双功能化合物靶向降解免疫检查点蛋白PD-L1

doi:10.6023/A24020042

摘要/Abstract

本研究利用整合素促进的靶蛋白溶酶体降解策略, 设计合成了识别整合素受体α_vβ₃的小分子Gua-Azide以替代c(RGDyK)环肽, 通过连接子使其与PD-L1抑制剂BMS-8相连, 形成双功能小分子降解剂. 该降解剂能够通过内吞-溶酶体途径靶向降解细胞膜上的PD-L1靶标蛋白, 为开发癌症免疫治疗相关小分子药物开辟了新途径.

关键词: 蛋白质靶向降解, IFLD策略, 双功能降解剂, PD-L1, 肿瘤免疫治疗

Programmed death ligand 1 (PD-L1) is a protein overexpressed in numerous tumor cells as an essential immune checkpoint that can facilitate immune escape of tumor cells through binding to PD-1 on T cells. Compared to antibody drugs targeting PD-L1 or PD-1, which are limited in clinical application due to their immunogenicity, high cost and low oral bioavailability, small molecule inhibitors exhibit better tissue and tumor penetration ability, as well as higher bioavailability. However, traditional small molecule inhibitors often face drug resistance issues and are difficult to inhibit protein-protein interactions. Targeted protein degradation (TPD) technologies achieve a more comprehensive suppression of protein activity by degrading the target protein, thereby eliminating its functions entirely. The strategy not only reduces the drug resistance but also enhances the therapeutic efficacy. In the previous study, we have developed the integrin-facilitated lysosome degradation (IFLD) strategy to degrade extracellular and cell membrane proteins by using bifunctional compounds containing a target protein-binding domain, and a cyclic RGD peptide as the integrin-binding domain, connected via a linker. The cyclic RGD peptide, which incorporates the sequence arginine-glycine-aspartic acid (RGD), has been extensively employed for the development of targeted drug delivery systems. It specifically recognizes the integrin α_vβ₃, which is overexpressed in a variety of tumor cells and plays a significant role in tumor-targeted drug delivery. Considering the instability of the RGD peptide, we designed and synthesized a small molecule compound, Gua-Azide, in this study to mimick the RGD (Arg-Gly-Asp) peptide to bind with α_vβ₃ integrin. Subsequently, we constructed a new IFLD degrader targeting PD-L1, BMS-Gua, through the conjugation of Gua-Azide with BMS-8, a small molecule with high binding affinity to PD-L1, by using click chemistry. The western blotting and immunofluorescence analysis verified that BMS-Gua, a bifunctional molecule degrader, could effectively induce the endocytosis and lysosomal degradation of PD-L1 through an integrin-dependent pathway, warranting further investigation for the development of tumor immunotherapy drugs.

Key words: targeted protein degradation, IFLD strategy, bifunctional degrader, PD-L1, cancer immunotherapy

引用此文

陈从丽, 师怀怀, 郝芮, 房丽晶, 徐华栋. 双功能化合物靶向降解免疫检查点蛋白PD-L1[J]. 化学学报, 2024, 82(6): 613-620.

Congli Chen, Huaihuai Shi, Rui Hao, Lijing Fang, Huadong Xu. Bifunctional Compound for Targeted Degradation of the Immune Checkpoint Protein PD-L1[J]. Acta Chimica Sinica, 2024, 82(6): 613-620.

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图/表 6

图1. c(RGDyK)与Gua-Azide的结构比较

Figure 1. Comparison of the structures of c(RGDyK) and Gua-Azide

图式1. Gua-Azide的合成路线

Scheme 1. Synthetic route of Gua-Azide

图2. Gua-Azide与c(RGDyK)在人工胃液中的稳定性

Figure 2. The stability of Gua-Azide and c(RGDyK) in simulated gastric fluidc

图3. 通过ITC测定Gua-Azide与整合素αvβ3之间的相互作用

Figure 3. Interactions between Gua-Azide and integrin αVβ3 was assessed by ITC

图式2. BMS-Gua的合成路线

Scheme 2. Synthetic route of BMS-Gua

图4. BMS-Gua靶向降解细胞膜上PD-L1蛋白. (A) Western blot分析经BMS-Gua和BMS-L1-RGD (25 nmol/L, 8 h)处理的MDA-MB-231细胞中PD-L1的蛋白水平. (B) Western blot分析BMS-Gua介导的PD-L1在指定时间和指定浓度下的降解. (C) Western blot分析25 nmol/L BMS-Gua与100 μmol/L Bafilomycin A1或5 μmol/L MG132处理24 h的MDA-MB-231细胞中PD-L1的蛋白水平. (D) Western blot分析Gua-Azide与整合素结合的竞争性抑制对BMS-Gua介导的PD-L1降解的影响. (E) 细胞膜上PD-L1(红色)降解的免疫荧光分析. 用BMS-Gua、BMS-8、Gua-Azide、BMS-8和Gua-Azide联合处理稳定表达PD-L1的HeLa细胞. (F) 免疫荧光分析Gua-Azide与整合素结合的竞争性抑制对BMS-Gua介导的PD-L1(红色)降解的影响. (G) 在人宫颈癌(HeLa)细胞中PD-L1(绿色)与早期核内体标记物(Rab5, 红色)共定位. 细胞核用DAPI标记(蓝色), 对于(E), (F)和(G), 比例尺, 10 μm

Figure 4. BMS-Gua mediated degradation of PD-L1 on the cell membrane. (A) Western blot analysis of PD-L1 protein levels in MDA-MB-231 cells treated with BMS-Gua and BMS-L1-RGD (25 nmol/L, 8 h). (B) Western blot analysis of BMS-Gua-mediated PD-L1 degradation at indicated concentration, or indicated time. (C) Western blot analysis of PD-L1 protein levels in MDA-MB-231 cells treated with 25 nmol/L BMS-Gua for 24 h along with 100 μmol/L Bafilomycin A1 or 5 μmol/L MG132. (D) Effects of competitive inhibition of Gua-Azide-integrin binding on BMS-Gua-mediated PD-L1 degradation, analyzed by Western blot. (E) Immunofluorescence analysis of PD-L1 (red) degradation on cell membrane. HeLa cells stably expressing PD-L1 were treated with BMS-Gua, BMS-8, Gua-Azide, as well as a combination of BMS-8 and Gua-Azide. (F) Effects of competitive inhibition of Gua-Azide-integrin binding on BMS-Gua mediated PD-L1 degradation (red), analyzed by fluorescence imaging. (G) Fluorescence signals indicating the colocalization of PD-L1 (green) with the early endosome marker (Rab5, red) in human cervical carcinoma (HeLa) cells. The nuclei were labeled by DAPI (blue). For (E), (F) and (G), scale bar, 10 μm

参考文献 24

[1]	Zhang, X. Q.; Cheng, C.; Hou, J. Y.; Qi, X. Y.; Wang, X.; Han, P.; Yang, X. M. Cell. Mol. Immunol. 2019, 16, 392.
[2]	Gou, Q.; Dong, C.; Xu, H. H.; Khan, B.; Jin, J. H.; Liu, Q.; Shi, J. J.; Hou, Y. Z. Cell Death Dis. 2020, 11, 955.
[3]	Su, W.; Tan, M. X.; Wang, Z. H.; Zhang, J.; Huang, W. P.; Song, H. H.; Wang, X. Y.; Ran, H. T.; Gao, Y. F.; Nie, G. J.; Wang, H. Angew. Chem., Int. Ed. 2023, 62, e202218128.
[4]	Yang, Z. J.; Huang, C. J.; Wang, M. X. Oncol. Prog. 2020, 18, 772. (in Chinese)
	(杨占菊, 黄长江, 王名雪, 癌症进展, 2020, 18, 772.)
[5]	Sathe, G.; Sapkota, G. P. Trends Pharmacol. Sci. 2023, 44, 786.
[6]	Sun, X. Y.; Gao, H. Y.; Yang, Y. Q.; He, M.; Wu, Y.; Song, Y. G.; Tong, Y.; Rao, Y. Signal Transduct. Tar. 2019, 4, 64.
[7]	He, M.; Cao, C. G.; Ni, Z. H.; Liu, Y. B.; Song, P. L.; Hao, S.; He, Y. N.; Sun, X. Y.; Rao, Y. Signal Transduct. Tar. 2022, 7, 181.
[8]	Zhang, C.; He, Y.; Sun, X.; Wei, W.; Liu, Y.; Rao, Y. Acta Mater. Med. 2023, 2, 409.
[9]	Pei, J. P.; Pan, X. L.; Wang, A. X.; Shuai, W.; Bu, F. Q.; Tang, P.; Zhang, S.; Zhang, Y. W.; Wang, G.; Ouyang, L. Chem. Commun. 2021, 57, 13194.
[10]	Li, Z. Y.; Zhu, C. G.; Ding, Y.; Fei, Y. Y.; Lu, B. X. Autophagy 2020, 16, 185.
[11]	Ji, C. H.; Kim, H. Y.; Lee, M. J.; Heo, A. J.; Park, D. Y.; Lim, S.; Shin, S.; Yang, W. S.; Jung, C. A.; Kim, K. Y.; Jeong, E. H.; Park, S. H.; Kim, S. B.; Lee, S. J.; Na, J. E.; Kang, J. I.; Chi, H. M.; Kim, H. T.; Kim, Y. K.; Kim, B. Y.; Kwon, Y. T. Nat. Commun. 2022, 13, 904.
[12]	Banik, S. M.; Pedram, K.; Wisnovsky, S.; Ahn, G.; Riley, N. M.; Bertozzi, C. R. Nature 2020, 584, 291.
[13]	Cotton, A. D.; Nguyen, D. P.; Gramespacher, J. A.; Seiple, I. B.; Wells, J. A. J. Am. Chem. Soc. 2021, 143, 593. doi: 10.1021/jacs.0c10008 pmid: 33395526
[14]	Zhang, H.; Han, Y.; Yang, Y. F.; Lin, F.; Li, K. X.; Kong, L. H.; Liu, H. X.; Dang, Y. J.; Lin, J.; Chen, P. R. J. Am. Chem. Soc. 2021, 143, 16377. doi: 10.1021/jacs.1c08521 pmid: 34596400
[15]	Wu, Y. Q.; Lin, B. Q.; Lu, Y. Z.; Li, L.; Deng, K. Y.; Zhang, S. H.; Zhang, H. M.; Yang, C. Y.; Zhu, Z. Angew. Chem., Int. Ed. 2023, 62, e202218106.
[16]	Zheng, J. W.; He, W. Y.; Li, J.; Feng, X. J.; Li, Y. Y.; Cheng, B. H.; Zhou, Y. M.; Li, M. Q.; Liu, K.; Shao, X. M.; Zhang, J. C.; Li, H. C.; Chen, L.; Fang, L. J. J. Am. Chem. Soc. 2022, 144, 21831.
[17]	Nieberler, M.; Reuning, U.; Reichart, F.; Notni, J.; Wester, H. J.; Schwaiger, M.; Weinmüller, M.; Räder, A.; Steiger, K.; Kessler, H. Cancers 2017, 9, 116.
[18]	Hatley, R. J. D.; Macdonald, S. J. F.; Slack, R. J.; Le, J.; Ludbrook, S. B.; Lukey, P. T. Angew. Chem., Int. Ed. 2018, 57, 3298.
[19]	Danhier, F.; Le Breton, A.; Préat, V. Mol. Pharmaceut. 2012, 9, 2961.
[20]	Chen, T. K.; Li, Q.; Liu, Z. L.; Chen, Y.; Feng, F.; Sun, H. P. Eur. J. Med. Chem. 2019, 161, 378.
[21]	Sulyok, G. A. G.; Gibson, C.; Goodman, S. L.; Hölzemann, G.; Wiesner, M.; Kessler, H. J. Med. Chem. 2001, 44, 1938. pmid: 11384239
[22]	Chai, Z. L.; Ran, D. N.; Lu, L. W.; Zhan, C. Y.; Ruan, H. T.; Hu, X. F.; Xie, C.; Jiang, K.; Li, J. Y.; Zhou, J. F.; Wang, J.; Zhang, Y. Y.; Fang, R. H.; Zhang, L. F.; Lu, W. Y. ACS Nano 2019, 13, 5591.
[23]	Wang, X.; Zhang, X. R.; Huang, Z. Y.; Fan, X. Y.; Chen, P. Acta Chim. Sinica 2021, 79, 406. (in Chinese) doi: 10.6023/A20110530
	(汪欣, 张贤睿, 黄宗煜, 樊新元, 陈鹏, 化学学报, 2021, 79, 406.) doi: 10.6023/A20110530
[24]	Guzik, K.; Zak, K. M.; Grudnik, P.; Magiera, K.; Musielak, B.; Törner, R.; Skalniak, L.; Dömling, A.; Dubin, G.; Holak, T. A. J. Med. Chem. 2017, 60, 5857.