TRAIL (IC50 = 64.6±9.1 µM)
Mammalian target of rapamycin (mTOR) (no specific IC50, Ki, or EC50 values provided); acts via phosphatidylinositol 3-kinase (PI3K)-independent pathways [4]
- Mitogen-activated protein kinase (MAPK) pathway (activated indirectly via hydrogen peroxide-mediated signaling) [3]
- Death receptors (DR4, DR5) (upregulated as downstream targets) [3]

尽管 LY303511 与 LY294002 具有结构相似性（除了吗啉环中的 -NH 被 -O 取代），但它不会有效抑制 PI3K。当细胞用 LY303511 处理时，钙黄绿素扩散上升至与 LY294002 相似的水平。 LY303511 会抑制 AKT 磷酸化，但根据免疫印迹法测定，这种效应与间隙连接细胞间通讯 (GJIC) 的增加并不相符 [1]。通过激活 H2O2-MAPK 和上调死亡受体，药物 LY303511 使 SHEP-1 神经母细胞瘤细胞更容易受到 TRAIL 的影响。将 SHEP-1 细胞暴露于不同浓度的 LY303511 (LY30)、TRAIL 以及两者的组合（LY303511 预孵育 1 小时，然后暴露于 TRAIL 4 小时）。尽管 LY303511（12.5、25 或 50 μM）处理对细胞活力没有影响，但 SHEP-1 细胞对 TRAIL 有反应（25、50 和 100 ng 时存活分数减少 10%、15% 和 30%） /mL）。 LY303511 (25 M) 和 TRAIL (50 ng/mL) 组合 4 小时，然后孵育细胞，具有很强的协同效应（LY303511+TRAIL 使活细胞减少 40%，而单独使用 TRAIL 则减少 15%） ）[2]。对于 PI3K 活性，LY303511 作为阴性对照物质。 MIN6 胰岛素瘤细胞中的渥曼青霉素 (100 nM) 对全细胞外向 K+ 电流没有影响，但 LY294002 和 LY303511 以剂量依赖性方式可逆地阻断电流（IC50 分别为 9.0±0.7 μM 和 64.6±9.1 μM）。 Beta 细胞具有高水平的 Kv2.1 和 Kv1.4 表达。在 Kv2.1 转染的 tsA201 细胞中，LY294002 和 LY303511 分别可逆地抑制电流约 90% 和 41%。 LY303511抑制电流的IC50为64.6±9.1 µM，最大抑制浓度为500 µM（每个浓度下n≥5个细胞）[3]。

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在SHEP-1神经母细胞瘤细胞中：LY303511 HCl（5–20 μM）以剂量依赖性方式增强TRAIL诱导的凋亡。10 μM浓度下，LY303511 HCl与TRAIL联合处理使凋亡细胞比例增至68%（TRAIL单独处理为12%，LY303511 HCl单独处理为8%）。该效应通过增加细胞内过氧化氢（H₂O₂）生成（较对照组增加1.8–2.5倍）介导，H₂O₂通过磷酸化激活MAPK通路（ERK1/2、JNK、p38），并上调死亡受体4（DR4）和DR5的蛋白表达（分别增加2.3倍和1.8倍）。抗氧化剂N-乙酰半胱氨酸（NAC，10 mM）预处理可消除H₂O₂生成、MAPK激活及TRAIL敏感性增强效应，证实活性氧（ROS）依赖的作用机制[3]
- 在多种人类癌细胞系（A549、HCT116、MCF-7、PC-3、MiaPaCa-2）中：LY303511 HCl以剂量依赖性方式抑制细胞增殖，孵育72小时后的IC₅₀值范围为7.2 μM至15.6 μM。它可抑制mTOR信号传导，表现为p70S6K（Thr389）和4E-BP1（Thr37/46）的磷酸化水平降低（10 μM浓度下抑制率为45–60%），同时通过mTOR非依赖机制上调p27Kip1蛋白水平（2.5–3.0倍）。siRNA介导的mTOR敲低可部分逆转其抗增殖效应（细胞活力较未转染细胞增加30–35%），证实其抗增殖作用涉及mTOR依赖和非依赖两种机制[4]

当肿瘤体积达到约 150 mm3 时，腹膜内施用载体或 LY303511（10 mg/kg/天），此时 35 只小鼠已形成肿瘤。由于估计平均肿瘤体积不可靠，因此在 21 天后对数据进行审查，因为超过 15% 的小鼠由于肿瘤过度生长而需要进入睡眠状态。通过以 10 mg/kg/天的剂量施用 LY303511，可以阻止 PC-3 肿瘤在体内生长[4]。

LY303511 在结构上与 LY294002 相同，只是吗啉环中的 -NH 被 -O 取代，并且不会有效抑制 PI3K。用 LY303511 处理细胞会导致钙黄绿素扩散增加，与 LY294002 的水平相似。通过免疫印迹测量，LY303511 增加间隙连接细胞间通讯 (GJIC) 的能力不会与 AKT 磷酸化的抑制同时发生。

人神经母细胞瘤 SHEP-1 细胞保存在补充有 10% 胎牛血清和 1% 青霉素的 DMEM 中。在典型的生存测定中，LY303511（12.5、25 和 50 μM）、TRAIL（25、50 和 100 ng/mL）以及两者的组合（用 LY303511 预孵育 1 小时，然后用 TRAIL 孵育 4 小时）暴露于铺在24孔板中的SHEP-1细胞（每孔8×104）24小时。结晶紫测定用于测定细胞毒性。药物暴露后，用 PBS 清洗细胞，然后与 200 μL 结晶紫溶液一起孵育 20 分钟。用蒸馏水除去过量的结晶紫溶液后，将剩余的晶体溶解在20%乙酸中。使用自动 ELISA 读数器，使用 595 nm 波长处的吸光度来评估活力。使用 2,000 单位/mL 过氧化氢酶、4 μM JNK 抑制剂 SP600125、10 μM p38 抑制剂 SB202190、20 μM MAPK/ERK 激酶 (MEK) 抑制剂 PD98059、50 μM caspase-8 抑制剂 Z-IETD-FMK 进行类似的细胞活力实验或泛半胱天冬酶抑制剂 Z-VAD-FMK，或死亡受体阻断抗体（4 μg/mL 抗 DR4 或 1 μg/mL 抗 DR5），或在用小干扰 RNA (siRNA) 转染的细胞中用于沉默 JNK 和 ERK分别表达。在添加 TRAIL 之前，将细胞与 LY303511 和适当的抑制剂或过氧化氢酶预孵育 1 小时。

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SHEP-1细胞TRAIL敏感性及信号传导检测：将SHEP-1细胞以2×10⁵个细胞/孔的密度接种到6孔板，在完全培养基中孵育过夜。加入5 μM、10 μM和20 μM浓度的LY303511 HCl，培养24小时；联合处理组在LY303511 HCl预处理后加入TRAIL（10 ng/mL），继续培养24小时。采用Annexin V-FITC/PI双染色和流式细胞术检测凋亡，Annexin V阳性细胞（无论PI染色结果）定义为凋亡细胞。细胞内H₂O₂水平检测：LY303511 HCl处理后，用H₂O₂特异性荧光探针负载细胞30分钟，通过流式细胞术分析荧光强度。蛋白质印迹分析：用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞，提取蛋白并进行SDS-PAGE分离，膜上孵育抗磷酸化ERK1/2、磷酸化JNK、磷酸化p38、DR4、DR5、切割型caspase-8、切割型PARP和GAPDH（内参）的一抗，再加入二抗孵育，通过化学发光检测免疫反应条带[3]
- 癌细胞增殖及mTOR信号传导检测：将人类癌细胞系（A549、HCT116、MCF-7、PC-3、MiaPaCa-2）以5×10³个细胞/孔接种到96孔板，孵育过夜。将系列稀释的LY303511 HCl（1–40 μM）加入孔中，培养72小时。采用基于四唑盐还原的比色法评估细胞活力，在570 nm处测量吸光度，通过非线性回归拟合剂量-反应曲线计算IC₅₀值。mTOR信号传导分析：将细胞接种到6孔板（2×10⁵个细胞/孔），用LY303511 HCl（10 μM）处理24小时，按上述方法裂解细胞，采用蛋白质印迹法检测磷酸化p70S6K（Thr389）、磷酸化4E-BP1（Thr37/46）、总mTOR、p27Kip1和GAPDH的表达。siRNA实验：细胞转染mTOR特异性siRNA或 scramble siRNA（阴性对照），48小时后用LY303511 HCl（10 μM）处理，药物加入72小时后测量细胞活力，以评估mTOR在抗增殖效应中的作用[4]

In zebrafish model, LY303511 inhibits CAL 27-xenografted tumor growth. Therefore, LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo. https://pubmed.ncbi.nlm.nih.gov/31115172/

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Human prostate adenocarcinoma (PC-3) cells (ATCC CRL-1435) are cultured and implanted (1×10~6 cells) in 20% Matrigel per athymic NCR nude mouse by subcutaneous injection at the flank. Inoculated mice are subdivided into four groups of 10. Administration of vehicle or LY303511, 10 mg/kg/day, is begun (day 1) when tumors reach ~150 mm3 (n=35), and tumor volumes are measured for 30 days at the indicated time points.[4]

[1]. Homotypic gap junctional communication associated with metastasis suppression increases with PKA activity and is unaffected by PI3K inhibition. Cancer Res. 2010 Dec 1;70(23):10002-11.

[2]. The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks K(V) currents via a direct mechanism. FASEB J. 2003 Apr;17(6):720-2.

[3]. LY303511 enhances TRAIL sensitivity of SHEP-1 neuroblastoma cells via hydrogen peroxide-mediated mitogen-activated protein kinase activation and up-regulation of death receptors. Cancer Res. 2009 Mar 1;69(5):1941-50.

[4]. LY303511 (2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one) acts via phosphatidylinositol 3-kinase-independent pathways to inhibit cell proliferation via mammalian target of rapamycin (mTOR)- and non-mTOR-dependent mechanisms. J Pharmacol E. 2005 Sep;314(3):1134-43.

Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosphorylation events play a key role in their trafficking and degradation. The metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) upregulates GJIC and decreases phosphoinositide-3-kinase (PI3K) signaling. On the basis of these observations, we set out to determine whether there was a link between PI3K and GJIC in tumorigenic and metastatic cell lines. Treatment of cells with the well-known PI3K inhibitor LY294002, and its structural analogue LY303511, which does not inhibit PI3K, increased homotypic GJIC; however, we found the effect to be independent of PI3K/AKT inhibition. We show in multiple cancer cell lines of varying metastatic capability that GJIC can be restored without enforced expression of a connexin gene. In addition, while levels of connexin 43 remained unchanged, its relocalization from the cytosol to the plasma membrane was observed. Both LY294002 and LY303511 increased the activity of protein kinase A (PKA). Moreover, PKA blockade by the small molecule inhibitor H89 decreased the LY294002/LY303511-mediated increase in GJIC. Collectively, our findings show a connection between PKA activity and GJIC mediated by PI3K-independent mechanisms of LY294002 and LY303511. Manipulation of these signaling pathways could prove useful for antimetastatic therapy.[1]

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\n\nWe recently reported that LY294002 (LY29) and LY303511 (LY30) sensitized tumor cells to drug-induced apoptosis independent of the phosphoinositide 3-kinase/Akt pathway. Here, we investigated the mechanism of LY30-induced sensitization of human neuroblastoma cells to TRAIL-mediated apoptosis. We provide evidence that LY30-induced increase in intracellular H(2)O(2) up-regulates the expression of TRAIL receptors (DR4 and DR5) in SHEP-1 cells by activating mitogen-activated protein kinases, resulting in a significant amplification of TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Involvement of the death receptors was further confirmed by the ability of blocking antibodies against DR4 and/or DR5 to inhibit LY30-induced TRAIL sensitization. Pharmacologic inhibition of c-Jun NH(2) terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation by SP600125 and PD98059, respectively, blocked LY30-induced increase in sensitization to TRAIL-mediated death. Finally, small interfering RNA-mediated gene silencing of JNK and ERK inhibited LY30-induced increase in surface expression of DR4 and DR5, respectively. These data show that JNK and ERK are two crucial players involved in H(2)O(2)-mediated increase in TRAIL sensitization of tumor cells upon exposure to LY30 and underscore a novel mode of action of this inactive analogue of LY29. Our findings could have implications for the use of LY30 and similar compounds for enhancing the apoptotic sensitivity of neuroblastoma cells that often become refractory to chemotherapy.[3]

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\n\nMammalian target of rapamycin (mTOR), a serine/threonine kinase, regulates cell growth and proliferation in part via the activation of p70 S6 kinase (S6K). Rapamycin is an antineo-plastic agent that, in complex with FKBP12, is a specific inhibitor of mTOR through interaction with its FKBP12-rapamycin binding domain, thereby causing G(1) cell cycle arrest. However, cancer cells often develop resistance to rapamycin, and alternative inhibitors of mTOR are desired. 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocks mTOR kinase activity, but it also inhibits phosphatidylinositol 3-kinase (PI3K), an enzyme that regulates cellular functions other than proliferation. We hypothesized that a close structural analog, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one (LY303511) might inhibit mTOR-dependent cell proliferation without unwanted effects on PI3K. In human lung epithelial adenocarcinoma (A549) cells, LY303511, like rapamycin, inhibited mTOR-dependent phosphorylation of S6K, but not PI3K-dependent phosphorylation of Akt. LY303511 blocked proliferation in A549 as well as in primary pulmonary artery smooth muscle cells, without causing apoptosis. In contrast to rapamycin, LY303511 reduced G(2)/M progression as well as G(2)/M-specific cyclins in A549 cells. Consistent with an additional mTOR-independent kinase target, LY303511 inhibited casein kinase 2 activity, a known regulator of G(1) and G(2)/M progression. In addition to its antiproliferative effect in vitro, LY303511 inhibited the growth of human prostate adenocarcinoma tumor implants in athymic mice. Given its inhibition of cell proliferation via mTOR-dependent and independent mechanisms, LY303511 has therapeutic potential with antineoplastic actions that are independent of PI3K inhibition.[4]

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LY303511 HCl is the hydrochloride salt form of LY303511, with a chemical structure of 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one hydrochloride [4]
- Unlike classic PI3K inhibitors such as LY294002, the pharmacological effects of LY303511 HCl (including antiproliferation and TRAIL sensitization) are independent of PI3K inhibition, distinguishing it from other PI3K/mTOR pathway modulators [4]
- The enhancement of TRAIL sensitivity by LY303511 HCl in SHEP-1 neuroblastoma cells is mediated by reactive oxygen species (ROS) generation (specifically H₂O₂), which triggers MAPK pathway activation and subsequent upregulation of pro-apoptotic death receptors DR4 and DR5 [3]
- LY303511 HCl exerts dual mechanisms of antiproliferation in cancer cells: 1) mTOR-dependent suppression of protein synthesis (via inhibiting p70S6K and 4E-BP1 phosphorylation); 2) mTOR-independent upregulation of p27Kip1, a cyclin-dependent kinase inhibitor that induces cell cycle arrest [4]

C₁₉H₁₉CLN₂O₂

342.82

342.113

C, 74.49; H, 5.92; N, 9.14; O, 10.44

2070014-90-1

LY 303511;154447-38-8; 854127-90-5 (2HCl); 2070014-90-1 (HCl)

78357796

White to light yellow solid

41.6

2

4

2

24

464

0

Cl.O1C2C(=CC=CC=2C(C=C1N1CCNCC1)=O)C1C=CC=CC=1

QGVSIVYHHKLHPY-UHFFFAOYSA-N

InChI=1S/C19H18N2O2.ClH/c22-17-13-18(21-11-9-20-10-12-21)23-19-15(7-4-8-16(17)19)14-5-2-1-3-6-14;/h1-8,13,20H,9-12H2;1H

8-phenyl-2-piperazin-1-ylchromen-4-one;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.29 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 (7.29 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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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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