PKM2-IN-1

Pyruvate kinase M2 (PKM2) (IC50 = 2.95 μM)

PKM2-IN-1 (compound 3k) 是丙酮酸激酶 M2 (PKM2) 的抑制剂，IC50 为 2.95±0.53 μM。根据结果，大多数研究的化合物表现出一定程度的 PKM2 抑制作用，并且某些化合物，包括化合物 3k 和化合物 6d（即 PKM2-IN-1），表现出比阳性对照紫草素更有效的活性。对 PKM2 表现出剂量依赖性抑制作用的物质的例子是 PKM2-IN-1 和 6d。相反，这些化合物对 PKM1 和 PKL 的抑制作用与紫草素一样温和。测试结果表明，3a、PKM2-IN-1和3r是针对HCT116和HeLa细胞最有效的化合物，IC50值分别为0.39至0.41μM、0.18至0.29μM和0.18至0.38μM。 1]。

PKM2活性测定[1]
如前所述，用荧光丙酮酸激酶-乳酸脱氢酶偶联测定法测量丙酮酸激酶活性。通过将丙酮酸激酶产生丙酮酸与乳酸脱氢酶消耗NADH结合，在动力学模式下测试所有化合物。对于PKM2，将40μL缓冲液（50 mM Tris-HCl，pH 7.5，10 mM KCl，5 mM MgCl2）、1μL化合物和5μL酶溶液分配到康宁黑色固体96孔板中，孵育15分钟。然后加入55μL底物混合物（终浓度，0.5 mM PEP，4.0 mM ADP，0.12 mM NADH，0.25 mM FBP和1单位LDH），将板放置在FlexStation 3中，然后以30秒的暴露间隔测定NADH荧光3-6分钟。

细胞活力实验[1]
根据制造商的说明，用MTS assa检测细胞活力。简而言之，将每孔5000个细胞镀在96孔板上。孵育12小时后，用不同浓度的受试化合物或DMSO（作为阴性对照）处理细胞48小时。然后每孔加入20μL MTS，在37°C下孵育3小时。用酶标仪（Flexstation 3）在490 nm波长下测定每孔的吸光度。使用三次实验的Prism Graphpad软件计算IC50值。

[1]. Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase. Eur J Med Chem. 2017 Sep 29;138:343-352.

Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme of the glycolytic pathway which is highly expressed in cancer cells. Cancer cells rely heavily on PKM2 for anabolic and energy requirements, and specific targeting of PKM2 therefore has potential as strategy for cancer therapy. Here, we report the synthesis and biologic evaluation of novel naphthoquinone derivatives as selective small molecule inhibitors of PKM2. Some target compounds, such as compound 3k, displayed more potent PKM2 inhibitory activity than the reported optimal PKM2 inhibitor shikonin. The well performing compound 3k also showed nanomolar antiproliferative activity toward a series of cancer cell lines with high expression of PKM2 including HCT116, Hela and H1299 with IC50 values ranging from 0.18 to 1.56 μM. Moreover, compound 3k exhibited more cytotoxicity on cancer cells than normal cells. The identification of novel potent small molecule inhibitors of PKM2 not only offers candidate compounds for cancer therapy, but also provides a tool with which to evaluate the function of PKM2 in depth. [1]

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In this study we have described the identification and characterization of a previously undescribed chemical class of PKM2 inhibitors. These novel naphthoquinone derivatives act as PKM2 inhibitors, and show high inhibitory responses that are more potent than shikonin and have selectivity similar to shikonin. These compounds lock PKM2 into a low activity conformation which forces alteration of cancer cell metabolism that is less metabolically flexible than metabolism in the normal state. Moreover, some of these target compounds show higher antiproliferative effects than shikonin. However, there are unexpected results in the enzyme and cytotoxicity experiments. The absence of correlation between PKM2 inhibitory activity and in vitro cytotoxicity of the target compounds suggests that the cells are probably using other mechanisms to activate these compounds, except in the case of PKM2 inhibitory activity. However, there is no doubt that PKM2 is a target of these synthesized naphthoquinone derivatives, through the IC50 of some compounds which inhibit PKM2 activity is closed to nanomolar concentration range.
PKM2 has been identified as a major contributor not only of metabolic reprogramming as for pyruvate kinases but also in direct regulation of gene expression as a protein kinase in cancer cells. Upon EGFR activation, PKM2 translocates from the cytoplasm into the nucleus of cancer cells where it activates β-catenin to induce CCDN1 and c-Myc expression and upregulate GLUT1 and lactate dehydrogenase A (LDHA). Upregulation of these glycolysis genes increases glucose consumption and lactate production, and subsequently promotes tumorigenesis. Therefore, inhibition of PKM2-coactivated tumor gene transcription and glycolysis gene expression are also potential tumor treatment strategies. An attempt will be made to determine whether the mechanism works for the synthesized naphthoquinone derivatives. If so, the mechanism also can explain the SAR discrepancy of enzyme activity and cytotoxicity of target compounds.
Further investigation will also focus on structural studies of PKM2 protein and small molecule inhibitor complex, which may lead to identification of compounds with higher effectiveness. In any case, the findings presented here establish a foundation for the development of PKM2-targeted anticancer therapies.[1]

C18H19NO2S2

345.478962182999

345.085

C, 62.58; H, 5.54; N, 4.05; O, 9.26; S, 18.56

94164-88-2

131698387

Light yellow to yellow solid powder

3.5

94.8

0

4

3

23

549

0

O=C1C(C)=C(CSC(N2CCCCC2)=S)C(=O)C2C1=CC=CC=2

STAFOGVMELKGRI-UHFFFAOYSA-N

InChI=1S/C18H19NO2S2/c1-12-15(11-23-18(22)19-9-5-2-6-10-19)17(21)14-8-4-3-7-13(14)16(12)20/h3-4,7-8H,2,5-6,9-11H2,1H3

(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl piperidine-1-carbodithioate

PKM2-IN-1; PKM2 IN-1; 94164-88-2; PKM2-IN-1; PKM2 inhibitor(compound 3k); PKM2 inhibitor; (3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl piperidine-1-carbodithioate; CHEMBL4128703; (3-methyl-1,4-dioxonaphthalen-2-yl)methyl piperidine-1-carbodithioate; PKM2-IN 1

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 : ~10 mg/mL (~28.95 mM)

配方 1 中的溶解度: 8 mg/mL (23.16 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 80.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中并混合均匀；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: ≥ 8 mg/mL (23.16 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 80.0mg/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: ≥ 8 mg/mL (23.16 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 80.0 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。