靶向SHP2E76A突变体的小分子降解剂有效抑制野生型和突变体SHP2肿瘤细胞增殖

doi:10.6023/A23050229

摘要/Abstract

SHP2 (Src homology 2 domain-containing protein tyrosine phosphatase-2)是由Ptpn11基因编码的非受体型蛋白酪氨酸磷酸酶, 通过RAS (rat sarcoma)-ERK (extracellular regulated protein kinases)信号通路的活化调控细胞生长、分化和凋亡, 并参与PD-1 (programmed cell death protein 1)/PD-L1(programmed cell death ligand 1)通路的免疫监控, 已成为有突破意义的抗癌药物新靶标. 靶向SHP2变构位点的抑制剂通过稳定SHP2非活性构象而抑制磷酸酶催化功能, 具有很好的成药性. 但是, SHP2功能获得性突变(gain of function, 简称GOF)导致一系列发育障碍疾病和肿瘤的发生, 并对SHP2野生型变构抑制剂产生耐药性. 本工作首次以靶向SHP2激活突变体的变构抑制剂为靶头, 基于PROTACs (proteolysis-targeting chimeras)技术, 设计合成了一系列全新SHP2小分子降解剂. 其中先导化合物3f和4d保持了对突变型SHP2^E76A的酶抑制活性, 而且对于野生型SHP2依赖的人食管鳞癌细胞KYSE-520和突变型SHP2^N58S人大细胞肺癌细胞NCI-H661都显示强效抗增殖活性, 比相应变构抑制剂的活性提高了5~10倍, 为治疗SHP2突变或活化导致的遗传性疾病或肿瘤提供了新的干预策略.

关键词: 蛋白酪氨酸磷酸酶, SHP2, PROTAC, 小分子降解剂, 功能获得性突变, SHP2^E76A突变体

Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase encoded by the Ptpn11 gene. SHP2 regulates cell growth, differentiation and apoptosis via modulating various signaling pathways, such as RAS/ERK signaling pathway, and participates in the PD-1/PD-L1 pathway governing immune surveillance, thus has been recognized as a breakthrough antitumor therapeutic target. By stabilizing the inactive closed conformation of SHP2, allosteric inhibitors have overcome the druggability issues and advanced to clinical trials. However, the gain-of-function mutation (GOF) of the Ptpn11 gene renders SHP2 in a pathological active conformation, causing a range of developmental disorders and tumors. Overactivated SHP2 mutants undergo structural and functional changes, conferring resistance to SHP2 wild-type allosteric inhibitors. Therefore, developing novel therapeutic modality that effectively inhibits pathogenic SHP2 mutants has become an urgent need for the treatment of SHP2 related diseases. Herein, we report the design, synthesis and biological evaluation of novel SHP2 small molecule degraders based on PROTACs (proteolysis-targeting chimeras) technology by employing an allosteric inhibitor 2 targeting SHP2-activated mutant as the warhead. These SHP2^E76A PROTACs have shown strong inhibitory activities in both enzyme and cell proliferation. Notably, the lead compounds 3f and 4d exhibit potent antiproliferative activities against both the wild-type SHP2 dependent human esophageal squamous carcinoma cell line KYSE-520 and the mutant SHP2^N58S human large-cell lung carcinoma cell line NCI-H661, with an improvement by 5 to 10-fold compared to the positive control 2. This study provides a new therapeutic intervention strategy for treating developmental disorders and tumors caused by SHP2 mutation or activation.

Key words: protein tyrosine phosphatase, SHP2, PROTAC, small molecule degraders, gain of function, SHP2^E76A mutein

引用此文

孔娇, 杜琳, 李向阳, 朱继东, 龙亚秋. 靶向SHP2^E76A突变体的小分子降解剂有效抑制野生型和突变体SHP2肿瘤细胞增殖[J]. 化学学报, 2023, 81(9): 1120-1128.

Jiao Kong, Lin Du, Xiangyang Li, Jidong Zhu, Ya-Qiu Long. Small Molecule Degraders Targeting the SHP2^E76A Mutant Effectively Inhibiting the Proliferation of Wild-type and Mutant SHP2 Dependent Tumor Cells[J]. Acta Chimica Sinica, 2023, 81(9): 1120-1128.

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图/表 7

图1. 代表性的SHP2变构抑制剂和PROTAC降解剂

Figure 1. Representative SHP2 allosteric inhibitors and SHP2 PROTAC degraders

图2. 基于分子对接建立的小分子变构抑制剂2与SHP2E76A蛋白的结合模式(PDB code: 5XZR[15], 绿色结构代表靶头化合物2), 及其设计的两类基于两种E3泛素连接酶配体的SHP2E76A PROTACs分子

Figure 2. The predicted binding mode of small molecule allosteric inhibitor 2 with SHP2 protein by autodocking (based on the cocrystal structure PDB code: 5XZR[15], the green structure represents the warhead 2), and the design of two classes of SHP2E76A PROTACs based on the two types of E3 ligase ligands

图3. SHP2 PROTAC分子的合成路线

Figure 3. The synthetic routes toward SHP2 PROTAC molecules

Compound	SHP2^E76AIC₅₀/(μmol•L^-1)	KYSE 520 IC₅₀^b/(μmol•L^-1)	NCI-H661 IC₅₀^b/(μmol•L^-1)
3a	3.12	3.14±0.27	>50
3b	0.91	2.33±0.15	21.43±1.81
3c	3.95	1.54±0.09	5.49±0.34
3d	4.15	2.54±0.12	6.58±1.32
3e	14.39	1.09±0.11	1.55±0.12
3f	1.67	0.66±0.07	1.16±0.15
2 (变构抑制剂)	0.71	6.23±0.45	3.38±0.11
1 (SHP099)	0.31	3.40±0.08	9.61±0.35

表1. 基于CRBN配体的SHP2 PROTAC分子的酶抑制活性和肿瘤细胞增殖抑制活性a

Table 1. SHP2 enzyme inhibitory and tumor cell proliferation inhibitory activities of SHP2 PROTAC molecules based on CRBN ligand a

图4. (a) 化合物3f对SHP2E76A酶抑制活性. 化合物3f与阳性对照化合物2分别处理(b) KYSE-520细胞和(c) NCI-H661细胞的细胞生长抑制活性. 化合物在不同浓度下处理细胞4 d, 并通过CCK-8方法测定. (d) 化合物3f在不同浓度下分别处理NCI-H661细胞12 h和24 h后, 用Western blotting分析细胞中SHP2蛋白水平

Figure 4. (a) The SHP2E76A enzyme inhibitory activity by compound 3f. Cell growth inhibitory activities by compound 3f and the positive control SHP2E76A inhibitor 2 against (b) KYSE-520 cells and (c) NCI-H661 cells, respectively. The compound was treated with cells at different concentrations for 4 d and measured using the CCK-8 method. (d) NCI-H661 cells was treated with compound 3f at different concentrations for 12 and 24 h, and the levels of SHP2 protein in the cells were analyzed using Western blotting

Cpd	R	SHP2^E76A IC₅₀/(μmol•L^-1)	KYSE520 IC₅₀^b/(μmol•L^-1)	NCI-H661 IC₅₀^b/(μmol•L^-1)
4a	H	28.1	10.19±1.15	13.21±1.45
4b	H	18.18	5.25±0.34	6.50±0.25
4c	H	4.06	1.50±0.17	6.54±0.18
4d	Me	1.47	0.78±0.05	0.82±0.09
4e	Me	NT^c	0.80±0.10	1.16±0.16
4f	Me	NT^c	1.55±0.07	3.09±0.23
4g	H	NT^c	4.94±0.25	6.89±0.22
4h	Me	NT^c	1.61±0.15	3.22±0.11
4i	Me	NT^c	1.29±0.09	2.12±0.09
4j	Me	NT^c	1.38±0.05	3.39±0.12
2 (变构抑制剂)		0.71	6.23±0.45	3.38±0.11
1 (SHP099)		0.31	3.40±0.08	9.61±0.35

表2. 基于VHL配体的SHP2 PROTAC分子的酶抑制活性和肿瘤细胞增殖抑制活性a

Table 2. Enzyme inhibitory and tumor cell proliferation inhibitory activities of SHP2 PROTAC molecules based on VHL ligand a

图5. 化合物4d与阳性对照化合物2分别处理(a) KYSE-520细胞和(b) NCI-H661细胞的生长抑制活性. 化合物在不同浓度下处理细胞4 d, 并通过CCK-8方法测定. 化合物4d在不同浓度下分别处理(c) KYSE-520细胞和(d) NCI-H661细胞24 h和48 h后, 用Western blotting分析细胞中SHP2蛋白水平, 使用ImageJ软件的灰色值分析对蛋白质水平进行定量

Figure 5. Compound 4d and positive control compound 2 showed growth inhibitory activity in (a) KYSE-520 cells and (b) NCI-H661 cells, respectively. The cells was treated with compounds at different concentrations for 4 d and measured using the CCK-8 method. (c) KYSE-520 cells and (d) NCI-H661 cells were treated with compound 4d at different concentrations for 24 and 48 h respectively, and the levels of SHP2 protein in the cells were analyzed using Western blotting. Protein levels were quantified using grey value analyses by ImageJ software

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