PAD4 (IC50 = 50 nM, in the absence of Calcium); PAD4 (IC50 = 250 nM, in the presence of 2 mM Calcium)
PAD4 (peptidylarginine deiminase 4); IC50 = 50 nM in the absence of calcium, IC50 = 250 nM in the presence of 2 mM calcium (FP binding assay) [1]; also inhibits PAD4 citrullination activity in a concentration-dependent manner in the presence of 0.2 mM calcium (NH3 release assay) [1]. Highly selective over PAD1, PAD2, PAD3, and PAD6 (no specific IC50 values provided for other family members) [1].

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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GSK484 free base (CAS#: 1652629-23-6) inhibits PAD4 with IC50 of 50 nM (no calcium) and 250 nM (2 mM calcium) in fluorescence polarization ligand binding assays [1]. It shows reversible binding to PAD4 as confirmed by mass spectrometry and dialysis [1]. In functional ammonia release assays using BAEE substrate (0.2 mM calcium), GSK484 inhibits PAD4-mediated citrullination in a concentration-dependent manner [1]. In mouse neutrophils, pre-treatment with 10 μM GSK484 dramatically diminishes ionomycin-induced histone H3 hypercitrullination and NET formation; dose-dependent reduction of these endpoints is observed [1]. In human neutrophils stimulated with S. aureus, GSK484 (10 μM) causes a clear, statistically significant reduction in diffused NETs compared with vehicle, while the remaining diffused NETs lack citrullination [1]. GSK484 shows no effect on viability of human or mouse neutrophils or S. aureus cultures [1]. In HEK293 cells stably expressing FLAG-tagged PAD4, 100 μM GSK484 inhibits citrullination [1]. In tumor-bearing mice, GSK484 treatment (4 mg/kg daily i.p. for one week) suppresses the elevated number of neutrophils undergoing NETosis in peripheral blood, as detected by Gr1 staining and Hoechst DNA staining showing granulocytes with externalized DNA tails [2].

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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In MMTV-PyMT mammary carcinoma and RIP1-Tag2 pancreatic neuroendocrine tumor-bearing mice, GSK484 free base (CAS#: 1652629-23-6) administered at 4 mg/kg daily by intraperitoneal injection for one week prevents cancer-associated kidney injury. Treated mice show no elevated plasma creatinine levels (all values within healthy range), significantly reduced total protein levels in urine compared to untreated tumor-bearing mice, and restored vascular perfusion of the kidneys (measured by FITC-lectin perfusion and CD31 staining). GSK484 treatment suppresses tumor-induced elevated renal expression of pro-inflammatory cytokines IL-1β and IL-6, as well as endothelial activation markers E-selectin, ICAM-1, and VCAM-1, to levels seen in healthy mice or below. Electron microscopy analysis shows normalization of podocyte foot processes in the kidneys after GSK484 treatment. A tendency to suppressed mesangial hypercellularity is observed after 7 days of treatment. No detectable signs of toxicity are reported. [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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Fluorescence polarization (FP) ligand binding assay: PAD4 is serially diluted in assay buffer (100 mM HEPES pH8, 50 mM NaCl, 5% glycerol, 1 mM CHAPS, 1 mM DTT) with varying calcium concentrations (0, 0.2, 2, and 10 mM) and 10 nM fluorescent tracer GSK215. After 50 min incubation, apparent Kd values are determined. For IC50 determination, test compounds including GSK484 free base (CAS#: 1652629-23-6) are serially diluted in DMSO (1% final) and tested at the same calcium concentrations in the presence of PAD4 (at calculated Kd for each calcium condition) and 10 nM GSK215. After 50 min incubation, IC50 values are calculated using a four-parameter logistic equation [1].
PAD4 functional ammonia release assay: PAD4 is diluted to 30 nM in assay buffer (100 mM HEPES, 50 mM NaCl, 2 mM DTT, 0.6 mg/mL BSA, pH8) and added to wells containing various concentrations of compound or DMSO vehicle (0.8% final). After 30 min pre-incubation at room temperature, the reaction is initiated by 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, pH8). The reaction is stopped after 60 min by addition of stop/detection buffer containing 50 mM EDTA, 2.6 mM α-phthalaldehyde and 2.6 mM DTT. After 90 min incubation at room temperature, fluorescence is measured (λex405/λem460) [1].
Steady state citrulline production assay: Varying concentrations of BAEE in reaction buffer (50 mM HEPES, 50 mM NaCl, 2 mM DTT, 10 mM CaCl2) are pre-incubated at 37°C for 10 min prior to addition of PAD isozyme (0.2-0.5 μM). After 6 min at 37°C, samples are frozen in liquid nitrogen and citrulline levels quantified using the COLDER assay. Initial rates are fitted to Michaelis-Menten equation. For inhibition studies, data are globally fitted to competitive, non-competitive, or mixed inhibition equations. Mixed mode of inhibition is observed for GSK484 [1].
Mass spectrometry reversibility studies: 10 μM test compound (including GSK484) is incubated with 2 μM PAD4 in the presence or absence of 10 mM calcium in assay buffer (100 mM HEPES pH8, 50 mM NaCl, 5% glycerol, 1 mM CHAPS) overnight. Samples are run on a time-of-flight mass spectrometer to determine covalent binding. GSK484 shows reversible binding (no covalent adduct) [1].
Dialysis reversibility studies: PAD4 (2 μM) is incubated with 100 μM GSK199 (structurally similar) for 1 h at 37°C. An aliquot is assayed for residual activity. The remaining enzyme is dialyzed against storage buffer for 18 h at 4°C and then reassayed. Reversibility is confirmed [1]; similar mechanism expected for GSK484 [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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Mouse neutrophil isolation and NET assay: Peripheral blood neutrophils from mice are isolated by density centrifugation followed by hypotonic lysis (purity >90%). Cells are incubated in RPMI with 10 mM HEPES at 37°C and allowed to adhere to glass-bottom wells for 15 min. GSK484 free base (CAS#: 1652629-23-6) or vehicle is added for 20 min prior to stimulation with 4 μM ionomycin for 2 h. Cells are fixed in 2% PFA, permeabilized, blocked, and stained with anti-citrullinated histone H3 antibody (0.3 μg/mL) followed by Alexa Fluor 488-conjugated secondary antibody. Nuclei are counterstained with Hoechst 33342. Fluorescent images are acquired and analyzed. GSK484 (10 μM) dramatically diminishes H3Cit+ cells and NET formation; dose-dependent reduction is observed [1].
Human neutrophil NET assay with S. aureus: Human neutrophils are isolated from peripheral blood using dextran density gradient and Ficoll (purity >90-95%). Cells are allowed to adhere to 96-well plates (2.5×10^4 cells/well) in RPMI for 20 min at 37°C, then pre-incubated with GSK484 or vehicle (0.1% DMSO) for 30 min. S. aureus is added at 5× MOI. After 3 h stimulation, cells are fixed with 2% PFA and stained with Hoechst 33342. Plates are imaged on an Opera confocal system and cells classified as lobulated, delobulated, or diffused NETs using an algorithm. GSK484 (10 μM) causes a clear, statistically significant reduction in diffused NETs compared with vehicle [1].
Human neutrophil citrullination imaging assay: Isolated human neutrophils (3.5×10^5/mL) are incubated with compounds in 384-well plates for 45 min at 37°C, then stimulated with 2 μM calcium ionophore for 60 min. Cells are fixed with 1.3% PFA, permeabilized, blocked, and stained with mouse anti-H3Cit monoclonal antibody (6 μg/mL) followed by Alexa Fluor 488 goat anti-mouse IgG, with Hoechst 33342 counterstaining. Plates are imaged on an IN Cell Analyzer 2000, and FITC intensity in neutrophil nuclei is used as a measure of H3 citrullination. GSK484 reduces H3 citrullination [1].
Neutrophil viability assay: Human and mouse neutrophils are incubated with compound alone for durations identical to NET studies, and viability is assessed using Cell Titer Glo. GSK484 shows no effect on viability [1].
HEK293 cell citrullination assay: HEK293 cells stably expressing N-terminal FLAG-tagged PAD4 are lysed in buffer with protease inhibitors. Lysates are pre-incubated with DMSO alone (2%), 100 μM GSK484, or controls for 20 min at 4°C. Citrullination reactions are performed for 30 min at 37°C in the presence of 2 mM calcium. Proteins are separated by SDS-PAGE, transferred to PVDF, and citrullinated proteins are chemically modified and detected using anti-modified citrulline antibody. GSK484 inhibits citrullination [1].
Cytospin analysis of neutrophils with extracellular DNA tails: Citrated blood from tumor-bearing mice treated with GSK484 (4 mg/kg daily i.p. for one week) is centrifuged, erythrocytes lysed, and remaining cells used for cytospin preparations. Slides are stained with anti-Gr1 antibody (1:300) to identify neutrophils and Hoechst to identify DNA. GSK484 treatment suppresses the elevated number of granulocytes with externalized DNA tails [2].

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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For GSK484 free base (CAS#: 1652629-23-6) treatment in tumor-bearing mice (MMTV-PyMT mammary carcinoma and RIP1-Tag2 pancreatic neuroendocrine carcinoma models): GSK484 is dissolved in 99.9% ethanol at a concentration of 25 mg/mL to generate a stock solution, then diluted 1:50 in 0.9% NaCl shortly before injection. Mice are treated daily by intraperitoneal injection at a dose of 4 mg/kg in a volume of 200 μL per mouse for one week (7 days) [2]. For analysis of renal function, blood is sampled by cardiac puncture using citrate as anticoagulant, and plasma is prepared by sequential centrifugation [2]. For perfusion analysis, FITC-conjugated lectin (100 μg in 100 μL PBS) is administered by retro-orbital injection and allowed to circulate for 2-3 min, followed by heart perfusion with 10 mL PBS and 10 mL 2% PFA for fixation. Kidneys are dissected and processed for cryosectioning [2]. For neutrophil analysis, blood is collected and cytospin preparations are made as described [2].

No detectable signs of toxicity observed in tumor-bearing mice treated with GSK484 free base (CAS#: 1652629-23-6) at 4 mg/kg daily intraperitoneal injection for one week [2]. GSK484 shows no effect on viability of human or mouse neutrophils in vitro at concentrations up to 10 μM [1]. No effect on S. aureus cultures [1]. No off-target activity against a panel of 50 unrelated proteins, including cysteine-utilizing enzymes and chromatin-modifying enzymes (e.g., histone deacetylases 1-11, <50% inhibition at 100 μM). Unlike GSK199, GSK484 shows no activation of HDACs 1-11 even at 100 μM [1].

[1]. Lewis\nHD, et al. Inhibition of PAD4 activity is sufficient to disrupt mouse\nand human NET formation. Nat Chem Biol. 2015 Mar;11(3):189-91.

[2]. Cedervall\nJ, et al. Pharmacological targeting of peptidylarginine deiminase 4\nprevents cancer-associated kidney injury in mice. Oncoimmunology. 2017\nApr 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 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 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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GSK484 free base (CAS#: 1652629-23-6) is a first-in-class highly selective and reversible PAD4 inhibitor. It binds to a low-calcium conformation of PAD4, inducing a novel β-hairpin structure of residues 633-645 that forms a hydrophobic lid over the inhibitor, accounting for its high specificity over other PAD family members (PAD1-3,6) because residue Phe634 is not conserved in other human PADs [1]. The binding mode is mixed-type inhibition with respect to substrate BAEE, as the inhibitor stabilizes an inactive, calcium-deficient enzyme conformation [1]. GSK484 prevents cancer-associated kidney injury in mice by suppressing tumor-induced intravascular NETosis, improving renal perfusion, reducing inflammation, and restoring glomerular structure (podocyte foot processes) [2]. It also reduces mesangial hypercellularity after longer treatment [2]. These findings suggest potential therapeutic applications for preventing renal failure, deep vein thrombosis, and metastasis in cancer patients [2].

C27H31N5O3

473.566745996475

473.243

1652629-23-6

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

86340175

Typically exists as solid at room temperature

3.785

2

5

5

35

780

2

O[C@@H]1CCN(C(C2C=C(C3=C(C=2)N=C(C2=CC4C=CC=CC=4N2CC2CC2)N3C)OC)=O)C[C@@H]1N

BDYDINKSILYBOL-WMZHIEFXSA-N

InChI=1S/C27H31N5O3/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/t19-,23+/m0/s1

[(3S,4R)-3-amino-4-hydroxypiperidin-1-yl]-[2-[1-(cyclopropylmethyl)indol-2-yl]-7-methoxy-1-methylbenzimidazol-5-yl]methanone

CHEMBL4539512; GSK-484; GSK 484; ((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; GTPL8577; SCHEMBL18247692; GSK 484;GSK-484;

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.)