysis showed that treatment with 20 μM GSK199 dramatically downregulated HCoV-OC43 nucleocapsid protein expression in infected MRC-5 cells at 72 hours post-infection. [2]
Viral Titer Reduction: Plaque assay demonstrated that GSK199 (20 μM) reduced viral titers by approximately 2 logs (99% reduction) in HCoV-OC43-infected MRC-5 cells. [2]
Comparison with PAD2 Inhibitor: The PAD2-specific inhibitor AFM30a showed only marginal inhibition of viral replication (<40% at 20 μM) and had minimal effect on viral protein expression and viral titer, confirming that the antiviral effect is specifically mediated through PAD4 inhibition. [2]
Cytotoxicity: MTT assays showed that GSK199 did not significantly reduce cell viability at concentrations up to 20 μM. Significant cytotoxicity was observed only at concentrations above 100 μM. CC50 = 133.08 μM in MRC-5 cells. Selectivity Index (SI) = CC50/IC50 = 224.94, indicating a wide therapeutic window. [2]
PAD4 Expression: Western blot analysis of MRC-5 cells showed that PAD2 and PAD4 are the only PAD isoforms expressed. Upon HCoV-OC43 infection, PAD4 protein levels increased significantly, while PAD2 levels remained unchanged. PAD1, PAD3, and PAD6 were undetectable. [2]
Citrullination Inhibition: HCoV-OC43 infection induced robust protein citrullination in MRC-5 cells as detected by Rh-PG probe. GSK199 treatment suppressed this citrullination, correlating with reduced viral replication. [2]

Cell Culture: Human lung fibroblast MRC-5 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37°C, 5% CO₂. [2]
Virus and Infection: HCoV-OC43 (ATCC VR-1558) was propagated on MRC-5 cells at 33°C. For antiviral assays, cells were pre-treated with GSK199 or vehicle (DMSO) for 1 hour before infection at MOI 0.1 or 1. After 2 hours virus adsorption at 33°C, inoculum was removed and fresh medium with inhibitor was added. Cells were incubated for 72 hours at 33°C. [2]
Cell Viability Assay (MTT): MRC-5 cells (3 × 10⁴/well in 96-well plates) were treated with various concentrations of GSK199 for 72 hours. MTT solution was added and incubated for 4 hours. Formazan crystals were dissolved, and absorbance was measured at 570 nm. CC50 was calculated. [2]
Viral Load Quantification (Real-Time RT-PCR): Viral RNA was extracted from cell lysates and supernatants using TRI Reagent. Reverse transcription and amplification were performed using Sensi Fast Probe No Rox One-step kit with primers and probe specific for HCoV-OC43 N gene. Cycling conditions: 10 min at 45°C, 20 min at 95°C, followed by 40 cycles of 5 sec at 95°C and 1 min at 60°C. Viral load was normalized to DMSO-treated controls. [2]
Plaque Assay: MRC-5 cells were inoculated with 10-fold serial dilutions of supernatants from infected cells. After 24 hours, cells were fixed with cold acetone-methanol (50:50) and immunostained with anti-HCoV-OC43 monoclonal antibody. Virus titer (PFU/mL) was calculated as number of plaques × 0.1 mL/dilution factor. [2]
Western Blot Analysis: Cells were lysed in RIPA buffer (50 mM Tris-HCl pH8.0, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl). Protein concentration was determined by Bradford method. Equal amounts of protein were subjected to SDS-PAGE, transferred to membranes, and probed with antibodies against HCoV-OC43 NP, PAD1, PAD2, PAD3, PAD4, PAD6, and β-actin. [2]
Citrullination Detection with Rh-PG: Equal amounts of protein were precipitated with 80% trichloroacetic acid and incubated with 0.1 mM rhodamine-phenylglyoxal probe for 30 min. Reaction was quenched with 100 mM L-citrulline for 30 min at 4°C. After centrifugation, pellets were washed with cold acetone and resuspended in SDS loading buffer. Gels were imaged at excitation 532 nm, emission 580 nm using ChemiDoc Imaging System. [2]

Cytotoxicity Profile: In MRC-5 cells, GSK199 showed no significant cytotoxicity at concentrations up to 20 μM. CC50 was calculated as 133.08 μM, giving a selectivity index (SI = CC50/IC50) of 224.94, indicating a wide therapeutic window. [2]
Safety Profile Reference: The study mentions that PAD inhibitors, including GSK199, have demonstrated good safety profiles in preclinical studies for various inflammatory conditions such as arthritis, colitis, and sepsis. [2]

[1]. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol. 2015 Mar;11(3):189-91.

[2]. Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2. Antiviral Res. 2022 Apr;200:105278.

[3]. LC-ESI-MS/MS Determination of GSK-199, A Novel Reversible PAD4 Inhibitor in Mice Plasma and its Application to a Pharmacokinetic Study in Mice. Pharm Anal Chem. 2017, 3(124): 40-1.

Background: GSK199 is a highly specific PAD4 inhibitor originally developed and characterized by Lewis et al. (2015). This study repurposes GSK199 as a host-targeting antiviral agent against human beta-coronaviruses. [2]
Mechanism of Action: HCoV-OC43 infection upregulates PAD4 expression in MRC-5 cells, leading to increased protein citrullination. This citrullination appears to be required for optimal viral replication. GSK199 inhibits PAD4 enzymatic activity, suppressing citrullination and thereby reducing viral replication, protein expression, and infectious particle production. [2]
Specificity Validation: The lack of antiviral effect with the PAD2-specific inhibitor AFM30a confirms that the observed antiviral activity is specifically mediated through PAD4 inhibition, not through off-target effects. [2]
Potential Mechanism (Proposed): The authors suggest that PAD4-mediated citrullination may inactivate interferon-stimulated gene proteins (such as IFIT1 and Mx1) that have antiviral activity. By inhibiting PAD4, GSK199 may restore the antiviral function of these ISGs. However, this mechanism is not fully validated in the current study. [2]
Therapeutic Potential: The high selectivity index (224.94) and potent antiviral activity make GSK199 an attractive candidate for further therapeutic development against beta-coronavirus infections, including SARS-CoV-2. The study also notes that GSK199 has shown efficacy in preclinical models of arthritis, supporting its safety profile. [2]
Comparison with Pan-PAD Inhibitors: While pan-PAD inhibitors (Cl-A, BB-Cl) also showed antiviral activity, GSK199 offers the advantage of PAD4-specific inhibition, potentially reducing off-target effects associated with inhibiting other PAD isoforms. [2]

C24H29CLN6O2

468.98

468.204

1549811-53-1

86340155

Light yellow to yellow solid powder

2

5

4

33

679

1

CCN1C(=CC2=C1N=CC=C2)C3=NC4=C(N3C)C(=CC(=C4)C(=O)N5CCC[C@H](C5)N)OC.Cl

KRGMIOKDGHBYQE-UNTBIKODSA-N

InChI=1S/C24H28N6O2.ClH/c1-4-30-19(12-15-7-5-9-26-22(15)30)23-27-18-11-16(13-20(32-3)21(18)28(23)2)24(31)29-10-6-8-17(25)14-29;/h5,7,9,11-13,17H,4,6,8,10,14,25H2,1-3H3;1H/t17-;/m1./s1

[(3R)-3-aminopiperidin-1-yl]-[2-(1-ethylpyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methylbenzimidazol-5-yl]methanone;hydrochloride

GSK199 GSK 199 GSK-199

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, 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 : ~125 mg/mL (~266.54 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.)