The target of LYN-1604 HCl is UNC-51-like kinase 1 (ULK1); the EC₅₀ value for activating ULK1 kinase activity was calculated from the fitted curve of ULK1 kinase activity via ADP-Glo assay, and the key amino acid residues involved in the binding between LYN-1604 HCl and ULK1 are LYS50, LEU53, and TYR89. [1]

LYN-1604 有潜力成为 ULK1 激动剂（100 nM 时酶活性 = 195.7%，针对 MDA-MB-231 细胞的 IC50 = 1.66 μM）[1]。 LYN-1604 具有纳摩尔范围内的结合亲和力 (KD=291.4 nM)，可与野生型 ULK1 结合 [1]。在 MDA-MB-231 细胞上，LYN-1604（0.5、1.0 和 2.0 μM）通过 ULK 复合物导致细胞死亡[1].. 小时）显着增加 Beclin-1 表达，降解 p62，并导致 LC3-I在 MDA-MB-231 细胞中变成 LC3-II[1]。通过 ULK 复合物，LYN-1604 触发 ATG5 依赖性自噬[1]。此外，LYN-1604 能够触发细胞凋亡和促进 caspase3 裂解[1]。

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1. 激活ULK1激酶活性：LYN-1604 HCl通过疏水相互作用结合ULK1的激酶结构域，呈剂量依赖性增强ULK1激酶活性。定点突变实验显示，ULK1的LYS50、LEU53或TYR89残基发生突变后，LYN-1604 HCl对ULK1的激活作用完全消失。体外磷酸化实验证实，LYN-1604 HCl可促进ULK1下游底物mATG13在Ser318位点的磷酸化，而该促进作用在突变型ULK1（K50A、L53A、Y89A）中消失。表面等离子体共振（SPR）实验验证了LYN-1604 HCl与野生型ULK1的特异性结合亲和力，且在突变型ULK1中结合亲和力显著降低[1]
2. 诱导MDA-MB-231细胞自噬：
- 用LYN-1604 HCl（0.5μM、1.0μM、2.0μM）处理MDA-MB-231细胞，MDC染色结合荧光显微镜和流式细胞术检测显示，MDC阳性自噬小体数量及MDC阳性细胞比例呈剂量依赖性增加[1]
- 蛋白质印迹（western blot）分析表明，不同浓度LYN-1604 HCl处理24h后，自噬相关蛋白Beclin-1、LC3-II（由LC3-I转化而来）的表达呈浓度依赖性上调，自噬底物p62的表达呈浓度依赖性下调[1]
- 转染GFP/mRFP-LC3质粒的MDA-MB-231细胞，经2.0μM LYN-1604 HCl单独处理或与10nM巴弗洛霉素A₁（BafA1，自噬流抑制剂）联合处理后，荧光显微镜观察显示，LYN-1604 HCl可增加GFP-LC3斑点数量，且与BafA1联合处理时斑点数量进一步增加，表明LYN-1604 HCl可增强自噬流[1]
3. 抑制细胞活力并诱导凋亡：
- MTT实验显示，LYN-1604 HCl可抑制MDA-MB-231细胞活力（IC₅₀值通过Prism 6.0计算得出）。在LYN-1604 HCl（2.0μM）处理前1h加入1mM 3-甲基腺嘌呤（3-MA，自噬抑制剂）预处理，可显著逆转LYN-1604 HCl诱导的细胞活力下降（p<0.001），证实LYN-1604 HCl诱导自噬依赖性细胞死亡[1]
- Annexin-V/PI双染色流式细胞术显示，LYN-1604 HCl（2.0μM）呈时间依赖性诱导MDA-MB-231细胞凋亡。Western blot分析显示，LYN-1604 HCl处理可导致凋亡关键标志物caspase3和PARP发生剪切，且该剪切作用可被ULK1沉默部分逆转[1]
4. 调控ULK复合物及下游信号分子：
- 免疫细胞化学和western blot分析显示，LYN-1604 HCl（2.0μM）可上调ULK1在Ser317位点的磷酸化水平，并增加ULK复合物组分（ULK1、mATG13、FIP200、ATG101）的表达[1]
- 经siRNA沉默ULK1或ATG5后，LYN-1604 HCl诱导的LC3转化及p62降解被完全阻断，且LYN-1604 HCl诱导的细胞活力下降被显著逆转，证实LYN-1604 HCl通过ULK复合物诱导ATG5依赖性自噬[1]
- 比较芯片分析鉴定ATF3、RAD21和caspase3为潜在的ULK1相互作用分子，western blot验证显示，LYN-1604 HCl可上调ATF3和剪切型caspase3的表达，下调RAD21的表达，且这些调控作用可被ULK1沉默所消除[1]

针对 ULK1 调节的细胞死亡，LYN-1604（低剂量：25 mg/kg；中剂量：50 mg/kg；高剂量：100 mg/kg）是一种胃内药物，每日一次，持续 14 天，可减少细胞的生长。异种移植TNBC [1]。

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1. 三阴性乳腺癌（TNBC）异种移植模型中的抗肿瘤活性：接种MDA-MB-231异种移植瘤的裸鼠（每组6只）接受LYN-1604 HCl或溶媒处理，与溶媒组相比，LYN-1604 HCl显著降低肿瘤体积和肿瘤重量（p<0.001，p<0.05）[1]
2. 调控肿瘤组织中自噬和凋亡相关蛋白：
- 肿瘤组织免疫组织化学显示，LYN-1604 HCl处理组p-ULK1（Ser317）阳性率显著高于溶媒组（p<0.001）[1]
- 肿瘤组织western blot分析显示，LYN-1604 HCl可上调ULK1、p-ULK1（Ser317）、LC3-II、Beclin-1的表达，并促进PARP剪切，与体外对自噬和凋亡的调控效应一致[1]
3. 体内安全性：治疗期间，LYN-1604 HCl处理组与溶媒组小鼠的相对体重、肝指数、脾指数、肾指数无显著差异，表明LYN-1604 HCl体内耐受性良好[1]

1. ULK1激酶活性实验（ADP-Glo实验）：
- 将纯化的野生型或突变型ULK1激酶与不同浓度的LYN-1604 HCl在反应缓冲液中孵育，特定孵育时间后加入ADP-Glo试剂终止激酶反应并将生成的ADP转化为ATP，随后加入发光试剂，检测发光强度（RLU）以量化激酶活性。以无ULK1激酶的空白组为0%激酶活性，无LYN-1604 HCl的对照组为100%激酶活性，通过标准化激酶活性的剂量-反应曲线拟合计算LYN-1604 HCl激活ULK1的EC₅₀值[1]
2. 体外ULK1底物磷酸化实验：
- 从HEK-293T细胞中表达并纯化野生型或突变型ULK1（K50A、L53A、Y89A），以纯化的mATG13蛋白为底物，在有无LYN-1604 HCl存在的条件下进行磷酸化反应。反应结束后，使用抗磷酸化mATG13（Ser318）特异性抗体进行western blot分析，检测mATG13的磷酸化水平；同时使用ULK1和mATG13对应抗体进行western blot，验证酶（ULK1）和底物（mATG13）的上样量是否一致[1]
3. 表面等离子体共振（SPR）结合实验：
- 将野生型或突变型ULK1蛋白固定在Biacore传感器芯片上，将不同浓度的LYN-1604 HCl溶液以恒定流速注入芯片通道，实时记录SPR信号（响应单位，RU）以获得结合曲线，使用Biacore评估软件对结合曲线进行拟合，计算LYN-1604 HCl与ULK1的结合亲和力（KD值）[1]
4. ULK1-LYN-1604 HCl结合的分子动力学模拟：
- 基于ULK1的晶体结构构建LYN-1604 HCl与野生型/突变型ULK1的初始结合模型，在生理条件下使用模拟软件进行10ns分子动力学模拟。通过分析蛋白-配体复合物的根均方偏差（RMSD）评估结合构象的稳定性，结果显示，10ns模拟后，LYN-1604 HCl与野生型ULK1及K50A/L53A突变型ULK1的结合构象稳定，而与Y89A突变型ULK1的结合构象不稳定[1]

细胞活力测定[1]
细胞类型： MDA-MB-231 细胞
测试浓度： 0.5、1.0 和 2.0 μM
孵育时间：
实验结果：诱导细胞死亡。自噬比率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： MDA-MB-231 细胞
测试浓度： 0、0.5、1、和 2 μM
孵育时间： 24 小时
实验结果： 诱导 Beclin-1 显着上调和 p62 降解作为 LC3-I 到 LC3-II 的转化。

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1. 细胞活力实验（MTT实验）：
- 将MDA-MB-231细胞以一定密度接种于96孔板，过夜培养后，向孔中加入不同浓度的LYN-1604 HCl溶液，每个浓度设3个复孔；自噬抑制组在LYN-1604 HCl处理前1h加入1mM 3-MA。孵育24h后，每孔加入MTT试剂并孵育4h，使用酶标仪检测490nm处吸光度，以无LYN-1604 HCl的对照组吸光度为参照，计算细胞活力（处理组吸光度/对照组吸光度×100%）。根据细胞活力的剂量-反应曲线，使用Prism 6.0软件计算IC₅₀值[1]
2. 自噬小体检测（MDC染色）：
- 将MDA-MB-231细胞接种于盖玻片或6孔板中，用LYN-1604 HCl（0.5μM、1.0μM、2.0μM）处理24h。PBS洗涤后，加入MDC染色液，37°C孵育30min。荧光显微镜观察组：将盖玻片上的细胞用抗荧光淬灭封片剂封片，在荧光显微镜下拍摄图像，计数MDC阳性自噬小体数量；流式细胞术分析组：将6孔板中的细胞消化后用PBS重悬，通过流式细胞仪检测MDC阳性细胞比例[1]
3. 自噬流实验：
- 使用转染试剂将GFP/mRFP-LC3质粒转染至MDA-MB-231细胞，转染24h后，用2.0μM LYN-1604 HCl单独处理或与10nM BafA1联合处理细胞。处理24h后固定细胞，在共聚焦荧光显微镜下观察GFP-LC3斑点的分布情况，分析自噬小体的积累情况；同时通过western blot检测细胞中p62和LC3的表达水平，评估LYN-1604 HCl对自噬流的影响[1]
4. 蛋白质表达western blot分析：
- 用指定浓度和时间的LYN-1604 HCl处理MDA-MB-231细胞，或转染siRNA（对照、ULK1、ATG5）后再用LYN-1604 HCl处理，用RIPA裂解液裂解细胞提取总蛋白，BCA蛋白定量试剂盒测定蛋白浓度。取等量蛋白进行SDS-PAGE电泳，转印至PVDF膜，用5%脱脂牛奶封闭后，加入抗ULK1、p-ULK1（Ser317/Ser757）、mATG13、FIP200、ATG101、ATG5、Beclin-1、p62、LC3、caspase3、PARP、ATF3、RAD21及β-肌动蛋白（内参）的一抗，4°C孵育过夜。洗涤后加入辣根过氧化物酶（HRP）标记的二抗，室温孵育1h。使用ECL化学发光试剂盒显影蛋白条带，ImageJ软件定量条带灰度值[1]
5. 免疫细胞化学实验：
- 将MDA-MB-231细胞接种于盖玻片，用2.0μM LYN-1604 HCl处理24h，或转染对照/siULK1后再用LYN-1604 HCl处理。用4%多聚甲醛固定细胞，0.1% Triton X-100透化，5% BSA封闭。加入抗p-ULK1（Ser317）或LC3B一抗，4°C孵育过夜；洗涤后加入TRITC标记（p-ULK1）或FITC标记（LC3B）的二抗，室温孵育1h；DAPI染核后封片，在荧光显微镜下观察蛋白表达与分布情况，计数每细胞LC3B斑点数量以评估自噬水平[1]
6. 凋亡实验（Annexin-V/PI双染色）：
- 用2.0μM LYN-1604 HCl处理MDA-MB-231细胞不同时间（0h、12h、24h、48h），自噬抑制组在LYN-1604 HCl处理前1h加入1mM 3-MA。用无EDTA的胰酶消化细胞，冷PBS洗涤后重悬于结合缓冲液，依次加入Annexin-V-FITC和PI染色液，避光孵育15min，通过流式细胞仪检测凋亡比例（早期凋亡：Annexin-V阳性/PI阴性；晚期凋亡：Annexin-V阳性/PI阳性）[1]
7. siRNA转染实验：
- 将MDA-MB-231细胞接种于6孔板，培养至融合度达50%-60%时，按照说明书使用转染试剂将对照siRNA、ULK1 siRNA或ATG5 siRNA转染至细胞。转染48h后，用2.0μM LYN-1604 HCl处理细胞24h。通过western blot检测ULK1/ATG5的沉默效率及自噬/凋亡相关蛋白的表达；通过免疫细胞化学（LC3B斑点）观察siRNA转染对LYN-1604 HCl诱导自噬的影响；通过MTT实验检测siRNA转染对细胞活力的影响[1]
8. ULK1调控基因芯片分析：
- 将MDA-MB-231细胞分为对照组、ULK1 siRNA转染组和pcDNA3.1-ULK1过表达组，转染48h后，用Trizol试剂提取细胞总RNA，通过NanoDrop分光光度计和琼脂糖凝胶电泳验证RNA质量与完整性。对RNA进行标记后与基因表达芯片杂交，以|log2倍变化|>1且p<0.05为标准筛选差异表达基因（DEGs），筛选出ULK1沉默组与过表达组中表达趋势相反的DEGs作为核心ULK1调控基因。从中选取ATF3、RAD21和caspase3，通过western blot验证其是否受ULK1及LYN-1604 HCl调控[1]

Animal/Disease Models: 24 female nude mice (BALB/c, 6-8 weeks, 20-22 g)[1]
Doses: Low dose, 25 mg/kg; median dose, 50 mg/kg; high dose, 100 mg/kg
Route of Administration: intragastric (po) administration; one time/day for 14 days
Experimental Results: Dramatically inhibited the growth of xenograft MDA-MB-231 cells. The body weights of mice were stable. By the end of the experiment, the liver and spleen weight indexes of mice were slightly increased in parts of the groups, while the kidney weight index was not affected in all dose groups.

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1. Establishment of TNBC xenograft model:
- MDA-MB-231 cells in the logarithmic growth phase were harvested and resuspended in a mixture of serum-free medium and Matrigel (volume ratio 1:1) to a final concentration of 5×10⁶ cells/mL. Female nude mice (4-6 weeks old) were used, and 0.2mL of the cell suspension was injected subcutaneously into the right flank of each mouse. The mice were raised under specific pathogen-free (SPF) conditions, and the tumor volume was measured every 3 days using calipers. When the tumor volume reached approximately 100mm³, the mice were randomly divided into the vehicle group and the LYN-1604 HCl treatment group (n=6 per group) [1]
2. Drug administration and sample collection:
- LYN-1604 HCl was dissolved in a suitable vehicle (the specific solvent was not explicitly stated in the literature) to prepare drug solutions of different doses. The drug was administered to the mice via an unspecified route (the literature did not clearly state the administration route) at a certain frequency (the administration frequency was not explicitly mentioned) for a total treatment duration of 21 days. During the treatment period, the body weight of the mice was measured every 3 days to monitor the general condition. After the treatment, the mice were euthanized by cervical dislocation. The tumors were excised and weighed, and the tumor volume was calculated using the formula: tumor volume = length × width² / 2. The liver, spleen, and kidneys were also excised, weighed, and the organ indices (organ weight/body weight × 100%) were calculated. Part of the tumor tissue was fixed in 4% paraformaldehyde for immunohistochemistry, and the remaining part was stored at -80°C for western blot analysis [1]
3. Immunohistochemistry of tumor tissues:
- The paraformaldehyde-fixed tumor tissues were dehydrated, embedded in paraffin, and sectioned into 4μm-thick slices. The slices were dewaxed in xylene and rehydrated in gradient ethanol. Antigen retrieval was performed by boiling the slices in citrate buffer. The slices were blocked with 3% H₂O₂ to eliminate endogenous peroxidase activity, then blocked with 5% BSA. A primary antibody against p-ULK1 (Ser317) was added and incubated overnight at 4°C. After washing, a biotin-conjugated secondary antibody was added and incubated for 1h at room temperature. The slices were then incubated with streptavidin-peroxidase complex, and the immunoreactive signals were visualized using a DAB chromogenic kit. The slices were counterstained with hematoxylin, dehydrated, cleared, and mounted. The positive ratio of p-ULK1 was quantified by counting the number of positive cells in 10 random high-power fields (×100 magnification) using Image-Pro Plus software [1]

1. In vitro toxicity: MTS assay showed that LYN-1604 HCl only inhibited the viability of MDA-MB-231 cells (tumor cells) and did not affect the viability of normal cells (the literature did not mention specific normal cell types) at the tested concentrations [1]
2. In vivo toxicity: After the treatment with LYN-1604 HCl, there was no significant difference in the relative body weight, liver index, spleen index, or kidney index between the treatment group and the vehicle group. No obvious pathological changes were observed in the liver, spleen, or kidney tissues (the literature did not provide detailed pathological section results), indicating that LYN-1604 HCl had no obvious acute toxicity to the major organs of the mice at the tested doses. [1]

[1]. Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo. Chem Sci. 2017 Apr 1;8(4):2687-2701.

1. Background information: ULK1 is a key regulatory protein that initiates autophagy. TCGA database analysis and tissue microarray (TMA) analysis showed that the expression of ULK1 was significantly downregulated in breast cancer tissues, especially in triple-negative breast cancer (TNBC) tissues. This downregulation leads to impaired autophagy function in tumor cells, which is conducive to tumor progression. Therefore, activating ULK1-modulated autophagy is considered a potential therapeutic strategy for TNBC [1]
2. Mechanism of action: LYN-1604 HCl acts as a specific agonist of ULK1. It binds to ULK1 through the LYS50, LEU53, and TYR89 residues, activates ULK1 kinase activity, and then promotes the formation of the ULK complex (ULK1-mATG13-FIP200-ATG101). The activated ULK complex induces ATG5-dependent autophagy, leading to autophagic cell death of tumor cells. At the same time, LYN-1604 HCl regulates the expression of ATF3, RAD21, and caspase3 through ULK1, and promotes apoptosis of tumor cells by activating the caspase3-PARP signaling pathway. Thus, LYN-1604 HCl exerts an antitumor effect by inducing both autophagy and apoptosis of TNBC cells [1]
3. Therapeutic potential: LYN-1604 HCl showed significant antitumor activity in both in vitro MDA-MB-231 cell models and in vivo TNBC xenograft models, and had good in vivo tolerability. It is a potential small-molecule drug candidate for the treatment of TNBC [1]
4. Chemical structure: The chemical structure of LYN-1604 HCl is shown in Figure 5A of the literature, and it belongs to the class of small-molecule compounds targeting ULK1 [1]

C₃₃H₄₄CL₃N₃O₂

621.08

619.249

C, 63.82; H, 7.14; Cl, 17.12; N, 6.77; O, 5.15

2216753-86-3

LYN-1604 dihydrochloride;2310109-38-5;LYN-1604;2088939-99-3

131801112

White to off-white solid powder

36

1

4

12

41

750

0

ClC1C=C(C=CC=1C(CN1CCN(C(CN(CC(C)C)CC(C)C)=O)CC1)OCC1C=CC2C=CC=CC=2C=1)Cl.Cl

KWFDUNOLEJSFBQ-UHFFFAOYSA-N

InChI=1S/C33H43Cl2N3O2.ClH/c1-24(2)19-37(20-25(3)4)22-33(39)38-15-13-36(14-16-38)21-32(30-12-11-29(34)18-31(30)35)40-23-26-9-10-27-7-5-6-8-28(27)17-26;/h5-12,17-18,24-25,32H,13-16,19-23H2,1-4H3;1H

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.03 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 (4.03 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 (4.03 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；
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。