靶向FAK的PROTACs的设计、合成及生物活性评价

doi:10.5246/jcps.2024.09.057

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (9): 767-782.DOI: 10.5246/jcps.2024.09.057

• 【研究论文】 • 下一篇

靶向FAK的PROTACs的设计、合成及生物活性评价

王瑞峰¹^,³^,⁴^,^#, 赵昕²^,^#, 侯宏保², 陈珂¹^,⁴, 刘水华¹^,⁴, 任茹月¹^,⁴, 刘云峰³^,^*(), 章毅¹^,²^,^*()

^1. 山西医科大学教育部慢性肾脏病医药基础研究创新中心, 山西太原 030001
^2. 山西医科大学基础医学院药理系, 山西太原 030001
^3. 山西医科大学第一医院内分泌科, 山西太原 030001
^4. 山西医科大学药学院, 山西太原 030001

收稿日期:2024-01-12 修回日期:2024-02-21 接受日期:2024-04-19 出版日期:2024-10-03 发布日期:2024-10-03
通讯作者: 刘云峰, 章毅

Cutting-Edge FAK-targeting PROTACs: design, synthesis, and biological evaluation

Ruifeng Wang¹^,³^,⁴^,^#, Xin Zhao²^,^#, Hongbao Hou², Ke Chen¹^,⁴, Shuihua Liu¹^,⁴, Ruyue Ren¹^,⁴, Yunfeng Liu³^,^*(), Yi Zhang¹^,²^,^*()

¹ Medicinal Basic Research Innovation Center of Chronic Kidney Disease, Ministry of Education, Shanxi Medical University, Taiyuan 030001, Shanxi, China
² Department of Pharmacology, Shanxi Medical University, Taiyuan 030001, Shanxi, China
³ Department of Endocrinology, First Hospital of Shanxi Medical University, Shanxi Medical University, Taiyuan 030001, Shanxi, China
⁴ Department of Pharmacy, Shanxi Medical University, Taiyuan 030001, Shanxi, China

Received:2024-01-12 Revised:2024-02-21 Accepted:2024-04-19 Online:2024-10-03 Published:2024-10-03
Contact: Yunfeng Liu, Yi Zhang
About author:
^# Ruifeng Wang and Xin Zhao contributed equally to this work.
Supported by:
National Natural Science Foundation of China (Grant No. 82204222, 81973378 and 82073909), China Postdoctoral Science Foundation (Grant No. 2022M722012), Shanxi Province Science Foundation for Youths (Grant No. 20210302124191), Open Fund from Medicinal Basic Research Innovation Center of Chronic Kidney Disease, Ministry of Education, Shanxi Medical University (Grant No. CKD/SXMU-2024-02).

摘要/Abstract

摘要：

黏着斑激酶(FAK)是一种细胞内的酪氨酸激酶, 具有激酶依赖的催化功能和激酶非依赖的支架功能, 这两种功能在癌症的发生、发展、转移等过程中至关重要。目前的激酶抑制剂只能抑制其催化功能, 而蛋白靶向降解嵌合体(PROTACs)可以将FAK蛋白降解来同时阻断两种功能。本研究设计合成了一类以defactinib衍生物化合物12为FAK蛋白的配体, 来那度胺类似物为E3连接酶配体的新型PROTAC, 其结构经核磁共振氢谱(¹H NMR)、核磁共振碳谱(¹³C NMR)和高分辨质谱(HRMS)确证。其中, 优选化合物16b对A549细胞中的FAK蛋白具有较强的降解活性(DC₅₀ = 6.16 ± 1.13 nM), 显著抑制A549细胞的增殖和克隆形成。与defactinib相比, 16b显示出更强的抑制A549细胞迁移和侵袭活性。此外, 研究表明16b通过CRBN介导的泛素蛋白酶体系统将FAK蛋白降解。

关键词: 黏着斑激酶, 蛋白靶向降解嵌合体, 迁移, 侵袭

Abstract:

Focal adhesion kinase (FAK) is an intracellular tyrosine kinase that plays a critical role in the occurrence, development, and metastasis of cancer through both its kinase-dependent catalytic functions and kinase-independent scaffolding functions. Current kinase inhibitors target only its catalytic activity, leaving the scaffolding functions unaffected. However, proteolysis targeting chimeras (PROTACs) offers a promising approach by degrading the entire FAK protein, thereby inhibiting both functions simultaneously. In this study, we designed and synthesized novel PROTAC degraders, utilizing a defactinib derivative (compound 12) as the FAK ligand and a lenalidomide analog as the E3 ligase ligand. The structures of these compounds were confirmed through ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS). Among the synthesized compounds, the optimized compound 16b exhibited potent degradation activity against FAK protein in A549 cells, with a DC₅₀ of 6.16 ± 1.13 nM, significantly inhibiting the proliferation and colony formation of these cells. Compared to defactinib, 16b showed enhanced inhibition of A549 cell migration and invasion. Furthermore, our research demonstrated that the rapid and effective FAK degradation induced by 16b was mediated by a CRBN-dependent proteasome mechanism.

Key words: FAK, PROTAC, Migration, Invasion

Supporting: /attached/file/20241006/20241006175219_831.pdf

王瑞峰, 赵昕, 侯宏保, 陈珂, 刘水华, 任茹月, 刘云峰, 章毅. 靶向FAK的PROTACs的设计、合成及生物活性评价[J]. 中国药学(英文版), 2024, 33(9): 767-782.

Ruifeng Wang, Xin Zhao, Hongbao Hou, Ke Chen, Shuihua Liu, Ruyue Ren, Yunfeng Liu, Yi Zhang. Cutting-Edge FAK-targeting PROTACs: design, synthesis, and biological evaluation[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(9): 767-782.

图/表 11

Figure 1. Schematic diagram of PROTAC strategy for FAK.

Figure 2. The predicted docked pose of defactinib in the FAK kinase catalytic domain (PDB: 3BZ3). Dashed red line represents hydrogen bond.

Figure 3. Design strategy for FAK-targeting PROTACs. (A) The molecular docking model of compound 12 with the FAK protein (PDB: 3BZ3) and the co-crystal binding mode of pomalidomide (PDB: 4CI3); (B) FAK-targeting PROTACs consist of three components: compound 12, Linkers, and E3 ligase binders.

Scheme 1. Synthesis of intermediates 4, 6 and 8a–8c. Reagents and conditions: a) 3-Aminopiperidine-2,6-dione, CH3COOH, NaOAc, 150 °C; b) tert-butyl glycine, DIPEA, DMF, 90 °C; c) HCl-ethyl acetate, CH2Cl2, 25 °C; d) corresponding bromocarboxylic acid, SOCl2, THF, 50 °C.

Scheme 2. Synthesis of compound 12. Reagents and conditions: a) tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate, ZnBr2, TEA, DCE/t-BuOH, 0 °C; b) N-(3-(aminomethyl)phenyl)-N-methylmethanesulfonamide, DIPEA, 1,4-dioxane, 100 °C; c) CF3COOH, CH2Cl2, 25 °C.

Scheme 3. Synthesis of compounds 15, 16a–16c and 19. Reagents and conditions: a) 1-Azido-2-(2-(2-iodoethoxy)ethoxy)ethane, K2CO3, CH3CN, 80 °C; b) triphenylphosphine, THF/H2O, 70 °C; c) 4, HATU, DIPEA, CH2Cl2, 25 °C; d) 8a–8c, K2CO3, KI, CH3CN, 80 °C; e) tert-butyl 3-(3-iodopropoxy)propanoate, K2CO3, CH3CN, 80 °C; f) CF3COOH, CH2Cl2, 30 °C; g) 6, HATU, DIPEA, CH2Cl2, 25 °C.

Figure 4. Assessing the FAK-degrading efficacy of the PROTACs through Western blotting analysis. (A) A549 cells were treated with the 10 and 100 nM of 15, 16a–16c and 19 for 24 h; (B) A549 cells were treated with the specified concentrations of 16b for 24 h, and data are representative of three independent experiments.

Figure 5. Antiproliferative activities of compound 16b and defactinib on A549 cells. (A) Cell viability was assessed using CCK-8 assay; (B) Clonogenic assay conducted on A549 cells treated with DMSO, 16b (5 μM), or defactinib (5 μM).

Figure 6. Wound healing assay. (A) The impact of 16b and defactinib on wound healing of A549 cells. The wounded area was captured immediately after introducing the wound and again after 24 h of treatment. (B) The migration ratio of A549 cells treated with defactinib and 16b. ***P < 0.001. n = 3.

Figure 7. Transwell assay was conducted to demonstrate the impact of defactinib (1 μM) and 16b (1 μM) on the invasion of A549 cells. Graphical representation of cells counts. ***P < 0.001. n = 3.

Figure 8. (A) FAK, CRBN, and proteasome-dependent FAK degradation by 16b. A549 cells were pre-treated separately with 5 µM defactinib, pomalidomide and MG-132 for 2 h, followed by 22 h treatment with compound 16b at 10 nM, with GAPDH serving as loading control. (B) FAK protein expression ratio vs control.

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靶向FAK的PROTACs的设计、合成及生物活性评价

Cutting-Edge FAK-targeting PROTACs: design, synthesis, and biological evaluation

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