GSK-LSD1 hydrochloride

LSD1/lysine specific demethylase 1 (IC50 = 16 nM)

GSK-LSD1 DiHClide 的选择性比其他密切相关的 FAD 利用酶（包括 LSD2 和单胺氧化酶 MAO-A、MAO-B）高出 1000 倍以上[1]。 GSK-LSD1 可抑制 KDM1A/LSD1 酶活性。 GSK-LSD1 诱导 U2OS 细胞中 LC3-II 的形成。电子显微镜显示GSK-LSD1处理后自噬体的形成。 GSK-LSD1 通过改变基因表达模式有效抑制多种癌细胞系的增殖[2]。

为了评估LSD1在体内的抑制活性，用GSK-LSD1处理植入1×10~5 MLL-AF9原代AML细胞的次级受体小鼠。在14天的治疗窗口内，每天以0.5mg/kg的剂量给药。只有在确认外周血植入后才开始治疗（补充图1A，可在血液网站上获得）。治疗后，杀死一些小鼠，并使用流式细胞术检测GFP作为MLL-AF9等位基因负荷的读数进行分析。GSK-LSD1治疗的小鼠骨髓（图1A）、外周血和脾脏中GFP+细胞的比例较低（补充图1B-C）。在GSK-LSD1治疗的情况下，包括脾脏重量在内的其他疾病负担指标显著降低（补充图1E）。经GSK-LSD1治疗的小鼠血小板计数显著下降（P=0.003；补充图1D），这与LSD1耗竭的靶向效应一致。18经GSK-LSD1治疗3天后，骨髓细胞的免疫表型显示，共表达c-kit和Mac-1的更原始的GFP+白血病细胞减少（图1B）。与对照组小鼠（中位生存期39天）相比，GSK-LSD1治疗的小鼠存活率（中位存活期78天）也显著提高（图1C）。令人惊讶的是，一小部分接受治疗的小鼠在移植后248天也没有检测到疾病。为了证实LSD1抑制对存活的这种影响，我们对从单独用载体或GSK-LSD1治疗3天的白血病小鼠中采集的MLL-AF9细胞进行了连续移植。将同等数量的从载体或GSK-LSD1处理的小鼠中纯化的GFP+细胞注射到亚致死照射的小鼠体内。与载体处理的小鼠相比，移植了从GSK-LSD1处理的小鼠中获得的细胞的第三受体小鼠的存活率有所提高。虽然移植了载体处理细胞的受体小鼠的中位存活期为23天，但用GSK-LSD1处理的白血病细胞攻击的小鼠的中位数存活期为51天（图1D）。植入GSK-LSD1治疗的白血病细胞的小鼠中，只有50%死于AML。剩下的50%移植了GSK-LSD1处理细胞的小鼠在移植后308天内保持健康，没有出现白血病的迹象。这些数据表明，在MLL-AF9驱动的急性髓系白血病（AML）的侵袭性模型中，LSD1抑制具有强大的抗白血病活性，可提高总体生存率，偶尔可导致疾病完全根除。https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/

细胞周期分析
通过用GSKLSD1体外处理48小时的细胞的BrdU染色进行细胞周期分析。使用BrdU流动试剂盒（BD Biosciences）。简而言之，在暴露于GSK-LSD1 48小时后，根据制造商的说明将细胞暴露于10µM BrdU 20分钟。之后，收获细胞，使其透性，并用APC标记的抗BrdU抗体染色，而白血病细胞为GFP+（含有pMSCV-ML-AF9-IRES-GFP质粒）。使用SYTOX™蓝死细胞染色法进行DNA染色。SYTOX Blue信号是以线性模式采集的。https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

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通过在添加了15%FBS、IL-3、IL-6和mSCF的IMDM中培养细胞，并单独添加载体或浓度为0.5µM的GSK-LSD1，体外处理MLL-AF9白血病细胞48小时。同样，白血病细胞用浓度为1µM的DOT1L抑制剂EPZ4777处理6天。根据制造商的说明进行菌落形成试验。简而言之，将500个细胞/皿铺在MC3434甲基纤维素中，并在孵育6天后对菌落数量进行评分。对于每个组，对3个独立的培养皿进行评分，并至少进行两次菌落测定。在第0天将GSK-LSD1以0.5µM的浓度加入MC3434半固体培养基中，六天后对菌落进行评分。https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

For in vivo treatment experiments, GSK-LSD1 was administered via intraperitoneal injections at a dose of 0.5 mg/kg daily. Treatment was initiated only after peripheral blood engraftment of MLL-AF9 leukemia cells was confirmed at a minimum chimerism of 0.1-1% GFPpositive cells for syngeneic murine MLL-AF9 leukemia cells or 12.3% ± 2.7 hCD45-positive cells for xenotransplantation experiments. Mice were treated for 3 days (Figure 1B), 2 weeks (Figure 1C) or 6 weeks (Figure 1G). Cytological staining was performed on cytospin preparations of suspension cells from in vitro culture (Figures 1E, 5E, 6D+F) or from peripheral blood of mice (Figure 1J) using the Deep Quick Stain kit. https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

Biochim Biophys Acta. 2017 Aug 8;1864(12):2428-2437.; http://www.thesgc.org/chemical-probes/LSD1.

Epigenetic factors and related small molecules have emerged to be strongly involved in autophagy process. Here we report that 2-PCPA and GSK-LSD1, two inhibitors of histone H3K4 demethylase KDM1A/LSD1, induce autophagy in multiple mammalian cell lines. The two small molecules induce accumulation of LC3II, formation of autophagosome and autolysosome, and SQSTM1/p62 degradation. 2-PCPA treatment inhibits cell proliferation through cell cycle arrest but does not inducing cell death. Exogenous expression of KDM1A/LSD1 impaired the autophagic phenotypes triggered by 2-PCPA. The autophagy induced by 2-PCPA requires LC3-II processing machinery. But depletion of BECN1 and ULK1 with siRNA did not affect the LC3-II accumulation triggered by 2-PCPA. 2-PCPA treatment induces the change of global gene expression program, including a series of autophagy-related genes, such as SQSTM1/p62. Taken together, our data indicate that KDM1A/LSD1 inhibitors induce autophagy through affecting the expression of autophagy-related genes and in a BECN1-independent manner.Biochim Biophys Acta. 2017 Aug 8;1864(12):2428-2437

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Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine-specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. A common feature of many of these targeted agents is their ability to induce myeloid differentiation, suggesting that multiple paths toward a myeloid gene expression program can be engaged to relieve the differentiation blockade that is uniformly seen in AML. We performed a comparative assessment of chromatin dynamics during the treatment of mixed lineage leukemia (MLL)-AF9-driven murine leukemias and MLL-rearranged patient-derived xenografts using 2 distinct but effective differentiation-inducing targeted epigenetic therapies, the LSD1 inhibitor GSK-LSD1 and the DOT1L inhibitor EPZ4777. Intriguingly, GSK-LSD1 treatment caused global gains in chromatin accessibility, whereas treatment with EPZ4777 caused global losses in accessibility. We captured PU.1 and C/EBPα motif signatures at LSD1 inhibitor-induced dynamic sites and chromatin immunoprecipitation coupled with high-throughput sequencing revealed co-occupancy of these myeloid transcription factors at these sites. Functionally, we confirmed that diminished expression of PU.1 or genetic deletion of C/EBPα in MLL-AF9 cells generates resistance of these leukemias to LSD1 inhibition. These findings reveal that pharmacologic inhibition of LSD1 represents a unique path to overcome the differentiation block in AML for therapeutic benefit.https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

1431367-51-9

1431368-48-7 (rel-free base) 1821798-25-7 (2HCl) 2102933-95-7 (rel-2HCl) 1431368-50-1 1431367-49-5 (rel-HCl) 1431367-51-9 (HCl)

61815034

Typically exists as solid at room temperature

CHEMBL3945250; 1431368-48-7; GSK-LSD1 HCl; SCHEMBL14880456; 1431367-51-9;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。