DNMT1

GSK-3685032（6天）的中位生长IC50值为0.64 μM，这表明它抑制大多数癌细胞系的发育[1]。由于在整个 6 天持续时间内生长 IC50 降低，GSK-3685032（0.1-1000 nM，第 1-6 天）在 3 天后表现出生长抑制 [1]。 GSK3685032（10-1000 nM，第 4 天）可增加免疫相关基因转录，呈剂量依赖性[1]。 DNMT1 蛋白的表达被 GSK3685032（3.2-10,000 nM，2 天）抑制 [1]。 GSK3685032 诱导 DNA 低甲基化和基因激活 [1]。

在皮下 MV4-11 或 SKM-1 异种移植模型中，GSK-3685032（1-45 mg/kg；皮下注射，每天两次，持续 28 天）可减缓肿瘤的形成 [1]。小鼠药代动力学参数汇总：GSK-3685032[1]；剂量、途径； Cmax（纳克/毫升）； AUC0-8小时（h*ng/mL）； DNAUC（h*kg*ng/mL/mg）；清除率（mL/min/kg）；体积（升/千克）； T1/2（小时）2毫克/公斤； IV 5103 2418 1209 13 1.3 1.8 2 毫克/千克； SC 252 921 461 NA NA 2.8 2 mg/kg, SC 5473 15400 513 NA NA ND

荧光耦合破碎光分析。[1]
如前所述，使用半甲基化发夹寡核苷酸检查DNMT的活性39。最终测定浓度由125 nM DNA寡核苷酸和（1）40 nM全长DNMT1、2μM SAM组成；（2） 600 nM DNMT3A/3L，2.5μM SAM；或（3）300 nM DNMT3B/3L，0.15μM SAM。DNMT1的反应在40分钟（26°C）后淬灭，DNMT3A/3L和DNMT3B/3L的反应在120分钟（37°C）后淬灭。将化合物（10点，三倍连续稀释，100%二甲亚砜）预先印在黑色反应板上（最终2%二甲基亚砜）。通过用1:4比例的全甲基/半甲基化发夹寡核苷酸（ATDBio定制合成的5-FAM-ATCTAG5-me-dCG5me-dCATCAGTTTTCTGATG5me-dCTAGA-Dabcyl-3和5-FAM-ATCSAG5-me-dCATCAGTTTCTGATG 5me-dCG5me-dcTAGA-Daccyl-3）代替DNMT反应，进行Gla1计数器筛选。对于可逆性研究，在DNMT1与化合物（10×IC50）预孵育20分钟后，加入底物后，复合物迅速稀释100倍。通过稀释后不同时间点的淬灭，在70分钟内评估DNMT1活性的恢复情况。如Ariazi等人所述，数据符合固定的稳态速度方程。

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组蛋白甲基转移酶和激酶选择性。[1]
使用专有的HotSpot技术对甲基转移酶47和激酶48的抑制剂选择性进行了评估GSK-3685032在单一浓度（10μM）下对激酶组进行了测试。甲基转移酶面板加PIM1、PKD2/PRKD2和DYRK2以IC50格式进行测试（10点，三倍连续稀释）。

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共价加合物的研究。[1]
在GSK3685032A（25μM）存在或不存在的情况下，将小鼠DNMT1（731–1602，5.6μM）与14-mer半甲基化DNA（25μM）在20mM Tris pH 7.5、50mM NaCl、5mM二硫苏糖醇、20%甘油和2.5%二甲亚砜中孵育20小时。然后将等分试样在0.05%TFA、0.1%甲酸溶液中稀释五倍，并注射20pmol蛋白质样品，在安捷伦6224 TOF LC-MS仪器上进行完整质量分析，并可能检测共价加合物。14-mer半甲基化DNA双链购自IDT（5-GGAGGC5me-dCGCCTGCT-3，带有补体链3-CCTCCGGCGGACGA-5）。

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光亲和标记。[1]
在25μM光反应抑制剂（GSK3844831或GSK3901839）的存在下，将小鼠DNMT1（731–1602，5.7μM）与14-mer半甲基化DNA（25μM）在20 mM Tris pH 7.5、50 mM NaCl、5 mM二硫苏糖醇、20%甘油、2.5%二甲亚砜中孵育。在紫外光（λ=350nm）下进行光解45分钟。完整质量分析用于监测光标记掺入水平。光标记的mDNMT1用胃蛋白酶和因子XIII进行蛋白水解消化。通过相对于未标记mDNMT1的差异图谱鉴定标记的蛋白质片段，并使用液相色谱（LC）和基于串联质谱（MS/MS）的测序确定标记的氨基酸。使用Mascot v.2.6在内部蛋白质序列数据库中搜索MS/MS数据，以确定共价标记的位置。

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DNMT1-DNA抑制剂复合物的形成。[1]
DNMT1-DNA复合物是通过将DNMT1-DNA-SAH以约1:5:10的摩尔比孵育2小时制备的，并通过GE HiTrap肝素HP柱进一步纯化。双链DNA（5-GAGGCMGCTCT-3和5-GCAGGZGGCCTC-3，其中M为5-甲基胞嘧啶，Z为zebulline）含有12个碱基对的半甲基化寡核苷酸，用zebulline代替靶胞苷。在结晶之前，纯化的DNMT1-DNA复合物在4°C下与抑制剂（GSK-3685032或GSK3830052在二甲亚砜中）一起孵育1小时，蛋白质DNA与抑制剂的摩尔比约为1:8。

细胞增殖测定[1]
细胞类型：15种白血病细胞、29种淋巴瘤细胞和7种多发性骨髓瘤细胞系，如EOL-1、Ki-JK、MM.IR细胞。
测试浓度： 0.01-100 μM
孵育时间： 6 天
实验结果： 证明细胞生长抑制大多数癌细胞系，中位生长 IC50 值为 0.64 μM。

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细胞增殖测定 [1]
细胞类型： MV4-11 细胞
测试浓度： 0.1-1000 nM
孵育时间：1-6天
实验结果：3天后出现生长抑制，并且在整个6天的时间过程中生长IC50逐渐减小。

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RT-PCR[1]
细胞类型： MV4-11 细胞
测试浓度： 10-10000 nM
<孵育时间：4天
实验结果：MV4-11细胞处理后，CXCL11、IFI27、HLA-DQA1和MAGEA4以剂量依赖性方式增加。

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蛋白质印迹分析[1]
细胞类型： GDM-1 细胞
测试浓度： 3.2-10,000 nM
孵育时间：2天
实验结果：DNMT1蛋白表达受到抑制

Animal/Disease Models: MV4-11 xenograft model (female CD1-Foxn1 mice, 12 weeks old) or SKM-1 xenograft model (NOD.CB17-Prkdc1NCrCrl mice, 8-11 weeks old) [1]
Doses: 1, 5, 15, 30, 45 mg/kg (10% Captisol adjusted to pH 4.5-5 with 1 M acetic acid, store at 4 °C for up to 1 week)
Route of Administration: SC, twice (two times) daily, continuous 4-week
Experimental Results: Demonstrated statistically significant dose-dependent tumor growth inhibition, with significant regression at ≥30 mg/kg.

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GSK-3685032 or vehicle (10% captisol adjusted to pH 4.5–5 with 1 M acetic acid, stored for up to 1 week at 4 °C) was administered subcutaneously, twice daily, at a dosing volume of 10 ml kg−1 (0.2 ml per 20 g of body weight). DAC (Sun Pharmaceutical Industries) was administered by intraperitoneal injection, three times per week, at a dosing volume of 10 ml kg−1. DAC was reconstituted with the appropriate amount of manufacturer’s diluent (68 mg of monobasic potassium phosphate and 11.6 mg of sodium hydroxide in 10 ml of water) to yield a dosing solution of 0.04 mg ml−1 immediately before administration (final dose of 0.4 mg kg−1).[1]

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Efficacy ofGSK-3685032 in an MV4–11 human systemic AML model in female NOD.CB17-Prkdcscid/NCrCrl mice was evaluated at Charles River Laboratories. To ablate bone marrow, animals (10 weeks old) were dosed with cyclophosphamide (150 mg kg−1) starting 3 d before injection of MV4–11 cells intravenously into the tail vein. Randomization by body weight and dosing commenced 21 d after implant. Animals (10 per group, 70 total) were dosed over 30 study days, where GSK-3685032 or vehicle was administered subcutaneously twice daily while DAC was dosed intraperitoneally two times per week. Body weight measurements were taken three times per week. After a single observation of >30% body weight loss or consecutive measurements of >25% body weight loss, the animal was euthanized. Clinical signs associated with tumor progression such as impairment of hind limb function or ocular proptosis also resulted in euthanasia. The study endpoint was 76 d.[1]

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A separate pharmacokinetic study was conducted in naïve animals (3 mice per group, 9 mice total), where mice received a single intravenous or subcutaneous dose of 2 mg kg−1 (intravenously, male CD-1 mice), 2 mg kg−1 (subcutaneously, male C57/BL6 mice) or 30 mg kg−1 (subcutaneously, female Nu/Nu mice) GSK-3685032 and composite blood samples were collected over 24 h post-dose. Blood concentrations were determined by HPLC–MS/MS and pharmacokinetic parameters were estimated from the mean blood concentration–time profiles using noncompartmental analysis with Phoenix WinNonlin v.6.3 (Certara). Area under the blood concentration–time curve was calculated using the linear trapezoidal rule for each incremental trapezoid up to the maximal concentration (Cmax), and the linear or log interpolation rule for each trapezoid thereafter. The dose-normalized area under the curve (AUC) was calculated by dividing the AUC0–8 h by the dose.[1]

[1]. Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia. Nat Cancer. 2021;2(10):1002-1017.

DNA methylation is a key epigenetic driver of transcriptional silencing and is abnormally regulated in cancer. Reversing DNA methylation using demethylating agents (such as cytidine analogs decitabine or azacitidine) has shown clinical efficacy in hematologic malignancies. These nucleoside analogs can be incorporated into replicating DNA and inhibit DNA cytosine methyltransferases DNMT1, DNMT3A, and DNMT3B through irreversible covalent interactions. These drugs are significantly toxic to normal blood cells, thus limiting their clinical dosage. This article reports the discovery of GSK3685032, a highly potent first-in-class selective inhibitor of DNMT1. Crystallographic studies show that GSK3685032 competes with the active site loop of DNMT1 for access to the hemimethylated DNA region between two CpG base pairs. GSK3685032 significantly reduces DNA methylation levels in vitro, activates transcription, and inhibits cancer cell growth. Compared with decitabine, GSK3685032 has better in vivo tolerability and therefore significantly reduces tumor incidence and prolongs survival in a mouse model of acute myeloid leukemia. [1]

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This article describes the discovery of GSK3685032, a first-in-class, highly potent, non-nucleoside, reversible, selective DNMT1 inhibitor. GSK3685032 selectively binds to DNMT1 through a unique interaction: the inhibitor competes with the DNMT1 active site loop for entry into hemimethylated DNA and interacts with the DNMT1-specific transcription activation domain (TRD). This binding leads to a rapid reduction in DNA methylation levels and significant transcriptional activation. Overall, the kinetics of DNA hypomethylation (1–2 days) and transcriptional activation (≥2 days) following GSK3685032 treatment suggest that the reduction in sensitive AML cell growth and survival (≥4 days) is directly associated with these early epigenetic changes. Consistent with the enhanced inhibition of DNMT1 enzyme, GSK3685032 exhibited stronger growth inhibition than previously reported non-nucleoside DNMT inhibitors (RG-108, SGI-1027, and MC3343) [1]. Although there are many similarities between GSK3685032 and DAC in in vitro experiments, it is noteworthy that GSK3685032 showed a normal dose-response, maintaining DNA demethylation and transcriptional activation at high doses, and achieving a higher maximum demethylation level compared to DAC, despite the latter being phenotypic more effective. These observations suggest that while the effects of DNA-integrating pan-DNMT inhibitors and non-covalent DNMT1 selective inhibitors partially overlap, conventional HMAs have dose-limiting toxicity due to their non-epigenetic mechanisms of action. Furthermore, due to the limited tolerance of DAC, there are significant differences in the target binding levels of these two classes of compounds in vivo. GSK3685032 achieves higher target binding rates and DNA hypomethylation levels, which translates into significantly stronger antitumor activity, with complete tumor regression and prolonged overall survival observed in multiple AML models. Therefore, GSK3685032 is a well-tolerated small molecule suitable for in vivo studies of the downstream effects of selective DNMT1 inhibition without the complex toxicities observed in DAC. These selective DNMT1 inhibitors offer reduced toxicity, improved tolerability, and enhanced pharmacokinetics, providing enhanced clinical opportunities for AML and potentially expanding to other tumor types, including solid tumors where traditional HMAs have limited activity.

C22H24N6OS

420.5306

420.173

C, 62.83; H, 5.75; N, 19.98; O, 3.80; S, 7.62

2170137-61-6

(R)-GSK-3685032;2170140-50-6;(S)-GSK-3685032;2170142-58-0

132233067

White to off-white solid powder

2.6

158

2

7

6

30

684

0

S(C([H])(C(N([H])[H])=O)C1C([H])=C([H])C([H])=C([H])C=1[H])C1=C(C#N)C(C([H])([H])C([H])([H])[H])=C(C#N)C(=N1)N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])[H]

KNKHRZYILDZLRE-UHFFFAOYSA-N

InChI=1S/C22H24N6OS/c1-2-16-17(12-23)21(28-10-8-15(25)9-11-28)27-22(18(16)13-24)30-19(20(26)29)14-6-4-3-5-7-14/h3-7,15,19H,2,8-11,25H2,1H3,(H2,26,29)

2-((6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide

GSK3685032; GSK-3685032; GSK3685032 HCl; SCHEMBL19716804; GSK-3685032 HCl; GTPL11750; BDBM491199; GSK 3685032

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 : ~25 mg/mL (~59.45 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.94 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (5.94 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 25.0 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: ≥ 2.5 mg/mL (5.94 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
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