GSK484-hydrochloride-GTPL8577-DataSheet-生命科学试剂-MedChemExpress

1、Hotline: 400-820-3792Inhibitors Agonists Screening Librarieswww.MedChemEGSK484 hydrochlorideCat. No.: HY-100514CAS No.: 1652591-81-5Synonyms: GTPL8577; AOB6992分式: CHClNO分量: 510.03作靶点: Protein Arginine Deiminase作通路: Epigenetics储存式: 4C, stored under nitrogen* In solvent : -80C, 6 months; -20C, 1 month

2、 (stored undernitrogen)溶解性数据体外实验 DMSO : 62.5 mg/mL (122.54 mM; Need ultrasonic)Mass Solvent1 mg 5 mg 10 mg Concentration制备储备液1 mM 1.9607 mL 9.8033 mL 19.6067 mL5 mM 0.3921 mL 1.9607 mL 3.9213 mL10 mM 0.1961 mL 0.9803 mL 1.9607 mL请根据产品在不同溶剂中的溶解度，选择合适的溶剂配制储备液，并请注意储备液的保存式和期限。体内实验 请根据您的实验动物和给药式选择适当的溶解案，

3、配制前请先配制澄清的储备液，再依次添加助溶剂(为保证实验结果的可靠性，体内实验的作液，建议您现现配，当天使；澄清的储备液可以根据储存条件，适当保存；以下溶剂前的百分 指该溶剂在您配制终溶液中的体积占)：1. 请依序添加每种溶剂： 10% DMSO 40% PEG300 5% Tween-80 45% salineSolubility: 2.08 mg/mL (4.08 mM); Clear solution2. 请依序添加每种溶剂： 10% DMSO 90% (20% SBE-CD in saline)Solubility: 2.08 mg/mL (4.08 mM); Clear soluti

4、on3. 请依序添加每种溶剂： 10% DMSO 90% corn oilSolubility: 2.08 mg/mL (4.08 mM); Clear solution; Need warming1/3 Master of Small Molecules 您边的抑制剂师www.MedChemEBIOLOGICAL ACTIVITY物活性 GSK484 hydrochloride种肽酰 精氨酸脱亚氨酶 4 (PAD4) 抑制剂。GSK484 hydrochloride 亲和与PAD4 结合，在不存在钙的情况下 IC50 为 50 nM。在 2 mM 钙的存在下，IC50 为 250 nM。IC

5、50 & Target IC50: 50 nM (PAD4, in the absence of Calcium), 250 nM (PAD4, in the presence of 2 mM Calcium) 1体外研究 GSK484 demonstrates high affinity binding to the low-calcium form of PAD4 with IC50s of 50 nM and 250 nMin the absence of Calcium (0 mM) and Calcium (2 mM), respectively. GSK484 also inhib

6、its PAD4citrullination (at 0.2 mM Calcium) of benzoyl-arginine ethyl ester (BAEE) substrate in a concentration-dependent manner, as detected using an NH3 release assay 1.体内研究 To address whether PAD4 inhibition can suppress cancer-associated kidney injury, MMTV-PyMT mice aretreated with the PAD4 inhi

7、bitor GSK484 at 4 mg/kg daily for one week. This dose suppress the elevatednumber of neutrophils undergoing NETosis in peripheral blood in mice with cancer. In parallel, the totalprotein level in urine from MMTV-PyMT mice is significantly reduced compared with untreated tumor-bearingmice, further su

8、pporting an improved functional status of the kidneys after GSK484 treatment. Administrationof GSK484 at a dose of 4 mg/kg daily during one week reverts signs of kidney dysfunction in tumor-bearingmice to the same extent as DNase I treatment, without any detectable signs of toxicity 2.PROTOCOLKinase

9、 Assay 1 PAD4 is serially diluted in the presence of 10 nM GSK215 in assay buffer (100 mM HEPES, pH 8, 50 mMNaCl, 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 are determined

10、using a singlesite saturation curve. For IC50 determination, test compounds (e.g., GSK484) are serially diluted in DMSO(1% final assay concentration) and tested at the same range of calcium concentrations in the presence ofPAD4 (at the calculated Kd for each calcium condition) and 10 nM GSK215 in th

11、e same assay buffer andvolume. Reactions are incubated for 50 min after which IC50 values are calculated using a four-parameterlogistic equation 1.MCE has not independently confirmed the accuracy of these methods. They are for reference only.Cell Assay 1 HEK293 cells stably expressing N-terminal FLA

12、G-tagged PAD1, PAD2, PAD3 or PAD4 are engineered byretroviral transduction. Cells are grown in 15 cm diameter plates to subconfluency in DMEM supplementedwith 10% Foetal Bovine Serum, harvested by centrifugation and washed once in PBS/2 mM EGTA. Cells arelysed in 50 mM Tris-Cl, pH 7.4, 1.5 mM MgCl2,

13、 5% glycerol, 150 mM NaCl, 25 mM NaF, 1 mM Na3VO4,0.4% NP40, 1 mM DTT with protease inhibitors. Lysates are pre-incubated for 20 min at 4C with DMSOalone (2%), 100 M of GSK199, GSK484, GSK106 or 200 M Cl-amidine. Citrullination reactions areperformed for 30 min at 37C in the presence of 2 mM calcium

14、. Extracts are loaded on to gels, proteinsseparated by SDSand transferred to PVDF membranes. Citrullinated proteins are then chemicallymodified and detected using anti-modified citrulline antibody. FLAG-PAD constructs are detected using anti-FLAG antibody 1.2/3 Master of Small Molecules 您边的抑制剂师www.M

15、edChemEMCE has not independently confirmed the accuracy of these methods. They are for reference only.Animal Mice 2Administration 2 The study includes two transgenic mouse models, the MMTV-PyMT mouse model for mammary carcinoma(FVB/n background) and the RIP1-Tag2 mouse model for pancreatic neuroendo

16、crine carcinoma (C57BL/6background). Mice are treated daily by intra-peritoneal injections of the PAD4 inhibitor GSK484 (4 mg/kg).GSK484 is dissolved in 99.9% ethanol at a concentration of 25 mg/mL to generate a stock solution andfurther diluted 1:50 in 0.9% NaCl shortly before injection of 200 L/mo

17、use 2.MCE has not independently confirmed the accuracy of these methods. They are for reference only.REFERENCES1. Lewis HD, et al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol. 2015 Mar;11(3):189-91.2. Cedervall J, et al. Pharmacological targeting of peptidylarginine deiminase 4 prevents cancer-associated kidne