Discovery of a SHP2 Degrader with In Vivo Anti-Tumor Activity

Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we designed and synthesized a novel series of proteolysis targeting chimeras (PROTACs) using a SHP2 allosteric inhibitor as warhead, with the goal of achieving SHP2 degradation both inside the cell and in vivo. Among these molecules, compound P9 induces efficient degradation of SHP2 (DC50 = 35.2 ± 1.5 nM) in a concentration- and time-dependent manner. Mechanistic investigation illustrates that the P9-mediated SHP2 degradation requires the recruitment of the E3 ligase and is ubiquitination- and proteasome-dependent. P9 shows improved anti-tumor activity in a number of cancer cell lines over its parent allosteric inhibitor. Importantly, administration of P9 leads to a nearly complete tumor regression in a xenograft mouse model, as a result of robust SHP2 depletion and suppression of phospho-ERK1/2 in the tumor. Hence, P9 represents the first SHP2 PROTAC molecule with excellent in vivo efficacy. It is anticipated that P9 could serve not only as a new chemical tool to interrogate SHP2 biology but also as a starting point for the development of novel therapeutics targeting SHP2.


Introduction
Protein tyrosine phosphatases (PTP) cooperate with protein tyrosine kinases (PTK) in regulating the level of protein tyrosine phosphorylation, which is crucial in cell signal transduction.Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by PTPN11, is a member of the PTP family [1] that regulates cellular proliferation, survival, migration, and differentiation by participating in numerous cell-signaling cascades, such as RAS-ERK1/2, PI3K-AKT and JAK-STAT pathways [2][3][4].Germline mutations in PTPN11 cause Noonan [5] and LEOPARD [6] syndromes, which have overlapping clinical features.Autosomal dominant activating mutations in PTPN11 fuel excess RAS/ERK1/2 signaling that drives certain human RASopathies and cancers [7].Somatic mutations of PTPN11 have been found in patients with myelodysplastic syndrome (10%), juvenile acute myeloid leukemia (AML) (5%), and B-cell acute lymphoblastic leukemia (7%) [8].PTPN11 mutations also occur in sporadic solid tumors including lung cancer, colon cancer, neuroblastoma, and melanoma [9].Moreover, accumulating evidence suggests that SHP2 may play an important role in immune evasion and in the T-cell programmed cell death/checkpoint pathway (PD1/PD-L1) [10,11].Regarded as an appealing target for human cancer therapies, significant endeavors have been dedicated to the development of SHP2 inhibitors [12][13][14].In 2016, Novartis reported the first potent, selective, and orally bioavailable SHP2 inhibitor SHP099, which targets an allosteric binding site in SHP2 and stabilizes the inactive conformation of the enzyme [15,16].Subsequently, several allosteric SHP2 inhibitors with superior pharmaceutical properties were developed and progressed to clinical trials for treating advanced or metastatic solid tumors [17].
Despite the clinical promise of the allosteric SHP2 inhibitors, recent studies have drawn attention to the possibility of non-mutational drug resistance mechanisms [18].Through the acute depletion of the target protein, proteolysis targeting chimeras (PRO-TACs) may overcome such resistance and provide an alternative and potentially more compelling approach to inhibiting SHP2.Over the past few years, the PROTAC approach has emerged as a powerful post-translational chemical technique targeting selective protein degradation [19].A typical PROTAC molecule consists of a ligand to the protein of interest, a ligand to an E3 ligase, and a linker between the two.PROTACs induce the proximity of target proteins with E3 ubiquitin ligases to form interactions between the two, leading to ubiquitination and subsequent degradation through the proteasome system.Compared with classical small molecule inhibitors, PROTAC degraders usually feature lower doses and higher selectivity and can often overcome mutation-caused drug resistance.With regard to SHP2, Ruess et al. have revealed that knockout of the PTPN11 gene in KRAS mutant human ductal adenocarcinoma (PDAC) cells results in a reduction in cell proliferation and PTPN11-knockout cells are uniquely susceptible to mitogen-activated protein kinase (MEK) inhibitors [20].Thus, small molecule-induced degradation of SHP2 could be an effective therapeutic strategy for human cancers, particularly those carrying a KRAS-mutation.Moreover, phosphatase-independent functions of SHP2 can also be explored and targeted using the degraders as unique tools [21].
Several PROTACs that leverage the SHP2 allosteric inhibitors as ligands were identified for SHP2 degradation in cells (Figure 1) [22].In 2020, Wang and coworkers reported the first SHP2 PROTAC D26, which recruits Von Hippel-Lindau (VHL) as the E3 ligase [23].Shortly thereafter, several Cereblon (CRBN)-based SHP2 PROTACs including ZB-S-29 [24], SP4 [25], and R1-5C [26] were also reported.Despite having promising activities in cell culture, most of the current SHP2 PROTACs did not exhibit in vivo efficacies, although D26 did show modest anticancer activities (<20% tumor growth inhibition) as a single agent in a xenograft mice model [27].This lack of in vivo activities limits the application of the SHP2 degraders.Herein, we describe our efforts in the discovery of a novel potent SHP2 degrader that features high efficacy in blocking tumor growth at the cellular level and in vivo in a xenograft mouse model.
human cancer therapies, significant endeavors have been dedicated to the development of SHP2 inhibitors [12][13][14].In 2016, Novartis reported the first potent, selective, and orally bioavailable SHP2 inhibitor SHP099, which targets an allosteric binding site in SHP2 and stabilizes the inactive conformation of the enzyme [15,16].Subsequently, several allosteric SHP2 inhibitors with superior pharmaceutical properties were developed and progressed to clinical trials for treating advanced or metastatic solid tumors [17].
Despite the clinical promise of the allosteric SHP2 inhibitors, recent studies have drawn attention to the possibility of non-mutational drug resistance mechanisms [18].Through the acute depletion of the target protein, proteolysis targeting chimeras (PROTACs) may overcome such resistance and provide an alternative and potentially more compelling approach to inhibiting SHP2.Over the past few years, the PROTAC approach has emerged as a powerful post-translational chemical technique targeting selective protein degradation [19].A typical PROTAC molecule consists of a ligand to the protein of interest, a ligand to an E3 ligase, and a linker between the two.PROTACs induce the proximity of target proteins with E3 ubiquitin ligases to form interactions between the two, leading to ubiquitination and subsequent degradation through the proteasome system.Compared with classical small molecule inhibitors, PROTAC degraders usually feature lower doses and higher selectivity and can often overcome mutation-caused drug resistance.With regard to SHP2, Ruess et al. have revealed that knockout of the PTPN11 gene in KRAS mutant human ductal adenocarcinoma (PDAC) cells results in a reduction in cell proliferation and PTPN11-knockout cells are uniquely susceptible to mitogen-activated protein kinase (MEK) inhibitors [20].Thus, small molecule-induced degradation of SHP2 could be an effective therapeutic strategy for human cancers, particularly those carrying a KRAS-mutation.Moreover, phosphatase-independent functions of SHP2 can also be explored and targeted using the degraders as unique tools [21].
Several PROTACs that leverage the SHP2 allosteric inhibitors as ligands were identified for SHP2 degradation in cells (Figure 1) [22].In 2020, Wang and coworkers reported the first SHP2 PROTAC D26, which recruits Von Hippel-Lindau (VHL) as the E3 ligase [23].Shortly thereafter, several Cereblon (CRBN)-based SHP2 PROTACs including ZB-S-29 [24], SP4 [25], and R1-5C [26] were also reported.Despite having promising activities in cell culture, most of the current SHP2 PROTACs did not exhibit in vivo efficacies, although D26 did show modest anticancer activities (<20% tumor growth inhibition) as a single agent in a xenograft mice model [27].This lack of in vivo activities limits the application of the SHP2 degraders.Herein, we describe our efforts in the discovery of a novel potent SHP2 degrader that features high efficacy in blocking tumor growth at the cellular level and in vivo in a xenograft mouse model.

Design of SHP2 PROTACs
The initial considerations for the rational design of PROTAC molecules include the target protein recruiting elements and the tethering site for anchoring the linkers.The co-crystal structure of SHP2 complexed with compound 1 (Figure 2A, PDB code 7JVN), a close analog of SHP099, showed that the terminal methyl group on the piperidine ring is solvent exposed and therefore is suitable for linker attachment (Figure 2B,C) [28].Furthermore, another analogous allosteric inhibitor compound 2, which has a longer alkyl group chain in the place of methyl group in 1, was reported to be a potent SHP2 inhibitor (IC 50 = 17 nM) [29].Based on these observations, we reasoned that we could tether the linker on the terminal ethyl group on the piperidine ring in 1 without diminishing the binding affinity to SHP2.In consideration of the synthetic complexity, we chose a potent and highly cell-active SHP2 inhibitor compound 3 (SHP2 IC 50 = 17 nM, pERK1/2 IC 50 = 88 nM) [28] as the starting point for a ligand for SHP2 protein recruitment in this study (Figure 2A).To test this hypothesis, we designed and synthesized compound 4 which contains hexanoic acid serving as an anchor in the place of methyl group in 1 (Figure 2D).The synthesis started with the alkylation of the 1-benzylpiperidine-4-carbonitrile ( 5) with the 6-bromohexanoic acid, followed by the protection of the carboxylic acid to give the ester 6.The reduction in this intermediate 6 with cobalt chloride and sodium borohydride and the subsequent Boc protection afforded the intermediate 7. Removal of the piperidyl benzyl group followed by the nucleophilic substitution to a chloro-pyrazine gave the intermediate 9, which was then converted to compound 4 by the deprotection of the carboxylic acid (intermediate 10) and the amine.As expected, compound 4 displayed a low IC 50 (90 nM) against full-length SHP2 in the in vitro enzymatic assay, suggesting that it is a suitable ligand for constructing SHP2 PROTAC molecules.

Design of SHP2 PROTACs
The initial considerations for the rational design of PROTAC molecules include the target protein recruiting elements and the tethering site for anchoring the linkers.The cocrystal structure of SHP2 complexed with compound 1 (Figure 2A, PDB code 7JVN), a close analog of SHP099, showed that the terminal methyl group on the piperidine ring is solvent exposed and therefore is suitable for linker attachment (Figure 2B,C) [28].Furthermore, another analogous allosteric inhibitor compound 2, which has a longer alkyl group chain in the place of methyl group in 1, was reported to be a potent SHP2 inhibitor (IC50 = 17 nM) [29].Based on these observations, we reasoned that we could tether the linker on the terminal ethyl group on the piperidine ring in 1 without diminishing the binding affinity to SHP2.In consideration of the synthetic complexity, we chose a potent and highly cell-active SHP2 inhibitor compound 3 (SHP2 IC50 = 17 nM, pERK1/2 IC50 = 88 nM) [28] as the starting point for a ligand for SHP2 protein recruitment in this study (Figure 2A).To test this hypothesis, we designed and synthesized compound 4 which contains hexanoic acid serving as an anchor in the place of methyl group in 1 (Figure 2D).The synthesis started with the alkylation of the 1-benzylpiperidine-4-carbonitrile ( 5) with the 6-bromohexanoic acid, followed by the protection of the carboxylic acid to give the ester 6.The reduction in this intermediate 6 with cobalt chloride and sodium borohydride and the subsequent Boc protection afforded the intermediate 7. Removal of the piperidyl benzyl group followed by the nucleophilic substitution to a chloro-pyrazine gave the intermediate 9, which was then converted to compound 4 by the deprotection of the carboxylic acid (intermediate 10) and the amine.As expected, compound 4 displayed a low IC50 (90 nM) against full-length SHP2 in the in vitro enzymatic assay, suggesting that it is a suitable ligand for constructing SHP2 PROTAC molecules.

Exploration of E3 Ligands and Linkers
With the desired SHP2 ligand in hand, we synthesized an initial set of PROTAC molecules using pomalidomide or lenalidomide as a ligand of the E3 ligase Cereblon (CRBN), which is one of the most productive E3 in the development of PROTACs [30].However, the synthesized PROTAC compounds did not induce significant degradation of SHP2 in cells.Further investigation of these PROTAC candidates revealed that they are steadily hydrolyzed and oxidized in cell mediums or in the presence of water at room temperature (Scheme S1).LCMS analysis showed that over 90% of the compounds (11 and 12) were converted into ring-opened products 13 within 3 h in the Dulbecco's Modified Eagle Medium (DMEM) cell media or in water at room temperature (Scheme S1).This observation is consistent with the fact that phthalimides can be smoothly hydrolyzed in the presence of water and an organic base [31], as these PROTAC compounds contain a basic aliphatic amino group.Given that this free amino group in the allosteric inhibitors is essential for the binding affinity to SHP2 [28], we reasoned that pomalidomide or lenalidomide-based CRBN ligands are not suitable for the development of in vivo efficacious SHP2 degraders in this study due to stability considerations.
We then turned to VHL, another E3 ligase that is commonly employed in PROTAC development [32].Since the linker length and compositions both play important roles in PROTAC design and have an essential influence on the degradation potency [33], we synthesized and evaluated an initial set of PROTACs with varied linear alkyl chains and polyethylene glycols using VHL-2 (colored in red) as the VHL ligand [34] to reveal the favorable linker length (Table 1).The biochemical enzymatic assay showed that these PROTAC candidates inhibited SHP2 with IC 50 values of 100~200 nM, which is similar to that of compound 4 (the SHP2 ligand used for the PROTAC construction with an IC 50 of 90 nM), indicating that installation of the linkers to compound 4 does not lead to loss of SHP2 binding (Table S1).Subsequently, these PROTAC candidates were surveyed by Western blotting in the HEK293 human embryonic kidney cell line for their ability to induce degradation of SHP2 protein at 1 and 10 µM (Table 1 and Figure S1).To our delight, compound SC11, which consists of a n-undecane linker between compound 4 and the VHL ligand (12-atom long), induced 27% and 64% degradation of SHP2 at 1 µM and 10 µM, respectively.Other compounds with different alkyl chain linkers (SC5, SC7, SC9) showed negligible to modest degradation at these concentrations, suggesting that SC11 has the most favored linker in this series.Nevertheless, compounds with polyethylene glycol linkers (P3, P4, P5) did not induce significant degradation of SHP2, although the linker in P3 has the same atom numbers as that of SC11.
To achieve higher efficiency of SHP2 degradation, we further optimized the length and chemical composition of the linkers by synthesizing and evaluating the second set of PROTACs with a total linker length similar to that in SC11 (Table 2 and Table S2).First, we synthesized SC10 with a n-decane chain to make the linker length 1 atom shorter than that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradation at 1 µM, suggesting that the 11-atom long carbon chain might be preferred to the 12-atom.With the aim of modulating lipophilicity, solubility, and rigidity and therefore improving the permeability of the PROTACs, we restrained the conformation of the linkers by introducing a positively charged piperazinyl group, yielding compounds P7 P8, P9, and P10.Western blotting data revealed that both P7 and P8 induce a higher degree of SHP2 degradation compared to their counter PROATCs with alkyl chain linkers (SC10 and SC11), while P9 achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 with a linker length of 14-atom appeared to be less effective than P9, indicating that a 13-atom length is optimal under the circumstances.Overall, the potency of P9 is significantly higher than any other PROTACs we obtained, making it the lead compound in our study.SC11, which consists of a n-undecane linker between compound 4 and the VHL (12-atom long), induced 27% and 64% degradation of SHP2 at 1 µM and 10 µM, r tively.Other compounds with different alkyl chain linkers (SC5, SC7, SC9) showe ligible to modest degradation at these concentrations, suggesting that SC11 has th favored linker in this series.Nevertheless, compounds with polyethylene glycol l (P3, P4, P5) did not induce significant degradation of SHP2, although the linker in the same atom numbers as that of SC11.tion of SHP2 protein at 1 and 10 µM (Table 1 and Figure S1).To our SC11, which consists of a n-undecane linker between compound 4 a (12-atom long), induced 27% and 64% degradation of SHP2 at 1 µM tively.Other compounds with different alkyl chain linkers (SC5, SC7 ligible to modest degradation at these concentrations, suggesting tha favored linker in this series.Nevertheless, compounds with polyeth (P3, P4, P5) did not induce significant degradation of SHP2, although the same atom numbers as that of SC11.tion of SHP2 protein at 1 and 10 µM (Table 1 and Figure S1).To our SC11, which consists of a n-undecane linker between compound 4 a (12-atom long), induced 27% and 64% degradation of SHP2 at 1 µM tively.Other compounds with different alkyl chain linkers (SC5, SC7 ligible to modest degradation at these concentrations, suggesting tha favored linker in this series.Nevertheless, compounds with polyeth (P3, P4, P5) did not induce significant degradation of SHP2, although the same atom numbers as that of SC11.To achieve higher efficiency of SHP2 degradation, we further o and chemical composition of the linkers by synthesizing and evaluati PROTACs with a total linker length similar to that in SC11 (Tables synthesized SC10 with a n-decane chain to make the linker length that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradati ing that the 11-atom long carbon chain might be preferred to the 12-at modulating lipophilicity, solubility, and rigidity and therefore impro ity of the PROTACs, we restrained the conformation of the linkers by tively charged piperazinyl group, yielding compounds P7 P8, P9, and ting data revealed that both P7 and P8 induce a higher degree of SHP pared to their counter PROATCs with alkyl chain linkers (SC10 an achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 of 14-atom appeared to be less effective than P9, indicating that a 13mal under the circumstances.Overall, the potency of P9 is significan other PROTACs we obtained, making it the lead compound in our stu To achieve higher efficiency of SHP2 degradation, we further o and chemical composition of the linkers by synthesizing and evaluati PROTACs with a total linker length similar to that in SC11 (Tables synthesized SC10 with a n-decane chain to make the linker length that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradati ing that the 11-atom long carbon chain might be preferred to the 12-at modulating lipophilicity, solubility, and rigidity and therefore impro ity of the PROTACs, we restrained the conformation of the linkers by tively charged piperazinyl group, yielding compounds P7 P8, P9, an ting data revealed that both P7 and P8 induce a higher degree of SHP pared to their counter PROATCs with alkyl chain linkers (SC10 a achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 of 14-atom appeared to be less effective than P9, indicating that a 13mal under the circumstances.Overall, the potency of P9 is significan other PROTACs we obtained, making it the lead compound in our stu To achieve higher efficiency of SHP2 degradation, we further o and chemical composition of the linkers by synthesizing and evaluati PROTACs with a total linker length similar to that in SC11 (Tables synthesized SC10 with a n-decane chain to make the linker length 1 that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradati ing that the 11-atom long carbon chain might be preferred to the 12-at modulating lipophilicity, solubility, and rigidity and therefore impro ity of the PROTACs, we restrained the conformation of the linkers by tively charged piperazinyl group, yielding compounds P7 P8, P9, and ting data revealed that both P7 and P8 induce a higher degree of SHP pared to their counter PROATCs with alkyl chain linkers (SC10 an achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 of 14-atom appeared to be less effective than P9, indicating that a 13mal under the circumstances.Overall, the potency of P9 is significan other PROTACs we obtained, making it the lead compound in our stu To achieve higher efficiency of SHP2 degradation, we further o and chemical composition of the linkers by synthesizing and evaluati PROTACs with a total linker length similar to that in SC11 (Tables synthesized SC10 with a n-decane chain to make the linker length 1 that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradati ing that the 11-atom long carbon chain might be preferred to the 12-at modulating lipophilicity, solubility, and rigidity and therefore impro ity of the PROTACs, we restrained the conformation of the linkers by tively charged piperazinyl group, yielding compounds P7 P8, P9, and ting data revealed that both P7 and P8 induce a higher degree of SHP pared to their counter PROATCs with alkyl chain linkers (SC10 an achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 of 14-atom appeared to be less effective than P9, indicating that a 13mal under the circumstances.Overall, the potency of P9 is significan other PROTACs we obtained, making it the lead compound in our stu To achieve higher efficiency of SHP2 degradation, we further o and chemical composition of the linkers by synthesizing and evaluati PROTACs with a total linker length similar to that in SC11 (Tables synthesized SC10 with a n-decane chain to make the linker length 1 that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradati ing that the 11-atom long carbon chain might be preferred to the 12-at modulating lipophilicity, solubility, and rigidity and therefore impro ity of the PROTACs, we restrained the conformation of the linkers by tively charged piperazinyl group, yielding compounds P7 P8, P9, and ting data revealed that both P7 and P8 induce a higher degree of SHP pared to their counter PROATCs with alkyl chain linkers (SC10 an achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 of 14-atom appeared to be less effective than P9, indicating that a 13mal under the circumstances.Overall, the potency of P9 is significan other PROTACs we obtained, making it the lead compound in our stu  To achieve higher efficiency of SHP2 degradation, we further optimized and chemical composition of the linkers by synthesizing and evaluating the s PROTACs with a total linker length similar to that in SC11 (Tables 2 and S synthesized SC10 with a n-decane chain to make the linker length 1 atom s that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradation at 1 µ ing that the 11-atom long carbon chain might be preferred to the 12-atom.Wit modulating lipophilicity, solubility, and rigidity and therefore improving the ity of the PROTACs, we restrained the conformation of the linkers by introdu tively charged piperazinyl group, yielding compounds P7 P8, P9, and P10.W ting data revealed that both P7 and P8 induce a higher degree of SHP2 degra pared to their counter PROATCs with alkyl chain linkers (SC10 and SC11 achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 with a li of 14-atom appeared to be less effective than P9, indicating that a 13-atom len mal under the circumstances.Overall, the potency of P9 is significantly high other PROTACs we obtained, making it the lead compound in our study.To achieve higher efficiency of SHP2 degradation, we further optimized and chemical composition of the linkers by synthesizing and evaluating the s PROTACs with a total linker length similar to that in SC11 (Tables 2 and S synthesized SC10 with a n-decane chain to make the linker length 1 atom s that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradation at 1 µ ing that the 11-atom long carbon chain might be preferred to the 12-atom.Wit modulating lipophilicity, solubility, and rigidity and therefore improving the ity of the PROTACs, we restrained the conformation of the linkers by introdu tively charged piperazinyl group, yielding compounds P7 P8, P9, and P10.W ting data revealed that both P7 and P8 induce a higher degree of SHP2 degra pared to their counter PROATCs with alkyl chain linkers (SC10 and SC11 achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 with a l of 14-atom appeared to be less effective than P9, indicating that a 13-atom len mal under the circumstances.Overall, the potency of P9 is significantly high other PROTACs we obtained, making it the lead compound in our study.To achieve higher efficiency of SHP2 degradation, we further optimized and chemical composition of the linkers by synthesizing and evaluating the s PROTACs with a total linker length similar to that in SC11 (Tables 2 and S2 synthesized SC10 with a n-decane chain to make the linker length 1 atom s that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradation at 1 µ ing that the 11-atom long carbon chain might be preferred to the 12-atom.Wit modulating lipophilicity, solubility, and rigidity and therefore improving the ity of the PROTACs, we restrained the conformation of the linkers by introdu tively charged piperazinyl group, yielding compounds P7 P8, P9, and P10.W ting data revealed that both P7 and P8 induce a higher degree of SHP2 degrad pared to their counter PROATCs with alkyl chain linkers (SC10 and SC11 achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 with a li of 14-atom appeared to be less effective than P9, indicating that a 13-atom len mal under the circumstances.Overall, the potency of P9 is significantly high other PROTACs we obtained, making it the lead compound in our study.To achieve higher efficiency of SHP2 degradation, we further optimized and chemical composition of the linkers by synthesizing and evaluating the s PROTACs with a total linker length similar to that in SC11 (Tables 2 and S2 synthesized SC10 with a n-decane chain to make the linker length 1 atom s that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradation at 1 µ ing that the 11-atom long carbon chain might be preferred to the 12-atom.Wit modulating lipophilicity, solubility, and rigidity and therefore improving the ity of the PROTACs, we restrained the conformation of the linkers by introdu tively charged piperazinyl group, yielding compounds P7 P8, P9, and P10.W ting data revealed that both P7 and P8 induce a higher degree of SHP2 degrad pared to their counter PROATCs with alkyl chain linkers (SC10 and SC11 achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 with a li of 14-atom appeared to be less effective than P9, indicating that a 13-atom len mal under the circumstances.Overall, the potency of P9 is significantly high other PROTACs we obtained, making it the lead compound in our study.To achieve higher efficiency of SHP2 degradation, we further optimized and chemical composition of the linkers by synthesizing and evaluating the s PROTACs with a total linker length similar to that in SC11 (Tables 2 and S synthesized SC10 with a n-decane chain to make the linker length 1 atom s that of SC11.After 16 h treatment, SC10 induced 36% SHP2 degradation at 1 µ ing that the 11-atom long carbon chain might be preferred to the 12-atom.Wit modulating lipophilicity, solubility, and rigidity and therefore improving the ity of the PROTACs, we restrained the conformation of the linkers by introdu tively charged piperazinyl group, yielding compounds P7 P8, P9, and P10.W ting data revealed that both P7 and P8 induce a higher degree of SHP2 degrad pared to their counter PROATCs with alkyl chain linkers (SC10 and SC11 achieved nearly complete depletion of SHP2 at 1 µM.Compound P10 with a li of 14-atom appeared to be less effective than P9, indicating that a 13-atom len mal under the circumstances.Overall, the potency of P9 is significantly high other PROTACs we obtained, making it the lead compound in our study.

Dose-and Time-Dependency Study of P9
To further evaluate the ability of P9 to induce SHP2 degradation, Western blotting was used to analyze the levels of SHP2 in HEK293 cells incubated with different concentrations of the degrader (Figure 3A).The results clearly illustrated that SHP2 protein levels were reduced in a dose-dependent manner, and quantification of the data gave the DC 50 (concentration required for 50% degradation of protein) of 35.2 ± 1.5 nM, and no significant hook effect was observed in the tested concentration range.To determine the kinetics of P9-mediated degradation of SHP2 in cells, we assessed the abundance of SHP2 protein at a series of time points after the addition of the compound (Figure 3B).Over 90% of SHP2 protein was degraded within 6 h of P9 treatment, and the maximal SHP2 depletion was achieved in 16 h.
To further evaluate the ability of P9 to induce SHP2 degradation, Western blotting was used to analyze the levels of SHP2 in HEK293 cells incubated with different concentrations of the degrader (Figure 3A).The results clearly illustrated that SHP2 protein levels were reduced in a dose-dependent manner, and quantification of the data gave the DC50 (concentration required for 50% degradation of protein) of 35.2 ± 1.5 nM, and no significant hook effect was observed in the tested concentration range.To determine the kinetics of P9-mediated degradation of SHP2 in cells, we assessed the abundance of SHP2 protein at a series of time points after the addition of the compound (Figure 3B).Over 90% of SHP2 protein was degraded within 6 h of P9 treatment, and the maximal SHP2 depletion was achieved in 16 h.

Selectivity Evaluation and Mechanism of Action Study of P9
To investigate the binding affinity of P9 to SHP2 and selectivity towards other PTPs, we determined its enzymatic IC50s against a panel of 15 representative PTP family members, including receptor-like, nonreceptor-like, and dual-specific PTPs (Table S3).The results indicated that P9 showed an IC50 of 95 nM for SHP2 and no significant inhibition towards other PTPs even at up to 10 µM P9 concentration.Next, the selectivity of P9 as a degrader for SHP2 over other PTP family members and different classes of enzymes was investigated (Figure 3C).Western blots showed that treatment of HEK293 cells with 1 µM P9 for 16 h led to almost complete degradation of SHP2, whereas none of the other PTPs including SHP1, PTP1B, TC-PTP, LYP, PRL1, and PRL2, were affected.In addition, under the same conditions, P9 did not induce any appreciable degradation of other signaling proteins including ERK1/2, AKT, and β-Actin.Taken together, the above results validated P9 as a selective SHP2 degrader.With the aim to verify the mechanism of action for P9, a series of control experiments were performed in cells (Figure 3D).Western blotting analysis demonstrated the addition of an excess amount of either the SHP2 inhibitor compound 3 or VHL ligand VHL-2 prohibited P9-induced SHP2 degradation, indicating that simultaneous binding of the degrader to both SHP2 and VHL ligase and therefore, the

Selectivity Evaluation and Mechanism of Action Study of P9
To investigate the binding affinity of P9 to SHP2 and selectivity towards other PTPs, we determined its enzymatic IC 50 s against a panel of 15 representative PTP family members, including receptor-like, nonreceptor-like, and dual-specific PTPs (Table S3).The results indicated that P9 showed an IC 50 of 95 nM for SHP2 and no significant inhibition towards other PTPs even at up to 10 µM P9 concentration.Next, the selectivity of P9 as a degrader for SHP2 over other PTP family members and different classes of enzymes was investigated (Figure 3C).Western blots showed that treatment of HEK293 cells with 1 µM P9 for 16 h led to almost complete degradation of SHP2, whereas none of the other PTPs including SHP1, PTP1B, TC-PTP, LYP, PRL1, and PRL2, were affected.In addition, under the same conditions, P9 did not induce any appreciable degradation of other signaling proteins including ERK1/2, AKT, and β-Actin.Taken together, the above results validated P9 as a selective SHP2 degrader.With the aim to verify the mechanism of action for P9, a series of control experiments were performed in cells (Figure 3D).Western blotting analysis demonstrated the addition of an excess amount of either the SHP2 inhibitor compound 3 or VHL ligand VHL-2 prohibited P9-induced SHP2 degradation, indicating that simultaneous binding of the degrader to both SHP2 and VHL ligase and therefore, the formation of a ternary complex was required for the SHP2 protein degradation.Furthermore, the ubiquitination-and proteasome-dependency of the degradation was assessed by the addition of the NEDD8 E1 ubiquitin-activating enzyme inhibitor MLN-4924 and the proteasome inhibitor MG-132 to the assay conditions.Pretreatment with either MLN-4924 or MG-132 markedly reduced the extent of SHP2 degradation, demonstrating that the E1 and 26S proteasomes were indeed implicated in the degradation.In addition, the CRBN ligand lenalidomide did not affect the SHP2 degradation induced by P9.Collectively, these observations illustrate that P9 is a genuine SHP2 PROTAC degrader.

Tumor Cell Growth Inhibition Study
SHP2 has been implicated as a major positive regulator of RTK/RAS/ERK1/2 signaling, which is aberrantly activated in a variety of human cancers [35].SHP2 inhibitors were found to suppress the phosphorylation level of ERK1/2 and tumor growth in multiple cancer cell lines including EGFR, PI3K, and KRAS mutant cancers [17].To determine the effect of our SHP2 degrader in a cellular context, we first evaluated the cellular effects of P9 in squamous cell carcinoma KYSE-520 (EGFR amplified) cells [16].Western blotting analysis showed that the SHP2 and pERK1/2 levels in KYSE-520 cells were reduced by P9 in a dose-dependent manner, with a SHP2 degradation DC 50 of ~130 nM and pERK1/2 inhibition EC 50 of ~240 nM (Figure 4A).Notably, the protein levels of PTP1B and SHP1, a close homologue of SHP2, were not affected by P9, which further demonstrates the selectivity of the degrader over other closely related PTP family members.Consistently, cell proliferation assay indicated that P9 restrains the growth of KYSE-520 with the IC 50 of 0.64 ± 0.13 µM, suggesting that the PROTAC attenuates the tumor growth by degrading SHP2 and therefore inhibiting the RAS/ERK1/2 signaling pathway (Figure 4B).
s 2023, 28, x FOR PEER REVIEW 7 4924 or MG-132 markedly reduced the extent of SHP2 degradation, demonstrating the E1 and 26S proteasomes were indeed implicated in the degradation.In addition CRBN ligand lenalidomide did not affect the SHP2 degradation induced by P9.Co tively, these observations illustrate that P9 is a genuine SHP2 PROTAC degrader.

Tumor Cell Growth Inhibition Study
SHP2 has been implicated as a major positive regulator of RTK/RAS/ERK1/2 sig ing, which is aberrantly activated in a variety of human cancers [35].SHP2 inhibitors w found to suppress the phosphorylation level of ERK1/2 and tumor growth in mult cancer cell lines including EGFR, PI3K, and KRAS mutant cancers [17].To determine effect of our SHP2 degrader in a cellular context, we first evaluated the cellular effec P9 in squamous cell carcinoma KYSE-520 (EGFR amplified) cells [16].Western blott analysis showed that the SHP2 and pERK1/2 levels in KYSE-520 cells were reduced b in a dose-dependent manner, with a SHP2 degradation DC50 of ~130 nM and pERK inhibition EC50 of ~240 nM (Figure 4A).Notably, the protein levels of PTP1B and SHP close homologue of SHP2, were not affected by P9, which further demonstrates the se tivity of the degrader over other closely related PTP family members.Consistently, proliferation assay indicated that P9 restrains the growth of KYSE-520 with the IC50 of ± 0.13 µM, suggesting that the PROTAC attenuates the tumor growth by degrading S and therefore inhibiting the RAS/ERK1/2 signaling pathway (Figure 4B).We next compared the activity of P9 to compound 3 on cancer cell growth inhibi in several cell lines previously demonstrated to be sensitive to SHP2 inhibition, includ KYSE-520 (EGFR amplified) [16], SKBR3 (HER2 amplified) [36], U2OS [37], M (PIK3CA E545K ) [36], H358 (KRAS G12C ) and A549 (KRAS G12S ) [36].Compared with the pa SHP2 allosteric inhibitor compound 3, P9 showed improved efficacy in inhibiting growth of these cancer cells in the colony formation assay (Figure 4C).These data gested that P9 is more potent than its parent SHP2 inhibitor compound 3 in blocking growth as measured by the colony formation assay.We next compared the activity of P9 to compound 3 on cancer cell growth inhibition in several cell lines previously demonstrated to be sensitive to SHP2 inhibition, including KYSE-520 (EGFR amplified) [16], SKBR3 (HER2 amplified) [36], U2OS [37], MCF7 (PIK3CA E545K ) [36], H358 (KRAS G12C ) and A549 (KRAS G12S ) [36].Compared with the parent SHP2 allosteric inhibitor compound 3, P9 showed improved efficacy in inhibiting the growth of these cancer cells in the colony formation assay (Figure 4C).These data suggested that P9 is more potent than its parent SHP2 inhibitor compound 3 in blocking cell growth as measured by the colony formation assay.

Evaluation of P9 in a Mouse Xenograft Model
SHP2 allosteric inhibitors have been shown to block tumor growth in mice by inhibiting the RTK/RAS/ERK1/2 signaling pathway [13][14][15][16][17]28].To assess the antitumor activity of P9 in vivo, we first carried out pharmacokinetics (PK) studies in mice, and the results are shown in Figure 5A.The data indicates that a single intraperitoneal injection of P9 at 25 and 50 mg/kg achieved a peak plasma concentration (C max ) of 1.2 ± 0.1 and 2.5 ± 0.2 µM with half-lives of 3.7 ± 0.7 and 3.0 ± 0.5 h, respectively (Tables S4 and S5).At both doses, the plasma concentrations of P9 were maintained above 0.5 µM, which is above its effective concentration for pERK1/2 inhibition (Figure 4A), for at least 6 h.Based on the pharmacokinetic analysis, we evaluated the tolerability and antitumor efficacy of P9 in a murine xenograft model of KYSE-520 cells exogenously.After 18 days of treatment, intraperitoneal administration of P9 alleviated tumor progression in a dose-dependent manner as determined by serial volumetric measurement (Figure 5B).A decrease in tumor burden was observed in the SHP2 degrader P9-treated mice at 25 mg/kg and nearly complete tumor regression was induced following injections of P9 at 50 mg/kg.Notably, the given compound was well tolerated in mice at the doses of 25 mg/kg or 50 mg/kg and the animal weight was preserved during the treatment procedure above (Figure 5C).Furthermore, Western blots showed that SHP2 and pERK1/2 levels are reduced to 34 ± 18% and 24 ± 12% of the control group in whole tumor homogenates after 50 mg/kg P9 treatment, respectively (Figure 5D), indicating that the degrader attenuates the tumor growth by inducing degradation of SHP2 and inhibiting RAS/ERK1/2 signaling pathway.Taken together, the SHP2 PROTAC P9 efficiently degrades SHP2 in the tumor tissue and effectively suppresses tumor growth in a KYSE-520 xenograft mice model.Considering that previously reported SHP2 degrader D26 alone only exerted moderate inhibition of xenograft tumor growth, P9 represents the best SHP2 degrader with robust in vivo anti-tumor activity so far.
Molecules 2023, 28, x FOR PEER REVIEW 8 of 20 effective concentration for pERK1/2 inhibition (Figure 4A), for at least 6 h.Based on the pharmacokinetic analysis, we evaluated the tolerability and antitumor efficacy of P9 in a murine xenograft model of KYSE-520 cells exogenously.After 18 days of treatment, intraperitoneal administration of P9 alleviated tumor progression in a dose-dependent manner as determined by serial volumetric measurement (Figure 5B).A decrease in tumor burden was observed in the SHP2 degrader P9-treated mice at 25 mg/kg and nearly complete tumor regression was induced following injections of P9 at 50 mg/kg.Notably, the given compound was well tolerated in mice at the doses of 25 mg/kg or 50 mg/kg and the animal weight was preserved during the treatment procedure above (Figure 5C).Furthermore, Western blots showed that SHP2 and pERK1/2 levels are reduced to 34 ± 18% and 24 ± 12% of the control group in whole tumor homogenates after 50 mg/kg P9 treatment, respectively (Figure 5D), indicating that the degrader attenuates the tumor growth by inducing degradation of SHP2 and inhibiting RAS/ERK1/2 signaling pathway.Taken together, the SHP2 PROTAC P9 efficiently degrades SHP2 in the tumor tissue and effectively suppresses tumor growth in a KYSE-520 xenograft mice model.Considering that previously reported SHP2 degrader D26 alone only exerted moderate inhibition of xenograft tumor growth, P9 represents the best SHP2 degrader with robust in vivo anti-tumor activity so far.

General Information
Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification.SHP099 [15] and compound 3 [28] were prepared following the literature procedures.Thin-layer chromatography was performed using glassprecoated Merck silica gel 60 F254 plates.Flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One (Biotage, Uppsala, Sweden).Normal phase column chromatography was effective concentration for pERK1/2 inhibition (Figure 4A), for at least 6 h.Bas pharmacokinetic analysis, we evaluated the tolerability and antitumor efficacy murine xenograft model of KYSE-520 cells exogenously.After 18 days of treatm peritoneal administration of P9 alleviated tumor progression in a dose-depende as determined by serial volumetric measurement (Figure 5B).A decrease in tum was observed in the SHP2 degrader P9-treated mice at 25 mg/kg and nearly co mor regression was induced following injections of P9 at 50 mg/kg.Notably, compound was well tolerated in mice at the doses of 25 mg/kg or 50 mg/kg and t weight was preserved during the treatment procedure above (Figure 5C).Fur Western blots showed that SHP2 and pERK1/2 levels are reduced to 34 ± 18% 12% of the control group in whole tumor homogenates after 50 mg/kg P9 trea spectively (Figure 5D), indicating that the degrader attenuates the tumor grow ducing degradation of SHP2 and inhibiting RAS/ERK1/2 signaling pathway.gether, the SHP2 PROTAC P9 efficiently degrades SHP2 in the tumor tissue and suppresses tumor growth in a KYSE-520 xenograft mice model.Considering t ously reported SHP2 degrader D26 alone only exerted moderate inhibition of tumor growth, P9 represents the best SHP2 degrader with robust in vivo antitivity so far.

General Information
Unless otherwise noted, all reagents were purchased from commercial sup used without further purification.SHP099 [15] and compound 3 [28] were pre lowing the literature procedures.Thin-layer chromatography was performed u precoated Merck silica gel 60 F254 plates.Flash column chromatography was p on Biotage prepacked columns using the automated flash chromatography sy tage Isolera One (Biotage, Uppsala, Sweden).Normal phase column chromatog effective concentration for pERK1/2 inhibition (Figure 4A), for at least 6 h.Based on the pharmacokinetic analysis, we evaluated the tolerability and antitumor efficacy of P9 in a murine xenograft model of KYSE-520 cells exogenously.After 18 days of treatment, intraperitoneal administration of P9 alleviated tumor progression in a dose-dependent manner as determined by serial volumetric measurement (Figure 5B).A decrease in tumor burden was observed in the SHP2 degrader P9-treated mice at 25 mg/kg and nearly complete tumor regression was induced following injections of P9 at 50 mg/kg.Notably, the given compound was well tolerated in mice at the doses of 25 mg/kg or 50 mg/kg and the animal weight was preserved during the treatment procedure above (Figure 5C).Furthermore, Western blots showed that SHP2 and pERK1/2 levels are reduced to 34 ± 18% and 24 ± 12% of the control group in whole tumor homogenates after 50 mg/kg P9 treatment, respectively (Figure 5D), indicating that the degrader attenuates the tumor growth by inducing degradation of SHP2 and inhibiting RAS/ERK1/2 signaling pathway.Taken together, the SHP2 PROTAC P9 efficiently degrades SHP2 in the tumor tissue and effectively suppresses tumor growth in a KYSE-520 xenograft mice model.Considering that previously reported SHP2 degrader D26 alone only exerted moderate inhibition of xenograft tumor growth, P9 represents the best SHP2 degrader with robust in vivo anti-tumor activity so far.

General Information
Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification.SHP099 [15] and compound 3 [28] were prepared following the literature procedures.Thin-layer chromatography was performed using glassprecoated Merck silica gel 60 F254 plates.Flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One (Biotage, Uppsala, Sweden).Normal phase column chromatography was effective concentration for pERK1/2 inhibition (Figure 4A), for at least 6 h.Based on the pharmacokinetic analysis, we evaluated the tolerability and antitumor efficacy of P9 in a murine xenograft model of KYSE-520 cells exogenously.After 18 days of treatment, intraperitoneal administration of P9 alleviated tumor progression in a dose-dependent manner as determined by serial volumetric measurement (Figure 5B).A decrease in tumor burden was observed in the SHP2 degrader P9-treated mice at 25 mg/kg and nearly complete tumor regression was induced following injections of P9 at 50 mg/kg.Notably, the given compound was well tolerated in mice at the doses of 25 mg/kg or 50 mg/kg and the animal weight was preserved during the treatment procedure above (Figure 5C).Furthermore, Western blots showed that SHP2 and pERK1/2 levels are reduced to 34 ± 18% and 24 ± 12% of the control group in whole tumor homogenates after 50 mg/kg P9 treatment, respectively (Figure 5D

General Information
Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification.SHP099 [15] and compound 3 [28] were prepared following the literature procedures.Thin-layer chromatography was performed using glassprecoated Merck silica gel 60 F254 plates.Flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One (Biotage, Uppsala, Sweden).Normal phase column chromatography was 50 mg/kg P9 intraperitoneal administration.

General Information
Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification.SHP099 [15] and compound 3 [28] were prepared following the literature procedures.Thin-layer chromatography was performed using glass-precoated Merck silica gel 60 F254 plates.Flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One (Biotage, Uppsala, Sweden).Normal phase column chromatography was performed using KP-SIL silica gel, and reverse phase column chromatography was performed using Teledyne Isco RediSepRf Gold columns (Teledyne, Thousand Oaks, CA, USA).High-performance liquid chromatography (HPLC) purification was performed using column: Phenomenex Kinetex C18 5 µm 150 × 21.2 mm; Eluent A: water + 0.1% formic acid (99%), Eluent B: methanol; DAD scan: 210-400 nm).Melting points were measured using an OptiMelt MPA100 Automated Melting Point System.The 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE 500 MHz spectrometer using chloroform-D (CDCl 3 ) or dimethyl sulfoxide (DMSO-d6) as the solvents.Chemical shifts are expressed in ppm (δ scale) and referenced to the residual protonated solvent.Peak multiplicities are reported using the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broad singlet).Mass spectra and purity data were obtained using an Agilent Technologies 6470 series, triple quadrupole LC-MS (Agilent, Santa Clara, CA, USA).The purity of all final tested compounds was determined to be >95% (UV, λ = 254 nm).High-resolution mass analysis was performed on an Agilent 6550 iFunnel Q-TOF mass LC-MS.DIFMUP was purchased from Thermo Fisher Scientific (catalog# D6567, Waltham, MA, USA).

Synthesis of CRBN-Based SHP2 PROTACs
General method (Scheme 2): To a stirred solution of carboxylic acid 10 (29.9 mg, 0.05 mmol) and DIPEA (28 µL, 0.15 mmol) in DMF (2 mL) at ambient temperature was added the corresponding primary amine L13 or L14 (0.075 mmol) and HATU (28 mg, 0.075 mmol).The reaction mixture was stirred for 1 h.After quenched with water (5 mL) and extracted with EtOAc (5 mL × 3), the organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure.To this residue was added DCM (2 mL), and trifluoracetic acid (0.2 mL) at 0 °C.The reaction was stirred for 6 h at ambient temperature, and the volatiles were removed under reduced pressure.The residue was purified by HPLC (40-85% gradient of MeOH in 5%TFA/H2O) to yield the corresponding product.

Synthesis of CRBN-Based SHP2 PROTACs
General method (Scheme 2): To a stirred solution of carboxylic acid 10 (29.9 mg, 0.05 mmol) and DIPEA (28 µL, 0.15 mmol) in DMF (2 mL) at ambient temperature was added the corresponding primary amine L13 or L14 (0.075 mmol) and HATU (28 mg, 0.075 mmol).The reaction mixture was stirred for 1 h.After quenched with water (5 mL) and extracted with EtOAc (5 mL × 3), the organic layers were washed with brine (15 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure.To this residue was added DCM (2 mL), and trifluoracetic acid (0.2 mL) at 0 • C. The reaction was stirred for 6 h at ambient temperature, and the volatiles were removed under reduced pressure.The residue was purified by HPLC (40-85% gradient of MeOH in 5%TFA/H 2 O) to yield the corresponding product.

Synthesis of VHL-Based SHP2 PROTACs
General method (Scheme 3): To a stirred solution of carboxylic acid 10 (29.9 mg, 0.05 mmol) and DIPEA (28 µL, 0.15 mmol) in DMF (2 mL) at ambient temperature was added the corresponding primary amine (0.075 mmol) and HATU (28 mg, 0.075 mmol).The reaction mixture was stirred for 1 h.After quenched with water (5 mL) and extracted with EtOAc (5 mL × 3), the organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure.To this residue was added DCM (2 mL), and trifluoracetic acid (0.5 mL) at 0 °C.The reaction was stirred for 6 h at ambient temperature, and the volatiles were removed under reduced pressure.The residue was purified by HPLC (40-90% gradient of MeOH in 5%TFA/H2O) to yield the corresponding product.

Synthesis of VHL-Based SHP2 PROTACs
General method (Scheme 3): To a stirred solution of carboxylic acid 10 (29.9 mg, 0.05 mmol) and DIPEA (28 µL, 0.15 mmol) in DMF (2 mL) at ambient temperature was added the corresponding primary amine (0.075 mmol) and HATU (28 mg, 0.075 mmol).The reaction mixture was stirred for 1 h.After quenched with water (5 mL) and extracted with EtOAc (5 mL × 3), the organic layers were washed with brine (15 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure.To this residue was added DCM (2 mL), and trifluoracetic acid (0.5 mL) at 0 • C. The reaction was stirred for 6 h at ambient temperature, and the volatiles were removed under reduced pressure.The residue was purified by HPLC (40-90% gradient of MeOH in 5%TFA/H 2 O) to yield the corresponding product.

Synthesis of VHL-Based SHP2 PROTACs
General method (Scheme 3): To a stirred solution of carboxylic acid 10 (29.9 mg, 0.05 mmol) and DIPEA (28 µL, 0.15 mmol) in DMF (2 mL) at ambient temperature was added the corresponding primary amine (0.075 mmol) and HATU (28 mg, 0.075 mmol).The reaction mixture was stirred for 1 h.After quenched with water (5 mL) and extracted with EtOAc (5 mL × 3), the organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure.To this residue was added DCM (2 mL), and trifluoracetic acid (0.5 mL) at 0 °C.The reaction was stirred for 6 h at ambient temperature, and the volatiles were removed under reduced pressure.The residue was purified by HPLC (40-90% gradient of MeOH in 5%TFA/H2O) to yield the corresponding product.

Cloning, Expression, and Purification of SHP2 Protein
The SHP2 protein (Aa 1-528) was cloned into a pET-21a(+) vector.Bacterial BL21(DE3) (Novagen) was used as an expression host, and the induction of protein expression was carried out in LB media with 1 mM IPTG at 18 • C overnight.Cell pellets were stored at −80 • C for subsequent protein purification.Protein purification was conducted at 4 • C. Frozen cell pellets were lysed by sonication in 40 mL cold lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM imidazole, and 1 mM PMSF) per liter cell pellet.Cell lysates were clarified by centrifuging for 15 min at 6000 rpm.The supernatant was incubated with HisPur Ni-NTA resin (Thermo Scientific, Waltham, MA, USA) for 2 h, and then packed onto a column and washed with 50 resin volume of buffer A (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole).The HIS-tagged proteins were eluted with Buffer B (50 mM Tris-HCl, pH = 8.0, 500 mM NaCl, 300 mM imidazole,).Pooled HIS-proteincontaining fractions were concentrated, loaded onto a HiLoad 26/600 Superdex 75 column (GE Healthcare Biosciences, Piscataway, NJ, USA), and eluted with storage buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol).Proteins used for inhibition assays were purified using Ni-NTA resin (Qiagen, Hilden, Germany) followed by size exclusion column chromatography (ÄKTA pure, Cytiva, Marlborough, MA, USA) and the purity was determined to be >95% by SDS-PAGE and Coomassie staining.The protein was aliquoted and stored at −80 • C.

PTP Inhibition Assay and Determination of IC 50 Values
PTP activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate in DMG buffer (50 mM DMG, pH 7.0, 1 mM EDTA, 150 mM NaCl, 2 mM DTT, 0.1 mg/mL BSA) at 25 • C. The assays were performed in 96-well plates.To determine the IC 50 values, the reaction was initiated by the addition of enzyme (final concentration = 10 nM) to a reaction mixture (0.2 mL) containing pNPP (at a final concentration close to the K m s of tested enzymes, in specific: 0.05 mM for CDC-14A; 0.5 mM for FAP-1; 2 mM for TC-PTP, PTP1B, STEP, LWM-PTP, and PTPα; 3 mM for SHP1; 4 mM for Laforin; 5 mM for LYP, CD45, and VHR; 6 mM for PTP-MEG2 and HePTP) with various concentrations of inhibitors.The reaction rate was measured using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices, San Jose, CA, USA).Data were fitted using the SigmaPlot Enzyme Kinetics Module (Systat Software, Inc., San Jose, CA, USA).

Cell Proliferation Assay
The cell proliferation inhibition EC 50 was determined by CCK-8 assay [40].KYSE-520 cells were seeded at 5 × 10 3 cells/well in 96 well plates.After 24 h, P9 was added to the medium starting at 80 µM in a 2-fold dilution rate of 0.16 µM.After 7 days, 10 µL of CCK-8 test solution (APExBIO Technology, Houston, TX, USA) was added into each well and incubated for 2 h at 37 • C. The optical density (OD) at 450 nm was measured with a microplate reader (Molecular Device, Sunnyvale, CA, USA).The cell viability rate at different concentrations of P9 treatment was determined with Prism, version 9.5.1 (GraphPad, San Diego, CA, USA).

Mouse Study
Experiments on mice were carried out in accordance with the regulations of the Institutional Animal Care and Use Committees at Purdue University.All mice were housed under pathogen-free conditions in the animal facility and received autoclaved water and food.Eight to ten weeks-old Nude mice were used in the study.
For pharmacokinetic studies, mice were administered a single dose of P9 at 25 or 50 mg/kg via IP injection.Blood samples were collected through the tail vein at indicated time points after injection.Isoflurane was used as an anesthetic.All blood samples were centrifuged at 1500 g for 5 min, and plasma was separated and stored at -80 • C until analysis by a validated method based on reversed-phase liquid chromatography coupled to mass-spectrometric detection (LC/MS) using a previously published procedure [41].
For the xenograft tumor study, KYSE-520 cells were suspended in PBS and a total of 3 × 10 6 cells (100 µL) were subcutaneously implanted into both the left and right flank using a 27-gauge needle.Tumor volume was calculated using the formula V = (W 2 × L)/2 for caliper measurements.Once the tumor volume reaches 200 mm 3 , daily intraperitoneal injection of either control, 25 mg/kg or 50 mg/kg of compound P9 was performed.Mice were sacrificed after injection for 18 days, and tumors were collected for biochemical analysis.

Conclusions
In summary, we have developed a new class of SHP2 degraders by recruiting a SHP2 allosteric inhibitor as the ligand and VHL as the E3 ubiquitin ligase.Thalidomide-based CRBN ligands were found incompatible with the allosteric inhibitors due to stability concerns.The linker length and composition were surveyed and compound P9 was identified as the most potent candidate among this class of SHP2 degraders.Mechanistic studies demonstrated that P9-induced SHP2 degradation requires ternary complex formation and ubiquitin-proteasome participation.P9 displayed a clear advantage over its parent SHP2 allosteric inhibitor in inhibiting the growth of a number of tumor cell lines including KYSE520.The in vivo studies indicate that P9, as a single agent, efficiently suppresses KYSE-520 xenograft tumor growth through SHP2 degradation and RAS/ERK1/2 inhibition.We believe that this SHP2 degrader could be used as a new chemical tool for further investigating the functions of SHP2 in physiological and pathological states.P9

Figure 2 .
Figure 2. Identification of a SHP2 ligand for PROTAC development.(A) Chemical structures of selected SHP2 allosteric inhibitors and the designed ligand 4. (B) Cocrystal structure of 1 (green stick) bound to the SHP2 allosteric pocket with the interacting structural water represented by a red sphere (PDB 7JVN).Hydrogen bonds are depicted by yellow dashes, and the cation-π interaction is shown with a purple dash.(C) Piperidine region of the scaffold bound to SHP2 in surface

Figure 2 .
Figure 2. Identification of a SHP2 ligand for PROTAC development.(A) Chemical structures of selected SHP2 allosteric inhibitors and the designed ligand 4. (B) Cocrystal structure of 1 (green stick) bound to the SHP2 allosteric pocket with the interacting structural water represented by a red sphere (PDB 7JVN).Hydrogen bonds are depicted by yellow dashes, and the cation-π interaction is shown with a purple dash.(C) Piperidine region of the scaffold bound to SHP2 in surface representation colored by element.The solvent-exposed methyl group exploited in PROTAC design is circled in red.(D) Synthetic route to the SHP2 ligand compound 4.

Figure 3 .
Figure 3. P9 is a genuine SHP2 PROTAC degrader.(A) P9 induces the degradation of SHP2 in a dose-dependent manner in HEK293 cells for 16 h of treatment.(B) P9 induces degradation of SHP2 in a time-dependent manner in HEK293 cells.(C) P9 treatment does not affect the protein level of other PTPs and common proteins in HEK293 cells.Cells were treated with 1 µM P9 for 16 h.(D) Control experiments show that P9-induced SHP2 degradation requires the formation of SHP2-P9-VHL ternary complex and is ubiquitination-and proteasome-dependent. HEK 293 cells were treated for 16 h with DMSO, 250 nM P9, or 250 nM P9 in combination with 1 µM 3, 10 µM MG132, 1 µM MLN4924, 40 µM VHL-2, or 40 µM Lenalidomide.

Figure 3 .
Figure 3. P9 is a genuine SHP2 PROTAC degrader.(A) P9 induces the degradation of SHP2 in a dose-dependent manner in HEK293 cells for 16 h of treatment.(B) P9 induces degradation of SHP2 in a time-dependent manner in HEK293 cells.(C) P9 treatment does not affect the protein level of other PTPs and common proteins in HEK293 cells.Cells were treated with 1 µM P9 for 16 h.(D) Control experiments show that P9-induced SHP2 degradation requires the formation of SHP2-P9-VHL ternary complex and is ubiquitination-and proteasome-dependent. HEK 293 cells were treated for 16 h with DMSO, 250 nM P9, or 250 nM P9 in combination with 1 µM 3, 10 µM MG132, 1 µM MLN4924, 40 µM VHL-2, or 40 µM Lenalidomide.

2. 6 .
Evaluation of P9 in a Mouse Xenograft Model SHP2 allosteric inhibitors have been shown to block tumor growth in mice by in iting the RTK/RAS/ERK1/2 signaling pathway [13-17,28].To assess the antitumor acti of P9 in vivo, we first carried out pharmacokinetics (PK) studies in mice, and the res

Figure 5 .
Figure 5. P9 suppresses tumor growth in vivo by inducing degradation of SHP2.(A) Pharmacokinetic curves of P9 in mice.(B) P9 treatment dose-dependently attenuates tumor growth in a KYSE-520 xenograft model.The mice were treated with daily intraperitoneal injection of • DMSO,
50 mg/kg P9. *** p ≤ 0.001.** p ≤ 0.01.(C) P9 treatment has no significant effect on mice body weight.(D) P9 induces SHP2 degradation and decreases pERK1/2 levels in KYSE-520 tumor homogenates.• DMSO intraperitoneal administration.Molecules 2023, 28, x FOR PEER REVIEW 8 of 20 ), indicating that the degrader attenuates the tumor growth by inducing degradation of SHP2 and inhibiting RAS/ERK1/2 signaling pathway.Taken together, the SHP2 PROTAC P9 efficiently degrades SHP2 in the tumor tissue and effectively suppresses tumor growth in a KYSE-520 xenograft mice model.Considering that previously reported SHP2 degrader D26 alone only exerted moderate inhibition of xenograft tumor growth, P9 represents the best SHP2 degrader with robust in vivo anti-tumor activity so far.

Table 1 .
Degradation results of first generation SHP2 PROTACs constructed using compound 4 and a VHL ligand 1 .

Table 1 .
Degradation results of first generation SHP2 PROTACs constructed using compoun a VHL ligand 1 .

Table 1 .
Degradation results of first generation SHP2 PROTACs constructed u a VHL ligand 1 .

Table 1 .
Degradation results of first generation SHP2 PROTACs constructed u a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs construct and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs construct and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs construct and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs construct and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs construct and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs constructed using compound 4 and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs constructed using and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs constructed using and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs constructed using and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs constructed using and a VHL ligand 1 .

Table 2 .
Degradation results of second generation SHP2 PROTACs constructed using and a VHL ligand 1 .