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Purity: ≥98%
GSK484 HCl is a novel and potent inhibitor of peptidylarginine deiminase 4 (PAD4). GSK484 demonstrates high binding affinity to PAD4 with an IC50 of 50 nM in the absence of Calcium. In the presence of 2 mM Calcium, notably lower potency (250 nM) is observed. PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
| Targets |
PAD4 (IC50 = 50 nM, in the absence of Calcium); PAD4 (IC50 = 250 nM, in the presence of 2 mM Calcium)
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| ln Vitro |
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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| ln Vivo |
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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| Enzyme Assay |
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. 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 |
| Cell Assay |
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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| Animal Protocol |
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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| References | |
| Additional Infomation |
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.[1]
Renal insufficiency is a frequent cancer-associated problem affecting more than half of all cancer patients at the time of diagnosis. To minimize nephrotoxic effects the dosage of anticancer drugs are reduced in these patients, leading to sub-optimal treatment efficacy. Despite the severity of this cancer-associated pathology, the molecular mechanisms, as well as therapeutic options, are still largely lacking. We here show that formation of intravascular tumor-induced neutrophil extracellular traps (NETs) is a cause of kidney injury in tumor-bearing mice. Analysis of clinical biomarkers for kidney function revealed impaired creatinine clearance and elevated total protein levels in urine from tumor-bearing mice. Electron microscopy analysis of the kidneys from mice with cancer showed reversible pathological signs such as mesangial hypercellularity, while permanent damage such as fibrosis or necrosis was not observed. Removal of NETs by treatment with DNase I, or pharmacological inhibition of the enzyme peptidylarginine deiminase 4 (PAD4), was sufficient to restore renal function in mice with cancer. Tumor-induced systemic inflammation and impaired perfusion of peripheral vessels could be reverted by the PAD4 inhibitor. In conclusion, the current study identifies NETosis as a previously unknown cause of cancer-associated renal dysfunction and describes a novel promising approach to prevent renal failure in individuals with cancer.[2] |
| Molecular Formula |
C27H32CLN5O3
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|---|---|
| Molecular Weight |
510.035
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| Exact Mass |
509.219
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| Elemental Analysis |
C, 63.58; H, 6.32; Cl, 6.95; N, 13.73; O, 9.41
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| CAS # |
1652591-81-5
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| Related CAS # |
1652629-23-6;1652591-81-5 (HCl);
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| PubChem CID |
86340151
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| Appearance |
White to light yellow solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
36
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| Complexity |
780
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CN1C2=C(C=C(C=C2OC)C(=O)N3CC[C@H]([C@H](C3)N)O)N=C1C4=CC5=CC=CC=C5N4CC6CC6.Cl
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| InChi Key |
MULKOGJHUZTANI-ADMBKAPUSA-N
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| InChi Code |
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
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| Chemical Name |
((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
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| Synonyms |
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.
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~245.08 mM)
H2O : ~50 mg/mL (~98.03 mM) |
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| Solubility (In Vivo) |
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. 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. View More
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. 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. 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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9606 mL | 9.8032 mL | 19.6063 mL | |
| 5 mM | 0.3921 mL | 1.9606 mL | 3.9213 mL | |
| 10 mM | 0.1961 mL | 0.9803 mL | 1.9606 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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