GSK484-hydrochloride-GTPL8577-DataSheet-生命科學(xué)試劑-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作靶點(diǎn): Protein Arginine Deiminase作通路: Epigenetics儲(chǔ)存式: 4C, stored under nitrogen* In solvent : -80C, 6 months; -20C, 1 month

2、 (stored undernitrogen)溶解性數(shù)據(jù)體外實(shí)驗(yàn) DMSO : 62.5 mg/mL (122.54 mM; Need ultrasonic)Mass Solvent1 mg 5 mg 10 mg Concentration制備儲(chǔ)備液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請(qǐng)根據(jù)產(chǎn)品在不同溶劑中的溶解度，選擇合適的溶劑配制儲(chǔ)備液，并請(qǐng)注意儲(chǔ)備液的保存式和期限。體內(nèi)實(shí)驗(yàn) 請(qǐng)根據(jù)您的實(shí)驗(yàn)動(dòng)物和給藥式選擇適當(dāng)?shù)娜芙獍福?/p>

3、配制前請(qǐng)先配制澄清的儲(chǔ)備液，再依次添加助溶劑(為保證實(shí)驗(yàn)結(jié)果的可靠性，體內(nèi)實(shí)驗(yàn)的作液，建議您現(xiàn)現(xiàn)配，當(dāng)天使；澄清的儲(chǔ)備液可以根據(jù)儲(chǔ)存條件，適當(dāng)保存；以下溶劑前的百分 指該溶劑在您配制終溶液中的體積占)：1. 請(qǐng)依序添加每種溶劑： 10% DMSO 40% PEG300 5% Tween-80 45% salineSolubility: 2.08 mg/mL (4.08 mM); Clear solution2. 請(qǐng)依序添加每種溶劑： 10% DMSO 90% (20% SBE-CD in saline)Solubility: 2.08 mg/mL (4.08 mM); Clear soluti

4、on3. 請(qǐng)依序添加每種溶劑： 10% DMSO 90% corn oilSolubility: 2.08 mg/mL (4.08 mM); Clear solution; Need warming1/3 Master of Small Molecules 您邊的抑制劑師www.MedChemEBIOLOGICAL ACTIVITY物活性 GSK484 hydrochloride種肽酰 精氨酸脫亞氨酶 4 (PAD4) 抑制劑。GSK484 hydrochloride 親和與PAD4 結(jié)合，在不存在鈣的情況下 IC50 為 50 nM。在 2 mM 鈣的存在下，IC50 為 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體外研究 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.體內(nèi)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 您邊的抑制劑師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