Small Molecule Degraders Targeting the SHP2E76A Mutant Effectively Inhibiting the Proliferation of Wild-type and Mutant SHP2 Dependent Tumor Cells

doi:10.6023/A23050229

Abstract

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

Cite this article

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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Fig. & Tab. 7

Figure 1. Representative SHP2 allosteric inhibitors and SHP2 PROTAC degraders

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

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

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

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

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

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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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