PRMT5

在 MTAP 缺失的 HCT116 分裂野生型细胞中，MRTX-1719（10 天）抑制 PRMT5 活性，IC50 为 8 nM [1]。在缺乏 MTAP 的 HCT116 细胞中，MRTX1719（10 天）的 IC50 值为 12 nM，但在具有野生型表型的 HCT116 细胞中为 890 nM [1]。

MRTX1719（12.5-100 mg/kg/天，面部，21 天）抑制 Lu-99 原位异种移植物中的肿瘤生长 [2]。药代动力学分析[2]模型途径剂量(mg/kg)Cltotal(mL/min/kg)Vdss(L/kg)t1/2(h)CD-1小鼠。 iv 3 83 6.3 1.5 Beagle 犬 iv 2 14 3.4 4.8 食蟹猴 iv 2 15 2.3 6.1 模型途径剂量 (mg/kg) Cmax (ug/mL) AUCinf (h*ug/mL) F (%) CD-1 小小鼠。 po 30 1.16 4.85 80 比格犬 po 10 1.40 7.47 59 食蟹猴 po 10 / / 41

使用Reaction Biology Corporation的FlashPlate系统进行PRMT5生物化学研究，使用PMRT5/MEP50复合物（#HMT-22-148：人重组PRMT5[基因库登录号NM_006109；氨基酸2-637（末端）]，具有N-末端Flag标签和人重组MEP50[基因库登记号NM_024102；氨基酸2-342（末端））]，带有N-末端His标签，在Sf9细胞表达系统中共表达）。FlashPlate底物是生物素组蛋白H4（1-15）肽。甲基供体是S-腺苷-L-[甲基-3H]甲硫氨酸。反应缓冲液为50mmol/L Tris-HCl（pH 8.5）、0.002%Tween20、0.005%牛血清白蛋白（BSA）、1mmol/L TCEP和1%二甲基亚砜（DMSO）。[1]
最终反应条件为3 nmol/L PRMT5/MEP50、40 nmol/L组蛋白H4）-生物素肽和1μmol/L SAM。在反应缓冲液中制备底物。将酶以40μL/孔的速度输送到底物溶液中。将化合物在DMSO中递送到反应混合物中，并在室温下预培养20分钟。将氚化的SAM输送到反应混合物中，以10μL/孔开始反应，并在室温下孵育反应2小时。为了停止反应，输送10μL/孔的600μmol/L冷SAM，并将50μL/井的停止反应转移到FlashPlate中，并在室温下孵育1小时。用PBS洗涤板，并在TopCount HTS微孔板闪烁和发光计数器上计数。在存在或不存在2μmol/L MTA的情况下，运行10剂量IC50，从1μmol/L开始连续稀释3倍。[1]

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生化甲基转移酶测定法[2]
该测定使用纯化的人PRMT5酶将S-腺苷-l-[甲基-3H]甲硫氨酸加组蛋白H4-l-精氨酸转化为S-腺苷-l-多半胱氨酸加组蛋白H4[甲基-3H]-l-精氨酸。使用链霉亲和素涂层的FlashPlates进行测定，FlashPlate含有嵌入板塑料中的闪烁体。组蛋白H4肽底物与生物素缀合，生物素与板的链霉亲和素包被的孔结合，将H4肽放置在孔和闪烁体的侧面附近。氚化甲基从S-腺苷-l-[甲基-3H]甲硫氨酸转移到结合的组蛋白H4肽产生放射性标记的组蛋白H3，通过在闪烁计数器中测量放射性来定量，以确定在存在和不存在化合物的情况下PRMT5酶的活性。测定反应也在MTA存在和不存在的情况下进行，以确定化合物是否表现出MTA协同活性。简言之，将化合物溶解在最高浓度为10mM的100%DMSO中。对于IC50测定，每种化合物的系列稀释液的初始起始浓度为50μM。缺乏化合物、PRMT5/MEP50复合物或各种反应组分的对照样品也与化合物测试样品平行制备和处理。SAH被用作测定验证的阳性对照。为了测量PRMT5的抑制活性，将3 nM PRMT5/MEP50复合物（反应生物学公司）与受试化合物在含有40 nM组蛋白H4肽（氨基酸1-15）-生物素缀合物的测定缓冲液中在室温下预孵育20分钟。通过添加1μM氚化SAM（终浓度）启动酶促反应，并使反应进行20分钟。停止反应，并使用闪烁计数器测定每个样品中结合的氚化H4肽的量。使用GraphPad Prism软件，根据加上和减去MTA的样品的每个10点剂量-反应曲线计算每个化合物的IC50值。

HCT116 MTAP-WT和MTAP del活力测定[2]
使用HCT116-MTAP-WT和HCT116-MTAP-del细胞系进行活力测定。平行分析对照样品。第0天，将250个HCT116-MTAP-WT或HCT116-MTAP-del细胞接种在补充有10%胎牛血清和pen/strep的McCoy’s 5A的96孔板中，并将细胞在37°C加5%CO2下孵育过夜。第二天，用DMSO载体对照或测试化合物的剂量反应处理细胞，并在37°C加5%CO2下孵育5天。在第6天，对细胞进行胰蛋白酶消化，并以1:20的比例将其分裂成含有相同浓度测试化合物的新鲜培养基的新的96孔板，并在37°C加5%CO2的条件下再孵育5天。在第11天，根据制造商的说明，使用CTG测定试剂盒测量细胞的活力。使用GraphPad PRISM软件计算处理10天后每种化合物的IC50值。

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HCT116 MTAP-WT和MTAP del SYM11细胞内蛋白质测定[2]
通过测量PRMT5依赖性对称二甲基精氨酸（SDMA）信号，使用HCT116-MTAP-WT和HCT116-MTAP-del细胞系进行细胞内蛋白质测定。平行分析对照样品。在第0天，将2000个HCT116-MTAP-WT和HCT116-MTAP-del细胞接种在补充有10%胎牛血清和pen/strep的McCoy’s 5A的96孔板中，并将细胞在37°C加5%CO2下孵育过夜。第二天，用DMSO载体对照或测试化合物的剂量反应处理细胞，并在37°C加5%CO2下孵育4天。处理4天后，通过向每个孔中加入50μL 4%多聚甲醛溶液来固定细胞，并在室温下孵育细胞20分钟。然后去除多聚甲醛溶液，加入150μL冰冷的甲醇，并将平板在−20°C下储存10分钟。经过这段时间后，去除甲醇，加入150µL奥德赛阻断缓冲液+0.05%Tween-20，并在室温下振荡孵育平板1小时。向每个测试孔中，加入50μL SYM11抗体，该抗体在奥德赛阻断缓冲剂+0.05%吐温-20中以1:500稀释，并将板置于4°C过夜。通过抽吸去除初级SYM11抗体溶液，并用含有0.1%吐温-20（PBST）的磷酸盐缓冲盐水洗涤孔3次。加入50μL等分试样的山羊抗兔IRDye 800CW二级抗体（Li Cor 926-32211），其稀释比例为1:800，核染色剂DRAQ5稀释比例为1:10000，加入奥德赛封闭缓冲液+DRAQ5+0.05%吐温-20中，并将板在室温下在黑暗中储存2小时。通过抽吸去除第二抗体溶液，并用PBST洗涤孔3次。SYM11信号和DRAQ5信号分别使用Li-Cor-Odyssey机器在800nM和700nM下进行定量。SYM11/DRAQ5比率用于计算SDMA的抑制作用，作为DMSO对照的百分比。

Animal/Disease Models: Lu-99 (MTAP/CDKN2A deleted human lung cancer cell line) xenograft tumor model [2]
Doses: 12.5, 25, 50 and 100 mg/kg/d, 21-day
Route of Administration: Oral tube feeding
Experimental Results: tumor volume reduction, tumor growth inhibition (TGI) of 86% at 50 mg/kg and 88% at 100 mg/kg.

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In Vivo Studies[1]
All mouse studies were approved by the Institutional Animal Care and Use Committee based on guidelines from the NIH. Mice were maintained under pathogen-free conditions, and food and water were provided ad libitum.[1]
Six- to 8-week-old female Hsd:Athymic Nude-Foxn1nu mice were injected subcutaneously with tumor cells in 100 μL of PBS and Matrigel matrix in the right hind flank of each mouse with 5e6 cells (LU99) or 1e6 cells (HCT116 parental and HCT116 MTAP del) 50:50 cells:Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 × L × W2, in which L refers to the length and W refers to the width of each tumor. When tumors reached the desired average study start tumor volume (LU99: 179 mm3 or 116 mm3; HCT116 MTAP WT: 140 mm3; HCT116 MTAP del: 185 mm3), mice were randomized into treatment groups. MRTX1719 was formulated in 0.5% methylcellulose (4,000 cps) + 0.2% Tween80 in water once per week and stored at room temperature protected from light. GSK3326595 was formulated in 0.5% methylcellulose (4,000 cps) + 0.2% Tween80 in water once per week and stored at 4°C protected from light. JNJ-64619178 was formulated in 20% HP-β-CD in water once per week and stored at room temperature protected from light. Mice were orally administered vehicle, MRTX1719, GSK3326595, or JNJ-64619178 at the indicated doses and schedules. Mice were monitored daily, with tumor volumes and body weights measured 2 or 3 times per week.[1]

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The PXF 537, PXF 2197, PXF 1118, PXF 541, and PXF 2442 mesothelioma PDX model experiments were conducted at Charles River with similar study designs to the LU99 and HCT116 tumor models. Briefly, NMRI nu/nu mice were inoculated subcutaneously with a 2- to 3-mm3 PDX fragment and randomized for treatment initiation when the mean tumor volumes reached ∼120 to 140 mm3. Mice were treated with vehicle or MRTX1719 (n = 3 per group) at the indicated dose and duration.[1]

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Xenograft Study[2]
The Lu-99 cell line (RCB1900) was obtained from RIKEN BioResource Research Center (Tsukuba, Japan) and grown in RPMI 1640 media containing 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, and 1% antibiotic–antimycotic. Mouse studies were conducted in compliance with all applicable regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) from the National Institutes of Health (NIH). Mice were maintained under pathogen-free conditions and food and water were provided ad libitum. 6–8-week-old female Hsd:Athymic Nude-Foxn1nu mice (Envigo, San Diego) were injected subcutaneously, with 5.0 × 106 Lu-99 tumor cells in 100 μL of PBS and Matrigel matrix in the right hind flank of each mouse (50:50 cells:Matrigel). Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 × L × W2, in which L refers to length and W refers to width of each tumor. When tumors reached an average tumor volume of ∼180 mm, mice were randomized into treatment groups. MRTX1719 was formulated in 0.5% methylcellulose (4000 cps) + 0.2% Tween 80 in water once per week and stored at room temperature protected from light. Mice were orally administered vehicle or MRTX1719 PO at the indicated doses and schedules. Mice were monitored daily, and tumor volumes and body weights were measured 2 or 3 times per week. Percent tumor growth inhibition (% TGI) was calculated using the following formula: (1 – (Final Drug Treated Tumor Volume – Initial Drug Treated Tumor Volume)/(Final Vehicle Treated Tumor Volume – Initial Vehicle Treated Tumor Volume)) × 100.[2]
Whole blood was collected to measure plasma concentrations of MRTX1719 and assess pharmacokinetic properties of MRTX1719 at specific time points postdose. Blood (∼50 μL) was collected via submandibular draw into a heparinized tube and immediately inverted to mix. Heparinized blood was then centrifuged at 4000 rpm for 6 min to separate plasma and cellular fractions. The separated plasma was transferred to a polypropylene 96-well plate and immediately stored at −20 °C until analysis. At the end of study, mice were humanely sacrificed, and tumors were surgically removed and immediately cut in half. One half was transferred to a Lysing Matrix A tube, and the other half was transferred to a screw-top microtube. These tubes were immediately submerged in liquid nitrogen to snap freeze the tissue. Frozen tumor fragments were stored at −80 °C until analysis.

[1]. MRTX1719 is an MTA-cooperative PRMT5 inhibitor that exhibits synthetic lethality in preclinical models and patients with MTAP deleted cancer. Cancer Discov. 2023 Aug 8;CD-23-0669.

[2]. Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers. J Med Chem. 2022 Feb 10;65(3):1749-1766.

[3]. Targeting the genetic and immunological drivers of cancer.

PRMT5-MTA抑制剂MRTX1719是一种口服生物利用度高的蛋白精氨酸甲基转移酶5 (PRMT5)-甲基硫代腺苷 (MTA) 复合物抑制剂，具有潜在的抗肿瘤活性。口服后，PRMT5-MTA抑制剂MRTX1719选择性地与甲基硫代腺苷磷酸化酶 (MTAP) 缺失的癌细胞中高表达的PRMT5-MTA复合物结合，从而特异性地抑制PRMT5在MTAP缺失的癌细胞中的功能，而不影响正常健康细胞。通过抑制PRMT5的甲基转移酶活性，组蛋白H2A、H3和H4中单甲基化和二甲基化精氨酸残基的水平降低。这调节了参与多种细胞过程（包括细胞增殖）的基因表达。这可能导致抗增殖基因表达增加和/或促进细胞增殖基因表达减少，从而导致快速增殖的癌细胞生长减缓。MRTX1719 还会导致 RNA 剪接失调和 pRb 水平降低。这些因素共同作用，特异性地降低 MTAP 缺失癌细胞的增殖并增加其凋亡。PRMT5 是一种 II 型甲基转移酶，催化组蛋白以及参与信号转导和细胞转录的多种其他蛋白质底物上 ω-N 单甲基精氨酸 (MMA) 和对称二甲基精氨酸 (sDMA) 的形成，对癌细胞和正常细胞的存活至关重要。它在多种肿瘤中过度表达。PRMT5 水平升高与患者生存期缩短相关。某些癌细胞中 MTAP 缺失，导致代谢产物 MTA 积累；MTA 与 PRMT5 结合并部分抑制其活性。

C23H18CLFN6O2

464.87942647934

464.12

C, 59.42; H, 3.90; Cl, 7.63; F, 4.09; N, 18.08; O, 6.88

2630904-45-7

(S)-MRTX-1719;2630904-44-6;MRTX-1719 hydrochloride

156151242

White to light yellow solid powder

2.7

118Ų

2

7

5

33

846

0

BZKIOORWZAXIBA-UHFFFAOYSA-N

InChI=1S/C23H18ClFN6O2/c1-31-22(20-15(8-26)19(33-12-3-4-12)7-17(24)21(20)25)16(10-28-31)11-2-5-13-14(6-11)18(9-27)29-30-23(13)32/h2,5-7,10,12H,3-4,9,27H2,1H3,(H,30,32)

2-(4-(4-(aminomethyl)-1-oxo-1,2-dihydrophthalazin-6-yl)-1-methyl-1H-pyrazol-5-yl)-4-chloro-6-cyclopropoxy-3-fluorobenzonitrile

MRTX1719; MRTX 1719; MRTX-1719;

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 (~215.11 mM)

配方 1 中的溶解度: ≥ 2 mg/mL (4.30 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 20.0 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 mg/mL (4.30 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 mg/mL (4.30 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 20.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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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。