STM2457

METTL3 (IC50 = 16.9 nM)

化合物 72，STM2457，IC50 为 8.699 μM，抑制 MOLM13 细胞增殖[1]。
我们鉴定和表征了一种高效、选择性的METTL3催化抑制剂(STM2457)及其与METTL3/METTL14结合的共晶结构。用STM2457治疗可导致AML生长减少，分化和凋亡增加。这些细胞效应伴随着m6A对已知的致白血病mrna水平的选择性降低，以及它们表达的减少，这与翻译缺陷一致[2]。

在METTL3在体外具有强药理抑制作用的积极证据之后，我们使用临床相关的AML模型进行了体内研究。最初，我们使用了3种不同基因型的人类AML患者来源的异种移植物(PDX)。每日使用STM2457治疗可导致体内移植和AML扩展受损，并显著延长小鼠寿命(图4a-d和扩展数据图6a-e)，且对小鼠体重无明显毒性或影响(扩展数据图6f)。治疗后骨髓和脾脏中人类CD45+细胞的减少也证实了抗白血病作用(图4e)。STM2457在体内有效抑制METTL3靶标，在蛋白水平上选择性减少关键的METTL3 m6A底物，而METTL3水平保持不变(扩展数据图6g)。此外，STM2457处理后，poly-A+富集RNA上的总m6A水平显著降低(扩展数据图6h)。与PDX模型平行，我们使用了一种小鼠MLL-AF9/Flt3Itd/+体内模型，在移植的AML细胞减少、脾脏大小减少、METTL3生物标志物选择性减少以及多聚a +富集RNA上m6A的减少等方面具有类似的抗白血病观察结果(扩展数据图7a-d)[2]。 最后，我们在体内评估了既定抗白血病剂量STM2457的潜在毒性。在骨髓来源的造血干细胞(hsc)和早期祖细胞(Lin−、Sca1+、Kit+)数量、外周血计数或小鼠体重方面未观察到显著变化(扩展数据图8a-e)。我们还证实了METTL3的有效催化抑制作用，因为STM2457处理后，poly-A+富集RNA上的m6A水平显著降低(扩展数据图8f)。这些数据表明，小分子抑制METTL3对AML的维持是有害的，但对正常的造血功能没有显著或持久的影响[2]。

METTL3/14 RF/MS甲基转移酶测定[2]
建立酶法测定抑制RNA甲基转移酶活性的IC50值。使用的酶是全长his标记的METTL3与全长flag标记的METTL14在杆状病毒表达系统中共表达。采用标准亲和层析纯化酶配合物。室温下384孔板进行酶促反应，终反应体积为20 μL，含20 mM TrisCl pH 7.6, 1 mM DTT, 0.01%吐温-20。将终浓度为5 nM的METTL3/14与不同浓度的化合物预孵育10分钟，然后加入终浓度为0.2 μM的合成RNA底物(5 ' p -uacacucgaucuggacuaaagcugcu -3 ')和终浓度为0.5 μM的s -腺苷蛋氨酸(SAM)。室温下再孵育60分钟，加入40 μL 7.5% TCA内标淬火。终止后，将板密封、离心并保存在4°C以待分析。
使用RapidFire™质谱(RF/MS)平台评估METTL3活性，以测量s -腺苷型同型半胱氨酸(SAH)产物。停止和稳定的分析板在Agilent RF300集成自动进样器/固相萃取(SPE)系统上进行分析，该系统与ABSciex 4000质谱联用，用于定量SAH，并归一化为两个内标的信号比。产物(SAH)的质量转变为384.9/135.9 Da。内标的过渡用于矩阵效应的归一化。[2]

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STM2457选择性分析[2]
通过检测一组甲基转移酶和激酶的抑制水平来评估STM2457的选择性。4种RNA甲基转移酶的抑制作用在汉堡Evotec AG公司进行了测试，使用的是与上述METTL3实验等效的RFMS方法。用STM2457稀释系列测定IC50，最高浓度为120 μM，并由此推断10μM化合物的抑制程度。此外，在10 μM STM2457条件下，用tritriated SAM进行放射性测定，评估了41组DNA和蛋白质甲基转移酶的抑制水平。

METTL3细胞水平靶点验证[2]
使用cell Pulse Assay (DiscoverX)通过热移测量STM2457的细胞靶标接合度。使用Fugene HD按照供应商的协议，用pICP-hMETTL3-eLP(人METTL3 Met1-Leu580)或pICP-mMETTL3-eLP(小鼠METTL3 Met1-Leu580)转染HeLa细胞。转染24小时后，将细胞冷冻并保存在液氮中，直到检测当天。在实验当天，将100 nL的化合物稀释液(11点3倍稀释液，实验最高浓度:25 μM)滴入384孔(PP，透明)的检测板中。转染后的细胞在37℃水浴中解冻，用不含酚红的Opti-MEM交换冷冻保护剂。每孔加入细胞悬液20 μL，最终细胞数为1360个/孔。板用铝箔密封，在37℃下孵育1小时。之后，在45°C水浴中倒置孵育15分钟，在室温下进一步孵育10分钟，最后短暂离心以收集每孔底部的液体。随后，每孔加入25 μL检测液(工作液:16.7 v/v EA试剂、16.7 v/v裂解缓冲液和66.7%底物试剂)，转移至384孔(PS，黑色，Flat Bottom)测量板。用铝箔封好，室温慢摇培养30分钟。最后，测定板在100 x g下离心1分钟，室温下使用EnVision多模板读取器测量发光。获得了3个生物重复的剂量响应曲线。<人力资源> AML细胞的流式细胞术分析[2]
用DMSO或STM2457处理细胞，并在指定时间点用抗小鼠CD11b PE/Cy5和抗人CD11b PE染色。数据分析使用LSRFortessa和FlowJo。
在指定的时间点，使用Annexin v检测载药(DMSO)或STM2457处理的人和/或小鼠AML细胞的凋亡水平。使用LSRFortessa仪器分析数据。 使用FITC BrdU Flow Kit或APC BrdU Flow Kit，使用溴脱氧尿苷(BrdU)在指定的时间点测量载药(DMSO)或STM2457处理的人类和/或小鼠AML细胞的细胞周期水平。数据分析采用LSRFortessa仪器。

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Western blot分析[2]
用DMSO或指定浓度的STM2457处理细胞，72小时后将细胞颗粒重悬于全细胞裂解缓冲液(50 mM Tris-HCl pH=8, 450 mM NaCl, 0.1% NP-40, 1mM EDTA)中，并添加1mM DTT、蛋白酶抑制剂和磷酸酶抑制剂。用Bradford法测定蛋白质浓度，每轨装载等量的蛋白质。在上样前，将样品加入SDS-PAGE样品缓冲液，并在每个样品中加入DTT。SDS-PAGE凝胶分离10-40 μg的蛋白，并印迹到聚偏二氟乙烯膜上。

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药物与细胞增殖试验[2]
将所有悬浮细胞接种于96孔板中，每孔5000 - 10000个细胞，一式三次，用载具或指定浓度的STM2457和STM2120 (0.04-50 μM)处理72小时。在第4天，使用新鲜培养基和化合物对所有井进行等量分割，使每口井的细胞密度与初始播种密度相匹配。第6天用CellTiter 96非放射性细胞增殖测定法测定，计算相对细胞增殖。所有化合物都溶解在DMSO中。

For PDX experiments related to Figure 4 and Extended Data 6, 6- to 10-week-old NSG female mice were injected with 106 patient-derived AML cells by intravenous injection. For primary transplants, indicated doses of STM2457 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 5 (PDX-2) or day 10 post-transplant (PDX1,3), once daily for total 12 or 14 days (12-14 treatments). Then bone marrow and spleen cells from these mice were freshly dissected (as mentioned above) and flow-cytometry analysis was performed after staining with various antibodies.[2]

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For primary and secondary transplantation experiments using primary murine MLL-AF9/Flt3ITD/+, 6- to 10-week-old NSG female mice were injected with 106 AML cells by intravenous injection. For primary transplants, indicated doses of STM2457 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 7 post-transplant, once daily for total 10 days. Then bone marrow cells from these mice was freshly dissected (as mentioned above) and blocked with anti-mouse CD16/32 (BD Pharmigen, cat. no. 101323) and 10% mouse serum. For the identification of L-GMP and CD93 populations, staining was performed using various antibodies.[2]

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Generation and bioluminescent imaging of primary murine and PDX models[2]
Generation of AML PDX models and lentiviral transduction for transgenic expression of enhanced firefly luciferase was performed as described in detail by Vick, et al. 38. For primary and secondary transplantation experiments using primary murine MLL-AF9/Flt3ITD/+, 6- to 10-week-old NSG female mice were injected with 106 AML cells by intravenous injection. Indicated doses of STM2457 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 10 post-transplant, once daily for total two weeks (14 treatments). STM2457 was dissolved in 20%(w/v) 2-hydroxyproply beta-cyclodextrin vehicle. At day 10 post-transplant, the tumor burdens of the animals were detected using IVIS Lumina II with Living Image version 4.3.1 software. Briefly, 100 μl of 30 mg/ml D-luciferin was injected into the animals intraperitoneally. Ten min after injection, the animals were maintained in general anesthesia by isoflurane and put into the IVIS chamber for imaging. The detected tumor burdens were measured and quantified by the same software. Diseased mice were assessed blindly by qualified animal technicians from the Sanger mouse facility. Mice were housed in specific pathogen-free conditions in the Wellcome Sanger Institute animal facilities. All cages were on a 12:12-h light:dark cycle (lights on, 07:30) in a temperature-controlled and humidity-controlled room. Room temperature was maintained at 72 ± 2 °F (22.2 ± 1.1 °C), and room humidity was maintained at 30–70%. The animals were culled when the tumor burden was 109 photons per second or higher. All animal studies were carried out in accordance with the Animals Act 1986, UK and approved by the Ethics Committee at the Sanger Institute. Randomization and blinding were not applied. All data in this section were plotted using GraphPad Prism (Version 9).[2]

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STM2457 pharmacokinetic analysis[2]
Three C57BL6/J mice were given IP injections of 30 mg/kg STM2457 and sampled serially up to 24 hours after dosing. Blood was collected from the tail vein at the indicated timepoints. Plasma was isolated by centrifugation, and 20 μL of plasma or blood was mixed with a precipitant solution of 120 μL acetonitrile and internal standard. Supernatant from this precipitation was diluted 1:1 v/v in water and 2.5 uL injections were characterised by LC-MS on a TSQ triple quadrupole mass spectrometer attached to an Accela pump and an HTS-CTC PAL autosampler. STM2457 was resolved on Hypersil Gold C18 solid phase (30 X 2.1 mm, 1.9 μm particles) with an increasing gradient of 5-95% B over 30 seconds. Mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B) and the flow was held at 1.0 ml/min. Blood to plasma ratio was determined using results from the appropriate samples, and the LLOQ was set at 10 ng/ml.

[1]. Mettl3 inhibitory compounds. WO2020201773A1.

[2]. Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021 Apr 26.

STM2457 is a secondary carboxamide resulting from the formal condensation of the carboxy group of 4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxylic acid with the primary amino group of 1-[2-(aminomethyl)imidazo[1,2-a]pyridin-6-yl]-N-(cyclohexylmethyl)methanamine. It is a highly potent and selective first-in-class small molecule inhibitor of METTL3 (N6-adenosine-methyltransferase 70 kDa subunit) enzyme activity and exhibits anti-leukaemic activity. It has a role as an EC 2.1.1.348 (mRNA m6A methyltransferase) inhibitor, an antineoplastic agent and an apoptosis inducer. It is an imidazopyridine, a secondary carboxamide, a secondary amino compound and a pyridopyrimidine.

C25H28N6O2

444.5288

444.23

C, 67.55; H, 6.35; N, 18.91; O, 7.20

2499663-01-1

155167581

White to yellow solid powder

2.7

91.1Ų

2

5

7

33

872

0

OBERVORNENYOLE-UHFFFAOYSA-N

InChI=1S/C25H28N6O2/c32-24-12-21(29-23-8-4-5-11-31(23)24)25(33)27-15-20-17-30-16-19(9-10-22(30)28-20)14-26-13-18-6-2-1-3-7-18/h4-5,8-12,16-18,26H,1-3,6-7,13-15H2,(H,27,33)

N-((6-(((cyclohexylmethyl)amino)methyl)imidazo[1,2-a]pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide

STM-2457; STM2457; CHEMBL5291234; STM-2457; N-((6-(((cyclohexylmethyl)amino)methyl)imidazo[1,2-a]pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide; N-[(6-{[(cyclohexylmethyl)amino]methyl}imidazo[1,2-a]pyridin-2-yl)methyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide; SCHEMBL22499068; GTPL11529; STM 2457

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 : ~50 mg/mL (~112.48 mM)
Ethanol : ~89 mg/mL (200.2 mM)
Water : Insoluble (<1 mg/mL)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.68 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 (4.68 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 (4.68 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: 5 mg/mL (11.25 mM) in 50% PEG300 50% 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。