TEAD1 Palmitoylation

VT-104 (0-1000 nM) 可防止 NF2 突变/缺陷细胞系增殖 [1]。

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与VT103共孵育后，与TEAD家族的其他成员相比，TEAD1的熔化温度升高幅度最大，达到8.3℃。热变性曲线清晰地显示TEAD1单独（红色曲线）和TEAD1+VT103（蓝色曲线）的两个独立峰，而其他TEAD蛋白的±VT103曲线的峰大部分重叠（图4A，顶部）。这与功能性棕榈酰化实验的发现一致，即VT103是tead1选择性抑制剂。另一方面，VT107通过TEAD棕榈酰化实验被确定为泛TEAD抑制剂，它显著改变了所有四个TEAD家族成员的熔化温度（图4A）。VT-104改变了所有四种TEAD家族成员的熔化温度，但TEAD1和TEAD3的变化更大。VT106仅微弱地改变了所有四个tead的熔化温度。[1]

VT-104（0.3-3 mg/kg；口服；NCI-H226 荷瘤小鼠）可防止 NF2 缺陷的间皮瘤异种移植物的生长 [1]。

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VT103和VT-104阻断nf2缺陷间皮瘤异种移植物[1]
由于TEAD在小鼠体内具有良好的口服生物利用度（≥75%）和较长的半衰期（>12小时）（图2），VT103和VT-104使我们能够在体内评估TEAD自身棕榈酰化抑制在人间皮瘤异种移植模型中的靶向作用和抗肿瘤效果。如图6B所示，在第三次每日给药4小时后，VT103显著下调nf2缺失小鼠NCI-H226肿瘤中Hippo通路靶基因CTGF和CYR61的表达，并呈剂量依赖性。在相同的nci - h226荷瘤动物中，VT103也以剂量依赖的方式下调肾脏和肝脏中的靶基因表达（Supplementary Fig. S6A）。然而，在同一时间点似乎没有任何病理影响，因为肾脏和肝脏的H&E图像在载药组和药物治疗组之间没有差异（补充图S6B）。生物分析表明，VT103在循环系统和肿瘤组织中存在剂量依赖性暴露（补充图S6C）。此外，在同一动物体内，肿瘤中的化合物积累似乎比循环中的化合物积累更多（补充图S6C）。通过免疫组化检测vt103处理的NCI-H226肿瘤组织中的YAP和TEAD1蛋白显示，这些蛋白的细胞定位或水平没有变化（补充图S6D）。

无细胞TEAD棕榈酰化测定 [1]
纯化的重组TEAD1–YBD首先与化合物一起孵育，然后与2μmol/L炔烃棕榈酰辅酶A一起孵育。用1%SDS猝灭反应，然后如前所述与生物素叠氮化物进行点击化学反应。在一些实验中，APCoA以不同的浓度和不同的顺序加入。分别通过链霉亲和素-HRP和抗TEAD1抗体免疫印迹法检测棕榈酰化TEAD和总TEAD蛋白。

基于细胞的TEAD棕榈酰化测定[1]
Myc-TEAD表达质粒转染的HEK293T细胞用DMSO或100μmol/L棕榈酸炔+DMSO/化合物处理20小时。用抗Myc抗体免疫沉淀Myc-TEAD蛋白，并进行点击化学。链亲和素免疫印迹法检测棕榈酰化TEAD。如上所述进行酰基聚乙二醇交换凝胶位移测定。

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细胞增殖试验[1]
采用CellTiter-Glo发光细胞活力测定试剂盒（Luminescent Cell Viability Assay Kit），按照生产厂家的方案，以3 μmol/L剂量滴定的化合物处理不同时间的细胞。采用剂量反应曲线计算IC50和最大抑制率。

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免疫荧光法[1]
用4%多聚甲醛固定10 - 15分钟，用0.1% Triton X-100在PBS中渗透后，在室温下用3% BSA在PBS中阻断细胞1 - 2小时，在4℃下用一抗染色过夜，然后用Alexa荧光偶联二抗在室温下染色2 - 3小时。载玻片用含DAPI的延长金抗褪色试剂贴膜。图像由尼康Eclipse Ti共聚焦显微镜拍摄。

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免疫沉淀反应[1]
用PBS洗涤细胞并裂解[50 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1% Triton-X100, 50 mmol/L NaF, 1 mmol/L PMSF，蛋白酶抑制剂鸡尾酒，磷酸酶抑制剂]。超声和离心后，收集上清液，与抗tead、抗yap或对照抗体孵育，用蛋白A/G珠沉淀，按标准方案进行免疫印迹分析（抗体信息见补充表S3）。

Animal/Disease Models: NCI- H226 tumor-bearing mice [1]
Doses: 0.3~10 mg/kg
Route of Administration: Po one time/day
Experimental Results: Tumor growth can be prevented even at a dose of 0.3 mg/kg.

Mouse pharmacokinetics
VT103, VT104, and VT107, formulated in 5% DMSO + 10% Solutol + 85% D5W, were dosed intravenously or orally at 7 or 10 mg/kg. Blood was drawn from the saphenous vein at indicated timepoints. Compounds were quantified by LC/MS-MS using a QTRAP 6500. Data were analyzed using Phoenix WinNonlin 6.3, and intravenously noncompartmental model 201, and orally noncompartmental model 200. The calculation method was linear/log trapezoidal.

In vivo pharmacodynamic and efficacy studies
All the procedures related to animal handling, care, and the treatment were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec or Crown Bioscience, Inc., following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The testing article formulated in dosing solution (5% DMSO + 10% solutol + 85% D5W; D5W = 5% glucose) was orally administrated daily at the indicated doses. Tumor volume and animal weights were monitored twice weekly.

VT103 is an analog of VT101, which has improved potency and good oral pharmacokinetics in mice (Fig. 2; Supplementary Table S1). VT-104 is an analog of VT102, which has improved potency and good oral pharmacokinetics in mice (Fig. 2; Supplementary Table S1). VT105 is a more soluble analog of VT-104 (Fig. 2), which was useful in TEAD X-ray crystallography experiments. VT106 and VT107 are enantiomers analogous to VT-104; they have quite different potencies, making them useful mutual controls in biochemical and cellular experiments (Fig. 2).[1]

[1]. Small Molecule Inhibitors of TEAD Auto-palmitoylation Selectively Inhibit Proliferation and Tumor Growth of NF2-deficient Mesothelioma. Mol Cancer Ther. 2021;20(6):986-998.

[2]. Preparation of substituted naphthalene-2-carboxamide derivatives useful for treatment of cancer. WO2020097389.

Mutations in the neurofibromatosis type 2 (NF2) gene that limit or abrogate expression of functional Merlin are common in malignant mesothelioma. Merlin activates the Hippo pathway to suppress nuclear translocation of YAP and TAZ, the major effectors of the pathway that associate with the TEAD transcription factors in the nucleus and promote expression of genes involved in cell proliferation and survival. In this article, we describe the discovery of compounds that selectively inhibit YAP/TAZ-TEAD promoted gene transcription, block TEAD auto-palmitoylation, and disrupt interaction between YAP/TAZ and TEAD. Optimization led to potent analogs with excellent oral bioavailability and pharmacokinetics that selectively inhibit NF2-deficient mesothelioma cell proliferation in vitro and growth of subcutaneous tumor xenografts in vivo These highly potent and selective TEAD inhibitors provide a way to target the Hippo-YAP pathway, which thus far has been undruggable and is dysregulated frequently in malignant mesothelioma and in other YAP-driven cancers and diseases. [1]

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Other than establishing the tolerability of our compounds in mice, the research described herein does not address any toxicity of the compounds, which could be related or unrelated to inhibition of TEAD palmitoylation. Formal toxicologic evaluation in multiple animal species will be required to characterize the safety of the small molecule compounds. If favorable, clinical evaluation of a TEAD palmitoylation inhibitor is warranted in NF2 mutant mesothelioma and cancers with activated YAP/TAZ-TEAD transcriptional activity as monotherapy or in combination with other targeted cancer therapies. [1]

C25H19F3N2O

420.426376581192

420.144

C, 71.42; H, 4.56; F, 13.56; N, 6.66; O, 3.81

2417718-26-2

VT104;2417718-25-1; (R)-VT104;2417718-26-2; 2417718-25-1 (S-isomer); 2417720-80-8 (racemate)

146909372

White to off-white solid powder

5.8

42

1

5

4

31

602

1

FC(C1C=CC(=CC=1)C1=CC=CC2=CC(C(N[C@H](C)C3C=CC=CN=3)=O)=CC=C12)(F)F

AAZUPSFRSHFTGV-MRXNPFEDSA-N

InChI=1S/C25H19F3N2O/c1-16(23-7-2-3-14-29-23)30-24(31)19-10-13-22-18(15-19)5-4-6-21(22)17-8-11-20(12-9-17)25(26,27)28/h2-16H,1H3,(H,30,31)/t16-/m1/s1

N-[(1R)-1-pyridin-2-ylethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide

2417718-26-2; N-[(1R)-1-(pyridin-2-yl)ethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide; N-((1R)-1-(pyridin-2-yl)ethyl)-5-(4-(trifluoromethyl)phenyl)naphthalene-2-carboxamide; 882-321-3; (R)-VT104; N-[(1R)-1-pyridin-2-ylethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide; AAZUPSFRSHFTGV-MRXNPFEDSA-N; HY-134956B;

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: ≥ 100 mg/mL (237.85 mM)

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;
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