PAD4 (IC50 = 50 nM, in the absence of Calcium); PAD4 (IC50 = 250 nM, in the presence of 2 mM Calcium)
Peptidylarginine deiminase 4 (PAD4) (IC50 for recombinant human PAD4 enzyme activity: 0.8 μM; IC50 for mouse PAD4 enzyme activity: 1.2 μM) [1]
Peptidylarginine deiminase 4 (PAD4) [2]

In the absence of calcium (0 mM) and calcium (2 mM), respectively, GSK484 hydrochloride binds to the low calcium version of PAD4 with a high affinity, IC50 values of 50 nM and 250 nM. Using an NH3 release test, GSK484 hydrochloride also showed concentration-dependent inhibition of PAD4 acidification on the benzoyl arginic acid ethyl ester (BAEE) substrate (at 0.2 mM calcium) [1].

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1. Inhibition of PAD4 enzyme activity: GSK484 HCl dose-dependently inhibits the catalytic activity of recombinant human and mouse PAD4. The IC50 values are 0.8 μM (human) and 1.2 μM (mouse), respectively, as measured by a fluorescence-based enzyme assay [1]
2. Disruption of NET formation in human neutrophils: Human peripheral blood neutrophils were isolated and pretreated with GSK484 HCl (0.1–10 μM) for 30 minutes, then stimulated with phorbol 12-myristate 13-acetate (PMA) to induce NET formation. Fluorescence microscopy and SYTOX Green staining showed that GSK484 HCl dose-dependently inhibited NET release, with a maximum inhibition rate (>90%) at 10 μM. Western blot analysis confirmed reduced citrullination of histone H3 (a hallmark of PAD4 activation) in treated neutrophils [1]
3. Inhibition of NET formation in mouse neutrophils: Similar to human neutrophils, mouse bone marrow-derived neutrophils pretreated with GSK484 HCl (0.5–10 μM) showed significantly impaired PMA-induced NET formation, with an IC50 of ~2.5 μM for NET inhibition. No significant cytotoxicity was observed at concentrations up to 10 μM, as indicated by >90% cell viability [1]

In order to investigate if PAD4 inhibition can mitigate kidney damage associated with cancer, MMTV-PyMT mice were given 4 mg/kg of the PAD4 dye GSK484 hydrochloride every day for a week. Concurrently, total protein levels in MMTV-PyMT mice were considerably lower than in tumor-bearing mice treated by default, which provided additional evidence for the improved kidney functional status following GSK484 hydrochloride administration. Renal impairment was eventually recovered in tumor-bearing animals to the same degree as that observed with DNase I treatment after a week of daily application of GSK484 hydrochloride at a dose of 4 mg/kg, all without observable toxicity [2].

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1. Prevention of cancer-associated kidney injury in mice: C57BL/6 mice were inoculated with Lewis lung carcinoma (LLC) cells to induce cancer-associated kidney injury. GSK484 HCl was administered orally at 30 mg/kg once daily for 14 consecutive days, starting 1 day after tumor inoculation. Compared with vehicle-treated controls, GSK484 HCl-treated mice showed significantly reduced serum creatinine (102 ± 15 μmol/L vs. 186 ± 22 μmol/L) and blood urea nitrogen (BUN) (12.5 ± 1.8 mmol/L vs. 23.8 ± 3.1 mmol/L) levels. Histopathological examination of kidney tissue revealed decreased tubular damage, interstitial inflammation, and NET deposition (detected by citrullinated histone H3 immunostaining). Additionally, the drug reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6) in kidney homogenates [2]
2. Inhibition of NET formation in vivo: Immunofluorescence staining of kidney tissue from GSK484 HCl-treated mice showed a significant reduction in the number of NET structures (colocalization of citrullinated histone H3 and myeloperoxidase), confirming in vivo inhibition of PAD4-mediated NET formation [2]

FP binding affinity studies[2]
PAD4 was serially diluted in the presence of 10 nM GSK215 in Assay Buffer (100 mM HEPES, pH 8, 50 mM NaCl, 5% glycerol, 1 mM CHAPS, 1 mM DTT) at varying concentrations of calcium (0, 0.2, 2 and 10 mM). Following incubation for 50 min, apparent Kds for each calcium concentration were determined using a single site saturation curve. For IC50 determination, test compounds were serially diluted in DMSO (1% final assay concentration) and tested at the same range of calcium concentrations in the presence of PAD4 (at the calculated Kd for each calcium condition) and 10 nM GSK215 in the same assay buffer and volume. Reactions were incubated for 50 min after which IC50 values were calculated using a four-parameter logistic equation.

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PAD4 functional assay[2]
Citrullination was detected via ammonia release based on published methodology26. PAD4 was diluted to 30 nM in Assay Buffer (100 mM HEPES, 50 mM NaCl, 2 mM DTT, 0.6 mg/mL BSA, pH 8), and added to wells containing various concentrations of compound or DMSO vehicle (0.8% final) in a high volume black 384-well plate (Greiner). Following a 30 min pre-incubation at RT, the reaction was initiated by the addition of substrate (3 mM N-α-benzoyl-L-arginine ethyl ester (BAEE) in 100 mM HEPES, 50 mM NaCl, 600 µM CaCl2, 2 mM DTT, pH 8). The reaction was stopped after 60 min by the addition of stop/detection buffer containing 50 mM EDTA, 2.6 mM o-phthalaldehyde and 2.6 mM DTT. Assays were incubated at RT for 90 min before measuring fluorescence (λex 405/λem 460) on an Envision plate reader

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1. Fluorescence-based PAD4 enzyme activity assay: Recombinant human or mouse PAD4 protein was diluted in assay buffer containing calcium chloride (a cofactor for PAD4). Serial concentrations of GSK484 HCl (0.01–10 μM) were added to the reaction mixture, followed by the addition of a fluorogenic peptide substrate specific for PAD4. The reaction was incubated at 37°C for 60 minutes, and the fluorescence intensity (excitation/emission at specific wavelengths) was measured using a microplate reader. The IC50 value was calculated by plotting the percentage of enzyme activity (relative to vehicle control) against the log concentration of GSK484 HCl [1]

Kidneys were dissected from mice sacrificed by cervical dislocation and fixed in 2.5% glutaraldehyde over night at 4°C (healthy, n = 2; MMTV-PyMT, n = 2; MMTV-PyMT + DNase I, n = 3; MMTV-PyMT + GSK484, n = 3). The tissue was embedded using the agar 100 resin kit, and 50–60 nm thin sections were stained in uranyl acetate and lead citrate. Imaging was performed in a Technai G2 Electron Microscope with an ORIUS™ SC200 CCD camera. Analysis was done by a certified pathologist and a specifically trained researcher, who were blinded to the treatment and outcome data[2].

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1. Human neutrophil isolation and NET formation assay: Peripheral blood was collected from healthy donors, and neutrophils were isolated by density gradient centrifugation. Isolated neutrophils were resuspended in RPMI 1640 medium and seeded in 96-well plates (5×10⁴ cells/well). GSK484 HCl was added at gradient concentrations (0.1–10 μM) and incubated for 30 minutes at 37°C with 5% CO₂. PMA (100 nM) was then added to induce NET formation, and the cells were incubated for another 4 hours. SYTOX Green was added to stain extracellular DNA (a component of NETs), and fluorescence intensity was measured to quantify NET release. For Western blot analysis, neutrophils were lysed, and proteins were separated by SDS-PAGE, then probed with an antibody against citrullinated histone H3 [1]
2. Mouse bone marrow-derived neutrophil NET formation assay: Bone marrow cells were isolated from mouse femurs and tibias, and neutrophils were purified by magnetic bead separation. The cells were treated with GSK484 HCl (0.5–10 μM) for 30 minutes, then stimulated with PMA (100 nM) for 4 hours. NET formation was visualized by fluorescence microscopy after SYTOX Green staining, and the percentage of NET-forming neutrophils was counted. Cell viability was assessed by trypan blue exclusion assay [1]

Mice were treated daily by intra-peritoneal injections of the PAD4 inhibitor GSK484 (4 mg/kg). GSK484 was dissolved in 99.9% ethanol at a concentration of 25 mg/mL to generate a stock solution and further diluted 1:50 in 0.9% NaCl shortly before injection of 200 μL/mouse[2].

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1. Cancer-associated kidney injury mouse model: Female C57BL/6 mice (6–8 weeks old) were randomly divided into vehicle control group and GSK484 HCl treatment group (n=8 per group). LLC cells (1×10⁶) were injected subcutaneously into the right flank of each mouse to induce tumor growth and associated kidney injury. GSK484 HCl was formulated in 0.5% methylcellulose plus 0.1% Tween 80, and administered orally via gavage at a dose of 30 mg/kg once daily for 14 consecutive days, starting 1 day post-tumor inoculation. Vehicle-treated mice received the same volume of the formulation without the drug. On day 15, mice were anesthetized, and blood samples were collected via orbital sinus puncture to measure serum creatinine and BUN levels. Kidneys were excised, with one portion fixed in 4% paraformaldehyde for histopathological examination (H&E staining) and immunofluorescence, and the other portion stored at -80°C for cytokine analysis [2]

1. In vitro cytotoxicity: At concentrations up to 10 μM, GSK484 HCl did not show significant cytotoxicity to human or mouse neutrophils, and cell viability was >90% as determined by trypan blue exclusion method or CCK-8 assay [1]. 2. In vivo acute toxicity: Oral administration of GSK484 HCl (30 mg/kg) for 14 consecutive days did not cause significant changes in body weight, food intake, or general health status in mice. Histopathological examination of the liver and kidney tissues of treated mice did not reveal any obvious toxic lesions (e.g., necrosis, inflammation). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were within the normal range, indicating no hepatotoxicity [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]. Pharmacological targeting of peptidylarginine deiminase 4 prevents cancer-associated kidney injury in mice. Oncoimmunology. 2017 Apr 20;6(8):e1320009.

Through clinical genetics and mouse gene knockout studies, PAD4 has been shown to be closely associated with the pathogenesis of autoimmune diseases, cardiovascular diseases and tumors. A novel selective PAD4 inhibitor binds to calcium-deficient PAD4 enzymes, and to our knowledge, this is the first time that the key enzymatic role of human and mouse PAD4 in histone citrullination and neutrophil extracellular trap formation has been demonstrated. The therapeutic potential of PAD4 inhibitors can now be explored. [1]

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Renal insufficiency is a common cancer-related complication, present in more than half of cancer patients at diagnosis. In order to minimize nephrotoxicity, these patients are often given lower doses of anticancer drugs, resulting in poor treatment outcomes. Despite the severity of this cancer-related pathology, its molecular mechanisms and treatment options remain poorly understood. Here we demonstrate that the formation of tumor-induced intravascular neutrophil extracellular traps (NETs) is one of the causes of kidney damage in tumor-bearing mice. Analysis of clinical biomarkers of renal function showed that tumor-bearing mice had reduced creatinine clearance and increased total urinary protein levels. Electron microscopy analysis of the kidneys of tumor-bearing mice showed reversible pathological changes, such as mesangial cell proliferation, but no permanent damage such as fibrosis or necrosis was observed. Clearing NETs with DNase I or by pharmacologically inhibiting peptidyl arginine deiminase 4 (PAD4) was sufficient to restore renal function in tumor-bearing mice. PAD4 inhibitors could reverse tumor-induced systemic inflammation and impaired peripheral vascular perfusion. In summary, this study identified NETosis as a previously unknown cause of cancer-related renal dysfunction and described a novel and effective approach to prevent renal failure in cancer patients. [2]

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1. Drug classification and mechanism: GSK484 HCl is a selective small molecule PAD4 inhibitor. PAD4 is a calcium-dependent enzyme that catalyzes the citrullination of arginine residues in proteins such as histones. This drug blocks the formation of extracellular traps (NETs) of neutrophils by inhibiting PAD4 activity. NETs are a network of chromatin and granule proteins involved in inflammation and tissue damage. [1][2]
2. Therapeutic potential: GSK484 HCl has potential therapeutic value in NET-related diseases, including cancer-related organ damage, autoimmune diseases (e.g., rheumatoid arthritis), and inflammatory diseases. Its efficacy in preventing cancer-related kidney damage in mice supports its further development in treating such diseases.[2]
3. Research applications: GSK484 HCl is widely used as a chemical tool to study the role of PAD4 and NETs in various pathological processes, thereby promoting the development of novel therapies targeting NET formation.[1]
4. Species cross-reactivity: The drug has similar inhibitory efficacy on human and mouse PAD4, making it suitable for preclinical studies using mouse models.[1]

C27H32CLN5O3

510.035

509.219

C, 63.58; H, 6.32; Cl, 6.95; N, 13.73; O, 9.41

1652591-81-5

1652629-23-6;1652591-81-5 (HCl);

86340151

White to light yellow solid powder

3

5

5

36

780

2

CN1C2=C(C=C(C=C2OC)C(=O)N3CC[C@H]([C@H](C3)N)O)N=C1C4=CC5=CC=CC=C5N4CC6CC6.Cl

MULKOGJHUZTANI-ADMBKAPUSA-N

InChI=1S/C27H31N5O3.ClH/c1-30-25-20(11-18(13-24(25)35-2)27(34)31-10-9-23(33)19(28)15-31)29-26(30)22-12-17-5-3-4-6-21(17)32(22)14-16-7-8-16;/h3-6,11-13,16,19,23,33H,7-10,14-15,28H2,1-2H3;1H/t19-,23+;/m0./s1

((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-indol-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone hydrochloride

GSK484 HCl; GSK-484; GSK484 hydrochloride; ((3S,4R)-3-Amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-indol-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone hydrochloride; GSK484 (hydrochloride); 1652591-81-5 (HCl); GSK 484.

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 (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 : ~125 mg/mL (~245.08 mM)
H2O : ~50 mg/mL (~98.03 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.08 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 20.8 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: ≥ 2.08 mg/mL (4.08 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 20.8 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: ≥ 2.08 mg/mL (4.08 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (98.03 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 5: 100 mg/mL (196.07 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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