EZM2302 (GSK-3359088)

CARM1 (IC50 = 6 nM)

当与 CARM1 结合时，EZM 2302 对其他组蛋白甲基转移酶表现出广泛的选择性，同时选择性抑制 CARM1 活性 (IC50=6 nM)。将 EZM 2302 应用于 MM 细胞系可防止细胞停滞以及 PABP1 和 SMB 甲基化，IC50 值在纳摩尔范围内（分别为 9 和 31 nM）。 EZM 2302 在体外抑制许多造血细胞系；第 14 天时，15 个细胞系中有 9 个的 IC50 值低于 100 nM [1]。

EZM 2302 在人肝细胞中表现出稳定性 (CL<3 mL/min/kg)，并在小鼠、大鼠和人类中表现出与血浆蛋白的中等结合，平均未结合分数依次为 0.66、0.46 和 0.74。大鼠和小鼠的血浆清除率（CL）分别为91 mL/min/kg和43 mL/min/kg。在 RPMI-8226 异种移植模型中使用 21 天后，EZM 2302 显示出剂量依赖性暴露和肿瘤生长抑制 (TGI)。第 21 天，所有 EZM 2302 给药组的肿瘤发展均明显慢于媒介物 [1]。

组蛋白甲基转移酶测定[1]
如先前报道的组蛋白甲基转移酶PRMT1/6/865和PRMT558那样测量CARM1活性。简言之，在开始反应之前，将CARM1与化合物在室温下预孵育30分钟。最终测定条件为0.25 nM CARM1、30 nM 3H-S-腺苷甲硫氨酸（SAM）和250 nM生物素化肽，缓冲液含有20 mM联苯胺、1 mM三（2-羧乙基）膦、0.005%牛皮明胶和0.002%吐温-20，pH 7.5。通过添加300μM未标记的SAM来猝灭测定。通过Topcount读取器上的Flashplate测量产生的3H标记肽的量。更多的细节可以在补充方法中找到。[1]

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蛋白质生产[1]
用于生物化学研究的FLAG-CARM1（氨基酸2-585）-His在293F哺乳动物细胞中表达，并使用FLAG亲和层析进行纯化。使用昆虫细胞生产用于x射线晶体学的His-TEV-CARM1（氨基酸134-479），并使用Ni亲和和尺寸排阻色谱进行纯化。详细方法见补充方法

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X射线晶体学[1]
使用蒸汽扩散法生长CARM1（氨基酸134-479）的晶体，并将化合物浸泡在预先形成的晶体中。结晶和结构测定方法，包括数据收集和细化统计，可在补充方法和补充表S1中找到。结构已经与以下PDB代码一起存储在蛋白质数据库中：化合物2:6ARV；EZM2302:6ARJ.

体外复合治疗[1]
线性/对数期生长的培养细胞在2–20mL的培养基中分裂为2e5个细胞/mL的接种密度，这取决于生长期结束时所需的产量。将化合物在DMSO中稀释并以0.2%的最终DMSO浓度添加到每个培养容器中。使细胞生长96小时。在每个处理期结束时，通过离心（5分钟，1200rpm）收获细胞，并用PBS冲洗细胞颗粒一次，然后冷冻在干冰上等待进一步处理。

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蛋白质印迹[1]
在含有0.1%SDS的1X RIPA缓冲液中，从细胞颗粒、快速冷冻的肿瘤异种移植物样品或外周组织制备裂解物。使用红外发射的第二抗体和ODYSSEY红外成像系统进行蛋白质印迹的定量。

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体外增殖测定[1]
使用先前描述的方法进行长期（14-15天）细胞增殖测定，并根据每个细胞系的生长特性调整初始接种密度。有关6天的增殖研究，请参阅补充方法。

In vivo xenograft studies[1]
All of the procedures related to animal handling, care and the treatment in this study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Chempartner following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Experimental protocols were approved by the IACUC of Shanghai Chempartner. For the in vivo efficacy studies, there were 8 mice per dose group and each mouse was inoculated subcutaneously at the right flank. All cells were suspended in a 0.2 mL mixture of base media and Matrigel at 1:1 for tumor development. RPMI-8226 cells were inoculated at 5 × 106 cells/mouse and treatment began when the mean tumor sizes reached 120 mm3 (28 days post-inoculation). CB-17 SCID Mice were assigned into groups using a randomized block design. EZM2302 or vehicle (0.5% methylcellulose in water) was administered orally BID at a dose volume of 37.5, 75, 150, or 300 mg/kg for 21 days. Body weights were measured twice a week for the duration of the study. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in cubic millimeters. Animals were euthanized 3 hours post-final dose, with blood and tissues collected for analysis.

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Pharmacokinetic Study in Mouse and Rat[1]
All of the procedures related to animal handling, care and the treatment in this study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Chempartner following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Experimental protocols were approved by the IACUC of Shanghai Chempartner. Male CD-1 mice (n = 9/group, 3 per time-point) and male Sprague-Dawley rats (n = 3) were treated with a single dose of EZM2302 at 2 mg/kg by intravenous (i.v.) injection and 10 mg/kg by oral gavage administration (p.o.; mouse only), formulated in 5% dextrose in water, pH 3.5. An additional group of rats, cannulated in both the jugular and portal veins were dosed by oral gavage (10 mg/kg, in 0.5% methylcellulose in water). Approximately 110 μL of blood was taken from the animals by retro-orbital bleeding (mouse), tail vein (rat i.v.) or both jugular and portal vein sampling (rat p.o.) at pre-specified time intervals. The 2 h samples were split for parallel determination of blood and plasma concentration (ex vivo ratio). Blood samples were collected into K2-EDTA tubes and centrifuged to obtain plasma. Following protein precipitation, EZM2302 concentrations were determined by LC-MS/MS analysis and data were analyzed using Phoenix WinNonlin 6.2 or higher.

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ADME Assays[1]
Hepatocyte stability, plasma protein binding and cytochrome P450 assays were performed using standard procedures. Details can be found in Supplementary Methods.

[1]. Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma. Sci Rep. 2017 Dec 21;7(1):17993.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases[1].

C29H37CLN6O5

585.094285726547

584.251

C, 59.53; H, 6.37; Cl, 6.06; N, 14.36; O, 13.67

1628830-21-6

90425581

White to off-white solid powder

3.4

126

2

10

9

41

873

1

CC1=C(N=C(N=C1N2CC3(C2)CCN(CC3)C(=O)OC)C4=C(C=CC(=C4)OC[C@@H](CNC)O)Cl)C5=C(ON=C5C)C

OWCOTUVKROVONT-HXUWFJFHSA-N

InChI=1S/C29H37ClN6O5/c1-17-25(24-18(2)34-41-19(24)3)32-26(22-12-21(6-7-23(22)30)40-14-20(37)13-31-4)33-27(17)36-15-29(16-36)8-10-35(11-9-29)28(38)39-5/h6-7,12,20,31,37H,8-11,13-16H2,1-5H3/t20-/m1/s1

Methyl (R)-2-(2-(2-chloro-5-(2-hydroxy-3-(methylamino)propoxy)phenyl)-6-(3,5-dimethylisoxazol-4-yl)-5-methylpyrimidin-4-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate

EZM2302 GSK3359088EZM-2302 GSK-3359088EZM 2302 GSK 3359088

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)

DMSO : ~100 mg/mL (~170.91 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.27 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 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (3.56 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澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: ≥ 2.08 mg/mL (3.56 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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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。