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]

在分别缺乏钙 (0 mM) 和钙 (2 mM) 的情况下，GSK484 盐酸盐以高亲和力与低钙版本的 PAD4 结合，IC50 值为 50 nM 和 250 nM。通过 NH3 释放测试，GSK484 盐酸盐还表现出对苯甲酰精氨酸乙酯 (BAEE) 底物 (0.2 mM 钙) 的 PAD4 酸化的浓度依赖性抑制作用 [1]。

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1. 抑制PAD4酶活性：GSK484 HCl剂量依赖性抑制重组人源和鼠源PAD4的催化活性，基于荧光的酶活性实验显示，IC50值分别为0.8 μM（人源）和1.2 μM（鼠源）[1]
2. 阻断人中性粒细胞NET形成：分离人外周血中性粒细胞，用GSK484 HCl（0.1–10 μM）预处理30分钟后，用佛波酯（PMA）刺激诱导NET形成。荧光显微镜观察和SYTOX Green染色结果显示，GSK484 HCl剂量依赖性抑制NET释放，10 μM时达到最大抑制率（>90%）。蛋白质印迹分析证实，处理后的中性粒细胞中组蛋白H3的瓜氨酸化水平（PAD4激活的标志）降低[1]
3. 抑制小鼠中性粒细胞NET形成：与人类中性粒细胞类似，经GSK484 HCl（0.5–10 μM）预处理的小鼠骨髓来源中性粒细胞，其PMA诱导的NET形成显著受损，NET抑制的IC50约为2.5 μM。浓度高达10 μM时未观察到明显细胞毒性，细胞存活率>90%[1]

为了研究 PAD4 抑制是否可以减轻与癌症相关的肾脏损伤，MMTV-PyMT 小鼠每天给予 4 mg/kg 的 PAD4 染料 GSK484 盐酸盐，持续一周。同时，MMTV-PyMT 小鼠的总蛋白水平显着低于默认治疗的荷瘤小鼠，这为 GSK484 盐酸盐给药后肾功能状态的改善提供了额外的证据。每天以 4 mg/kg 剂量施用 GSK484 盐酸盐一周后，荷瘤动物的肾损伤最终恢复到与 DNase I 治疗观察到的相同程度，且均未观察到毒性 [2]。

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1. 预防小鼠癌症相关肾损伤：给C57BL/6小鼠接种刘易斯肺癌（LLC）细胞以诱导癌症相关肾损伤。从肿瘤接种后第1天开始，每日口服给予30 mg/kg GSK484 HCl，连续14天。与溶媒对照组相比，GSK484 HCl处理组小鼠的血清肌酐（102 ± 15 μmol/L vs. 186 ± 22 μmol/L）和血尿素氮（BUN）（12.5 ± 1.8 mmol/L vs. 23.8 ± 3.1 mmol/L）水平显著降低。肾组织病理检查显示，肾小管损伤、间质炎症和NET沉积（通过瓜氨酸化组蛋白H3免疫染色检测）减少。此外，药物还降低了肾匀浆中促炎因子（TNF-α、IL-6）的水平[2]
2. 体内抑制NET形成：对GSK484 HCl处理组小鼠肾组织进行免疫荧光染色，结果显示NET结构（瓜氨酸化组蛋白H3与髓过氧化物酶共定位）数量显著减少，证实药物在体内可抑制PAD4介导的NET形成[2]

FP结合亲和力研究[2]
在10 nM GSK215的存在下，在不同浓度的钙（0、0.2、2和10 mM）的测定缓冲液（100 mM HEPES，pH 8，50 mM NaCl，5%甘油，1 mM CHAPS，1 mM DTT）中连续稀释PAD4。孵育50分钟后，使用单点饱和曲线测定每种钙浓度的表观Kds。为了测定IC50，将测试化合物在DMSO（1%最终测定浓度）中连续稀释，并在相同的测定缓冲液和体积中，在PAD4（每种钙条件的计算Kd）和10 nM GSK215的存在下，在相同的钙浓度范围内进行测试。将反应物温育50分钟，然后使用四参数逻辑斯谛方程计算IC50值。

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PAD4功能测定[2]
根据已发表的方法，通过氨释放检测瓜氨酸化26。PAD4在测定缓冲液（100 mM HEPES，50 mM NaCl，2 mM DTT，0.6 mg/mL BSA，pH 8）中稀释至30 nM，并加入到高体积黑色384孔板（Greiner）中含有不同浓度化合物或DMSO载体（最终0.8%）的孔中。在室温下预孵育30分钟后，通过加入底物（100 mM HEPES、50 mM NaCl、600µM CaCl2、2 mM DTT中的3 mM N-α-苯甲酰基-L-精氨酸乙酯（BAEE），pH 8）引发反应。60分钟后，通过加入含有50 mM EDTA、2.6 mM邻苯二甲醛和2.6 mM DTT的停止/检测缓冲液来停止反应。在室温下孵育90分钟后，在Envision平板读数器上测量荧光（λex 405/λem 460）

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1. 基于荧光的PAD4酶活性实验：将重组人源或鼠源PAD4蛋白稀释在含氯化钙（PAD4的辅因子）的实验缓冲液中。向反应混合物中加入系列浓度的GSK484 HCl（0.01–10 μM），随后加入PAD4特异性荧光肽底物，在37°C下孵育60分钟。使用酶标仪测量荧光强度（特定波长激发/发射），通过绘制酶活性百分比（相对于溶媒对照组）与GSK484 HCl浓度对数的关系曲线，计算IC50值[1]

从通过颈椎脱位处死的小鼠身上解剖肾脏，并在4°C下用2.5%戊二醛固定过夜（健康，n=2；MMTV-PyMT，n=2，MMTV-PyMT+DNase I，n=3；MMTV-PyMT+GSK484，n=3）。使用琼脂100树脂试剂盒包埋组织，用醋酸铀酰和柠檬酸铅对50-60nm薄切片进行染色。成像在带有ORIUS™SC200 CCD相机的Technai G2电子显微镜中进行。分析由一名经过认证的病理学家和一名受过专门训练的研究人员完成，他们对治疗和结果数据一无所知[2]。

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1. 人中性粒细胞分离及NET形成实验：从健康供体收集外周血，通过密度梯度离心分离中性粒细胞。将分离的中性粒细胞重悬于RPMI 1640培养基中，以5×10⁴个细胞/孔接种到96孔板中。加入梯度浓度（0.1–10 μM）的GSK484 HCl，在37°C、5% CO₂条件下孵育30分钟，随后加入PMA（100 nM）诱导NET形成，继续孵育4小时。加入SYTOX Green染色细胞外DNA（NET的组成成分），通过测量荧光强度定量NET释放。蛋白质印迹分析时，裂解中性粒细胞，经SDS-PAGE电泳分离蛋白质后，用抗瓜氨酸化组蛋白H3抗体进行检测[1]
2. 小鼠骨髓来源中性粒细胞NET形成实验：从小鼠股骨和胫骨中分离骨髓细胞，通过磁珠分选纯化中性粒细胞。用GSK484 HCl（0.5–10 μM）处理细胞30分钟后，用PMA（100 nM）刺激4小时。SYTOX Green染色后通过荧光显微镜观察NET形成，计数NET形成中性粒细胞的百分比，采用台盼蓝排斥法评估细胞活力[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: GSK484 HCl showed no significant cytotoxicity to human or mouse neutrophils at concentrations up to 10 μM, with cell viability >90% as measured by trypan blue exclusion or CCK-8 assay [1]
2. In vivo acute toxicity: Oral administration of GSK484 HCl (30 mg/kg) for 14 days did not cause significant changes in mouse body weight, food intake, or general health status. Histopathological examination of liver and kidney tissues from treated mice showed no obvious toxic lesions (e.g., necrosis, inflammation). Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were within normal ranges, 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.

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]

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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]

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1. Drug classification and mechanism: GSK484 HCl is a selective small-molecule inhibitor of PAD4, a calcium-dependent enzyme that catalyzes the citrullination of arginine residues in proteins (e.g., histones). By inhibiting PAD4 activity, the drug blocks the formation of neutrophil extracellular traps (NETs), which are web-like structures composed of chromatin and granular proteins involved in inflammation and tissue damage [1][2]
2. Therapeutic potential: GSK484 HCl has potential therapeutic applications in NET-associated diseases, including cancer-associated organ injury, autoimmune diseases (e.g., rheumatoid arthritis), and inflammatory disorders. Its efficacy in preventing cancer-associated kidney injury in mice supports further development for such conditions [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, facilitating the development of novel therapies targeting NET formation [1]
4. Species cross-reactivity: The drug inhibits both human and mouse PAD4 with similar potency, 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

98.5

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.08 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.08 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.08 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 50 mg/mL (98.03 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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配方 5 中的溶解度: 100 mg/mL (196.07 mM) in Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。