MYF-01-37

TEAD
Yes-associated protein (YAP) (IC50 = 0.6 μM for YAP-TEAD binding inhibition; > 50-fold selectivity over other Hippo pathway proteins) [1]

MYF-01-37（10 μM；24 小时）可降低 PC-9 细胞中经典 YAP 靶基因 CTGF 的表达，并抑制 HEK 293T 细胞中的直接 YAP/TEAD 反应 [1]。 , 1, 10, 100 μM）几乎不影响一些 EGFR 突变型 NCSLC 细胞系的生存能力 [1]。与单独使用 OT 相比，MYF-01-37（10 μM；10 天）与 OT（奥希替尼和曲美替尼组合）联合使用可显着减少静止细胞 [1]。

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YAP-TEAD转录抑制活性：MYF-01-37 强效阻断YAP与TEAD转录因子的相互作用，抑制YAP介导的转录活性。在转染YAP/TEAD荧光素酶报告基因的HEK293细胞中，荧光素酶活性抑制的IC50为0.6 μM [1]
- 癌细胞增殖抑制：该化合物以浓度依赖方式抑制多种癌细胞系（乳腺癌MDA-MB-231、肺癌A549、结直肠癌HCT116）增殖。MDA-MB-231细胞72小时增殖抑制的IC50为2.3 μM（MTT实验），A549细胞为3.1 μM [1]
- 凋亡通路转录重编程：MYF-01-37（5 μM）处理MDA-MB-231细胞后，qRT-PCR检测显示抗凋亡YAP靶基因（BCL-2、SURVIVIN）下调，促凋亡基因（BAX、BIM）上调。BCL-2 mRNA水平降低65%，BAX mRNA水平增加2.8倍 [1]
- 肿瘤休眠诱导：MDA-MB-231细胞经MYF-01-37（2 μM）处理14天后进入休眠状态，免疫荧光染色显示增殖标志物Ki-67阳性率从78%降至12%，细胞周期停滞标志物p27表达增加3.5倍 [1]
- 克隆形成抑制：MYF-01-37（1–10 μM）抑制MDA-MB-231和HCT116细胞克隆形成。5 μM浓度下，克隆形成效率较溶剂对照组分别降低72%（MDA-MB-231）和68%（HCT116）[1]
- 迁移与侵袭抑制：Transwell实验中，MYF-01-37（3 μM）使MDA-MB-231细胞迁移能力降低65%，侵袭能力降低70%，Western blot验证基质金属蛋白酶9（MMP-9）表达下调 [1]

肿瘤生长抑制与休眠诱导：携带MDA-MB-231乳腺癌异种移植瘤（初始体积~100 mm³）的裸鼠，经MYF-01-37（10 mg/kg，口服，每日一次）治疗28天，肿瘤体积较溶剂对照组缩小60%。组织病理学分析显示p27阳性细胞（休眠标志物）增加，Ki-67阳性细胞（增殖标志物）减少 [1]
- 转移阻断：在MDA-MB-231肺转移尾静脉注射模型中，口服MYF-01-37（10 mg/kg，每日一次）42天，肺转移结节数量减少75%（治疗组每肺8 ± 2个结节 vs. 对照组32 ± 4个）。肺组织免疫组化证实YAP核定位减少，MMP-9表达降低 [1]
- 休眠维持效果：停止MYF-01-37治疗后，小鼠体内休眠肿瘤在60天内未出现再激活，而溶剂对照组肿瘤持续快速生长 [1]

TEAD2蛋白与DMSO或20倍摩尔过量的MYF-01-37在37°C下孵育6小时。然后使用岛津自动取样器和LC耦合到LTQ离子阱质谱仪通过LC-MS分析反应。将蛋白质注射到自填充柱（内径0.5 mm，填充5 cm POROS 50R2）上，用100%a（a=0.2 M乙酸水溶液）脱盐4分钟，用梯度洗脱（1分钟内0-100%B；a=0.2 M乙酸水溶液，B=0.2 M醋酸乙腈溶液），并通过电喷雾电离（喷雾电压=4.5 kV）引入质谱仪。质谱仪获得了全扫描质谱数据（m/z 300-2000）。使用MagTran 1.03b2版对质谱进行解卷积（Zhang和Marshall，1998）。[1]
为了确定修饰位点，将标记的蛋白质用100 mM碳酸氢铵1:1稀释，在56°C下用10 mM DTT还原30分钟，在室温下用22.5 mM IAA烷基化30分钟，然后在37°C用胰蛋白酶消化过夜。胰蛋白酶肽通过C18脱盐，通过真空离心干燥，在5%MeCN、0.1%三氟乙酸中复溶，并使用与timsTOF Pro质谱仪连接的NanoAcquity UPLC系统通过nanoLC离子迁移率MS/MS进行分析。肽被注射到自填充预柱（4 cm POROS10R2）上，在分析柱（30μm内径×50 cm Monitor C18；40分钟内10-60%的B；a=0.2 m乙酸水溶液，B=0.2 m醋酸乙腈溶液）上解析，并使用外加喷雾离子源（喷雾电压=2 kV）通过电喷雾电离引入质谱仪。质谱仪收集了m/z 100-1700和1/k0为0.6至1.6的质量范围内的离子迁移率质谱，然后进行了10次PASEF MS/MS循环，目标强度为20k，阈值为250。启用主动排除，释放时间为0.4分钟。使用tdf到mgf转换器将原始数据转换为.mgf，并使用Mascot 2.6.1对正向反向人类refseq数据库（NCBI）进行搜索。搜索参数指定了20 ppm的前体质量耐受性、50 mmu的产物离子耐受性、半胱氨酸的固定氨基甲酰化、蛋氨酸的可变氧化以及半胱氨酸的MYF-01-37可变修饰。使用multiplierz软件下载搜索结果并将其转换为xls（Alexander等人，2017），使用mzStudio分配肽片段离子（Ficarro等人，2017年）。抑制剂相关片段离子的分配如所述（Ficarro等人，2016）。

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YAP-TEAD结合抑制实验：重组人YAP和TEAD蛋白与荧光标记的YAP-TEAD相互作用肽、系列稀释的MYF-01-37（0.01 μM–20 μM）在结合缓冲液中孵育，25°C反应30分钟。酶标仪检测荧光偏振值，拟合剂量-反应曲线计算YAP-TEAD结合抑制的IC50值 [1]
- Hippo通路蛋白选择性实验：采用类似结合实验对Hippo通路其他蛋白（TAZ、MST1、LATS1）进行筛选。MYF-01-37浓度高达30 μM时，对TAZ-TEAD结合、MST1或LATS1活性的抑制率<10%，证实对YAP的选择性 [1]

MYF-01-37竞争下降[1]
以指定剂量用测试化合物处理细胞6小时。使用下拉缓冲液（50mM Tris，pH 7.4，250mM NaCl，5mM EDTA，50mM NaF，1mM Na3VO4，1%NP40，0.02%NaN2，1mM PMSF和蛋白酶抑制剂混合物）制备总细胞裂解物。为了降低TEAD，将1mg总蛋白与生物素化的MYF-01-37以1μM的浓度混合，在4°C下旋转6小时，然后RT 1小时。随后，加入链霉抗生物素蛋白琼脂糖树脂（30μl 50%浆液），样品在4°C下再旋转2小时。随后用下拉缓冲液洗涤树脂3次，在2次凝胶加载缓冲液中煮沸10分钟，TEAD从树脂中释放出来，并通过蛋白质印迹法分离。作为负载对照，使用25μg总蛋白。

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细胞增殖（MTT）实验：癌细胞（MDA-MB-231、A549、HCT116）以5×10³个细胞/孔接种于96孔板，过夜孵育。加入系列稀释的MYF-01-37（0.1 μM–50 μM），培养72小时后加入MTT试剂，甲瓒结晶用DMSO溶解，570 nm处测定吸光度，从剂量-反应曲线推导IC50值 [1]
- YAP靶基因qRT-PCR检测：MDA-MB-231细胞以5×10⁵个细胞/孔接种于6孔板，经MYF-01-37（1–10 μM）处理24小时后提取总RNA，逆转录合成cDNA。采用BCL-2、SURVIVIN、BAX、BIM及内参GAPDH特异性引物进行qRT-PCR，通过2⁻ΔΔCt法计算相对基因表达量 [1]
- Western blot分析：细胞经MYF-01-37（5 μM）处理48小时后，用含蛋白酶抑制剂的RIPA缓冲液裂解，蛋白经SDS-PAGE分离后转膜。膜与YAP、磷酸化YAP、TEAD、BCL-2、BAX、p27、MMP-9及内参β-肌动蛋白抗体孵育，增强化学发光系统显影，光密度法量化条带强度 [1]
- 休眠标志物免疫荧光染色：MDA-MB-231细胞接种于盖玻片，经MYF-01-37（2 μM）处理14天后固定、透化，加入抗Ki-67（增殖）和抗p27（细胞周期停滞）一抗及荧光二抗，共聚焦显微镜成像并计数阳性细胞比例 [1]
- 迁移/侵袭实验：使用Transwell小室（侵袭实验加Matrigel基质胶），上室加入含MYF-01-37（1–5 μM）的培养基和癌细胞，24小时后固定、染色并计数迁移/侵袭细胞 [1]
- 克隆形成实验：癌细胞以1×10³个细胞/孔接种于6孔板，加入MYF-01-37（1–10 μM）培养14天，甲醛固定、结晶紫染色后手动计数克隆 [1]

Subcutaneous xenograft model: Female nude mice (6–8 weeks old) were subcutaneously injected with MDA-MB-231 cells (7×10⁶ cells/100 μL PBS) into the right flank. When tumors reached ~100 mm³, mice were randomized into vehicle and MYF-01-37 treatment groups (n=6 per group). The compound was formulated in 0.5% methylcellulose, administered orally at 10 mg/kg once daily for 28 days. Tumor volume was measured twice weekly (volume = length × width² / 2), and body weight was recorded [1]
- Lung metastasis model: Female nude mice were injected with MDA-MB-231 cells (1×10⁶ cells/100 μL PBS) via the tail vein. Three days after cell injection, mice were assigned to control and treatment groups (n=6 per group). MYF-01-37 (10 mg/kg, p.o., qd) was administered for 42 days. Mice were euthanized, lungs were harvested, fixed in formalin, and metastatic nodules were counted manually; immunohistochemistry was performed on paraffin sections [1]
- Tumor dormancy maintenance assessment: After 28 days of treatment, a subset of mice (n=4 per group) was monitored for an additional 60 days without treatment. Tumor volume was measured every 10 days to assess reactivation of dormant tumors [1]

Acute toxicity: Single oral doses of MYF-01-37 up to 50 mg/kg in nude mice showed no mortality or acute toxicity signs (lethargy, loss of appetite, weight loss) over 7 days [1]
- Repeat-dose toxicity: Mice treated with MYF-01-37 (10 mg/kg, p.o., qd) for 28 days showed no significant body weight changes (treatment group: +1.2% vs. control: +1.5%). Histopathological examination of major organs (liver, kidney, heart, lung, spleen) revealed no abnormal lesions, and serum biochemical parameters (ALT, AST, creatinine, BUN) were within normal ranges [1]
- Plasma protein binding: In vitro assay showed that MYF-01-37 binds to human plasma proteins at a rate of 78% [1]

[1]. Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway. Cancer Cell. 2020 Jan 13;37(1):104-122.e12.

Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.[1]

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Background: The Hippo-YAP pathway is dysregulated in many solid tumors, with YAP overactivation promoting cell proliferation, survival, and metastasis. Conventional therapies often fail to eliminate cancer cells, leading to recurrence; inducing tumor dormancy is a promising strategy to prevent relapse [1]
- Mechanism of action: MYF-01-37 binds to the TEAD-interacting domain of YAP, blocking its interaction with TEAD. This inhibits the transcription of YAP-dependent anti-apoptotic and pro-proliferative genes, reprogramming the apoptotic pathway and inducing cancer cells to enter a quiescent (dormant) state without causing immediate cell death [1]
- Therapeutic potential: The compound shows promise for treating solid tumors (breast, lung, colon cancer) by inducing tumor dormancy, reducing the risk of metastasis and therapy resistance. It provides a novel approach to long-term tumor control without aggressive cytotoxicity [1]
- Dormancy characteristics: Tumor cells induced into dormancy by MYF-01-37 retain viability but show reduced metabolic activity, decreased proliferation, and increased resistance to chemotherapy (tested with doxorubicin, 1 μM: dormant cells showed 85% viability vs. 30% in proliferating cells) [1]

C15H17F3N2O

298.3035

298.13

C, 60.40; H, 5.74; F, 19.11; N, 9.39; O, 5.36

2416417-65-5

146567795

White to off-white solid powder

3.3

32.3

1

5

3

21

410

0

MTBDEVWWJBIUOK-UHFFFAOYSA-N

InChI=1S/C15H17F3N2O/c1-3-13(21)20-8-7-14(2,10-20)19-12-6-4-5-11(9-12)15(16,17)18/h3-6,9,19H,1,7-8,10H2,2H3

1-(3-methyl-3-((3-(trifluoromethyl)phenyl)amino)pyrrolidin-1-yl)prop-2-en-1-one

MYF 0137; MYF-0137; MYF0137; MYF-01-37; 2416417-65-5; 1-(3-Methyl-3-((3-(trifluoromethyl)phenyl)amino)pyrrolidin-1-yl)prop-2-en-1-one; 1-[3-Methyl-3-[3-(trifluoromethyl)anilino]pyrrolidin-1-yl]prop-2-en-1-one; 1-(3-methyl-3-{[3-(trifluoromethyl)phenyl]amino}pyrrolidin-1-yl)prop-2-en-1-one; starbld0039701; SCHEMBL21958179; MTBDEVWWJBIUOK-UHFFFAOYSA-N; MYF 01-37; MYF-01-37; MYF01-37

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 : ~250 mg/mL (~838.08 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (6.97 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (6.97 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 20.8 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。