Drugging Protein Tyrosine Phosphatases through Targeted Protein Degradation

Abstract

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that regulate protein tyrosine phosphorylation levels of a large variety of proteins in cells. Anomalies in protein tyrosine phosphorylation have been associated with the development of numerous human diseases, leading to a heightened interest in PTPs as promising targets for drug development. However, therapeutic targeting of PTPs has faced skepticism about their druggability. Besides the conventional small molecule inhibitors, proteolysis-targeting chimera (PROTAC) technology offers an alternative approach to target PTPs. PROTAC molecules utilize the ubiquitin-proteasome system to degrade specific proteins and have unique advantages compared with inhibitors: 1) PROTACs are highly efficient and can work at much lower concentrations than that expected based on their biophysical binding affinity; 2) PROTACs may achieve higher selectivity for the targeted protein than that dictated by their binding affinity alone; and 3) PROTACs may engage any region of the target protein in addition to the functional site. This review focuses on the latest advancement in the development of targeted PTP degraders and deliberates on the obstacles and prospective paths of harnessing this technology for therapeutic targeting of the PTPs.

Graphical Abstract

Protein tyrosine phosphatases (PTPs) are an important class of enzymes and potential targets for various human diseases such as cancer, diabetes, and obesity. Proteolysis-targeting chimera (PROTAC) technology features unique advantages compared with traditional inhibitors and provides an alternative approach to target PTPs. This concept highlights the recent progress in the development of targeted degradation of PTPs.

Introduction

Reversible protein tyrosine phosphorylation, mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is one of the most important cellular regulatory mechanisms.^[1] This dynamic process governs various cellular functions such as survival, proliferation, migration, differentiation, and energy metabolism. Therefore, aberrations in protein tyrosine phosphorylation are associated with a multitude of human diseases. Endeavors in drug discovery aimed at modulating abnormal protein tyrosine phosphorylation-mediated events to date have concentrated on PTKs, with several dozen kinase inhibitor drugs already approved by the FDA.^[2] On the other hand, the PTPs, constituting a large family of enzymes with structural diversity and complexity comparable to PTKs, remain underexplored as drug targets.^[3] Illuminating the functions of PTPs is crucial for comprehending the normal physiology controlled by protein tyrosine phosphorylation and how tyrosine phosphorylation--mediated signaling pathways are disrupted in pathological conditions. Indeed, dysregulation of PTP activity has been linked to various human health conditions, including cancer, diabetes/obesity, autoimmune dysfunctions, and neurological diseases and disorders.^[4]

To develop PTP inhibitors as chemical probes and leads for drug development, the phosphotyrosine (pTyr)-binding catalytic site has attracted particular attention over the past decades.^[5] However, these efforts were frequently frustrated by the highly charged and conserved nature of the PTP active site.^[6] Since the positively charged active sites allow for high-affinity binding of negatively charged pTyr mimetics, potent orthosteric PTPinhibitors (active site-directed, competitive inhibitors) often possess a high level of negative charges, limiting their cell permeability, bioavailability, and potential as drug candidates. The significant conservation of the active site across PTPs also presents an additional challenge, as potent orthosteric inhibitors frequently interact with multiple PTPs. Despite these obstacles, efforts to drug PTPs continued due to the growing recognition of numerous PTPs as clinically significant targets. Moreover, several new active site-directed inhibitors with promising bioactivities have been developed.^[7,8] For example, Abbvie/Calico reported an orally available, highly potent, and selective active site PTPN2/N1 dual inhibitor, AC484, which is currently being evaluated in clinical trials as a new strategy for cancer immunotherapy.^[9]

In addition to the progress in competitive, orthosteric PTP inhibitor development, there has been a surge of innovative approaches to target these enzymes.^[10] Recently, several Proteolysis targeting chimera (PROTAC)^[11,12] degraders for PTPs were reported (Table 1). PROTACs are bifunctional molecules comprised of a ligand specific to the target protein (blue), a linker (black), and an E3 ligase binding unit (red). They leverage the ubiquitin-proteasome system within the cell to accomplish the selective degradation of the desired proteins. Compared to the conventional occupancy-based inhibitors, the event-driven PROTACs offer distinct advantages, including extended efficacy through target elimination, the requirement of only sub-stoichiometric concentrations as a result of their catalytic nature, and enhanced selectivity driven by the obligatory formation of a ternary complex that brings the target protein into proximity of the E3 ligase for efficient target protein ubiquitination and subsequent proteasome-mediated degradation.^[13,14] Consequently, this approach has the potential to surmount the challenges associated with targeting PTPs and forge a fresh direction. Herein, we highlight the recent progress in targeted PTP degradation and examine the obstacles and future directions for expanding the technology beyond the current stage.

Table 1.

Reported PROTAC degraders of PTPs ^[a]

^[a]The PTP ligands in the PROTAC structures are colored in blue, and the E3 ligands are colored in red.

SHP2 PROTACs based on allosteric inhibitors

Src homology-2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a classical non-receptor PTP and a convergent node of multiple signaling pathways.^[15,16] Unlike most PTPs, which typically play tumor-suppressive roles, SHP2 is a rare PTP that exhibits oncogenic activities by participating in several oncogenic signal cascades, such as RAS/MAPK, JAK/STAT, and PI3K/AKT.^[17] SHP2 has received a lot of attention lately in relation to human diseases, especially cancer.^[18–21] Apart from its critical roles in oncogenic signaling pathways, numerous research efforts have demonstrated the significant impact of SHP2 in controlling the functions of immune cells within the tumor microenvironment, making it also an attractive target for cancer immunotherapy.^[22]

SHP2 activity is regulated through an intramolecular auto-inhibited state in which several N-SH2 residues insert into the PTP domain’s catalytic cleft.^[23] When SHP2’s SH2 domains bind to the pTyr residues of interacting proteins, the enzyme transitions to an open, active conformation. In light of this mechanism, small molecules that can stabilize the closed inactive conformation of SHP2 have been discovered through high throughput screening and rational modifications.^[24] Since the report of the first potent, selective, and bioavailable SHP2 allosteric inhibitor (an inhibitor that binds to a specific site on an enzyme other than the active site) SHP099 by Norvatis in 2016,^[25,26] numerous efficacious SHP099-like inhibitors have been disclosed with high lipophilic efficiency, dramatically enhanced potency, and favorable pharmacokinetic properties.

While SHP2 allosteric inhibitors demonstrate promise for clinical use, recent research has raised concerns about the development of drug resistance through non-mutational mechanisms.^[27] Targeted protein degradation technologies offer an alternative approach to inhibit SHP2 that could potentially circumvent this drug resistance issue.^[28] In 2020, Wang and colleagues pioneered the creation of the first SHP2 PROTAC degrader, D26 (Table 1), by tethering a SHP2 ligand compound 5 (SHP2^WT IC₅₀ = 98.7 nM) to the Von Hippel-Lindau (VHL) ligand VHL-1.^[29] D26 showed excellent SHP2 degradation efficiency with DC₅₀ values of 2.6 and 6.0 nM in acute myeloid leukemia MV4;11 and esophageal cancer KYSE520 cells, respectively (Figure 1). SHP2-D26 was found to be over 30 times more effective than SHP099 in suppressing ERK1/2 phosphorylation (p-ERK1/2) and cell growth in the KYSE520 and MV4;11 cancer cell lines. Subsequent studies exploring D26’s therapeutic potential in non-small cell lung cancer (NSCLC) cells revealed that D26 effectively increased Bim levels while decreasing Mcl-1 levels accompanied by the induction of cell apoptosis.^[30] Furthermore, the combination of an EGFR inhibitor, osimertinib (AZD9291), and D26 significantly inhibited the growth of osimertinib-resistant NSCLC xenografts, although D26 alone only exerted moderate inhibition efficacy.

Figure 1.

SHP2 degrader D26 effectively degrades SHP2 and suppresses the p-ERK pathway. KYSE520 (A) or MV4;11 (B) cells were treated as indicated with SHP2-D26 or SHP099 for 48 h. The protein levels of SHP2 (Bethyl Lab. A301–544), ERK (#9102, CST), and phospho-ERK (#4370, CST) were determined by Western blotting. GAPDH was used as a loading control. Adapted with permission from Ref. [29]. Copyright 2020 American Chemical Society.

In addition to D26, two other effective SHP2 degraders, SP4^[31] and ZB-S-29^[32], were developed by linking the Cereblon (CRBN) ligand thalidomide to the allosteric inhibitors SHP099 and TNO155^[33], respectively, using PEG linkers of varying lengths (Table 1). SP4, in particular, showed significant inhibition of HeLa cells, being 100 times more potent than SHP099, leading to SHP2 degradation and cell apoptosis. Another advancement was the use of the clinical trial candidate SHP2 inhibitor RMC-4550^[34] to create a SHP2 degrader R1-5C with pomalidomide and a PEG linker (Table 1).^[35] R1-5C induced SHP2 degradation and hindered the growth of leukemic cell lines by inhibiting MAPK signaling, offering a new approach to treating ERK1/2-dependent cancers. More recently, our group reported the discovery of a SHP2 degrader P9 constructed with a reported SHP2 allosteric inhibitor and a VHL ligand (Table 1).^[36] P9 induces efficient degradation of SHP2 (DC₅₀ = 35.2 ± 1.5 nM) in HEK293 cells and demonstrated robust anti-proliferation activity in KYSE520 cells through SHP2 degradation and p-ERK1/2 inhibition. Enhanced anti-tumor effects in various cancer cell lines were observed compared to its parent allosteric inhibitor compound 3 (Figure 2A). Notably, P9 administration resulted in almost total regression of tumors in a KYSE520 xenograft mouse model due to the substantial SHP2 depletion and the inhibition of p-ERK1/2 in the tumor tissue (Figure 2B).

Figure 2.

Anti-tumor activity of the SHP2 degrader P9. (A) Colony formation assay of KYSE-520, SKBR3, U2OS, MCF7, H358, and A549 treated with DMSO, P9, or compound 3. (B) P9 treatment dose–dependently attenuates tumor growth in a KYSE–520 xenograft model. The mice were treated with daily intraperitoneal injections of • DMSO, ■ 25 mg/kg P9, or □ 50 mg/kg P9. *** p ≤ 0.001. ** p ≤ 0.01. Adapted with permission from Ref. [36]. Copyright 2023 MDPI.

PTP1B and TC-PTP dual PROTAC based on an active site inhibitor

Protein tyrosine phosphatase 1B (PTP1B, encoded by PTPN1) and its closely related paralogue T-cell protein tyrosine phosphatase (TC-PTP, encoded by PTPN2) share more than 72% amino acid sequence identity within their catalytic domains.^[37] PTP1B and TC-PTP are well recognized for their nonredundant roles in regulating cellular processes mediated by insulin and leptin.^[38–40] Recent studies reveal that PTP1B and TC-PTP perform coordinative functions in attenuating IFN-γ signaling and tumor cell antigen presentation.^[41–47] Furthermore, PTP1B and TC-PTP also play independent roles in attenuating T-cell activation.^[48,49] Consequently, concomitant blockage of PTP1B and TC-PTP concurrently may produce synergistic effects for several therapeutic applications, including obesity, type II diabetes, and cancer immunotherapies.

Recently, our group utilized the PROTAC strategy to transform an active site-directed, phosphonodifluoromethyl phenylalanine (F2pmp)-based PTP1B and TC-PTP dual inhibitor DI-03 into a highly efficacious and selective dual degrader DU-14 (Table 1).^[50] Despite having a high molecular weight (~1473.4) and a negatively charged F2pmp moiety, DU-14 induces efficient PTP1B and TC-PTP degradation with low nanomolar DC₅₀s in multiple cell lines (Figure 3A). As expected, DU-14 amplifies IFN-γ induced JAK1/2-STAT1 signaling and antigen presentation in tumor cells (Figure 3B and 3C). DU-14 also boosts TCR and IL-2 signaling, leading to enhanced activation of CD8+ T cells. NotablyTop of Form Notably, DU-14 shows excellent pharmacokinetic properties and effectively suppresses the growth of syngeneic MC38 tumors in immunocompetent mice (Figure 3D); which is associated with the increased infiltration of CD8⁺ T-cells.

Figure 3.

Characterizations and anti-tumor activities of the PTP1B/TC-PTP dual degrader DU-14. (A) Immunoblots of cell lysates from HEK293 and MC38 cells treated with DU-14 at indicated concentrations for 16 hours showed dose-dependent degradation of PTP1B and TC-PTP achieved by low nanomolar concentrations. (B) Immunofluorescence showing DU-14 dramatically enhanced IFN-γ mediated STAT1 phosphorylation (Green) and nucleus translocation. U2OS cells were treated with DMSO or 0.2 μM DU-14 for 16 hours and stimulated with 20 ng/ml IFN-γ for 30 minutes. (C) MC38 cells treated with DU-14 exhibited elevated expression of MHC-I. MC38 cells were treated with DMSO (Blue Peak) or 500 nM DU-14 (Red Peak) for 16 hours for PTP1B and TC-PTP degradation, then stimulated with 20 ng/ml mouse IFN-γ for 48 hours to induce MHC-I expression. (D) MC38 tumor growth after daily treatment with saline (•) versus 25 (▪) or 50 mg kg⁻¹ (▴) DU-14 (n=8). Adapted with permission from Ref. [50]. Copyright 2023 Wiley-VCH.

TC-PTP PROTACs based on active site inhibitors

It has been shown that deletion of TC-PTP in tumor cells promotes antigen presentation,^[42,43] while the loss of the phosphatase in T cells stimulates the activation of T cells, which directs immune cells to eliminate tumor cells.^{[44,45,47,48]} Thus, therapeutic targeting of TC-PTP represents an exciting approach to increasing tumor antigen presentation and alleviating the inhibitory constraints on immune cells in the tumor microenvironment for improved immunotherapy. Furthermore, the specific functions of TC-PTP in cancer development, anti-tumor immunity, and autoimmunity responses are yet to be clearly understood.^[51] To develop highly specific small molecule TC-PTP inhibitors as chemical tools to facilitate further biological studies and clinical translation of TC-PTP targeting, our group recently developed the first highly potent and selective TC-PTP PROTAC degrader TP1L^[52] using a previously reported (F2pmp)-based TC-PTP active site inhibitor compound 7 (Table 1).^[53] TP1L induces degradation of TC-PTP in multiple cell lines with low nanomolar DC₅₀s and shows >110-fold degradation selectivity over PTP1B in HEK293 cells, which is far more selective than the parent inhibitor 7 (Figure 4A). Increases in IFN-γ signaling due to TP1L treatment were demonstrated by the elevated levels of phosphorylated STAT1 and MHC-1 expression in HEK293 cells, while these levels were not changed by the TP1L treatment in TC-PTP knockout HEK293 cells (Figure 4B and 4C). In Jurkat T cells, TP1L activates TCR signaling through TC-PTP degradation and subsequent increase of LCK phosphorylation level (Figure 4D). Moreover, in a 4M5.3 CAR-T cell and KB tumor cell co-culture model, TP1L promotes tumor-killing efficacy through the stimulation of CAR-T cells.

Figure 4.

Characterizations of the selective TC-PTP degrader TP1L. (A) Immunoblots of cell lysates from HEK293 cells treated with TP1L at indicated concentrations for 16 hours showed dose-dependent and selective degradation of TC-PTP. (B) Immunofluorescence showing TP1L dramatically enhanced IFN-γ mediated STAT1 phosphorylation (green) and nucleus translocation through degradation of TC-PTP. Wild-type and TC-PTP deleted HEK293 cells were treated with DMSO or 0.5 μM TP1L for 16 hours and stimulated with 20 ng ml⁻¹ IFN-γ for 30 minutes. (C) TP1L promotes MHC-1 expression in HEK293 cells. Wildtype and TC-PTP knockout HEK293 cells were treated with DMSO (blue and light green peak) or 500 nM TP1L (orange and dark green peak) for 16 hours for TC-PTP degradation, then stimulated with 20 ng ml⁻¹ mouse IFN-γ for 48 hours to induce MHC-I expression. TCKO = TC-PTP knockout. (D) Immunoblots of cell lysates showed that TP1L dose-dependently degrades TC-PTP and promotes phosphorylation of LCK in Jurkat T-cells. Cells were treated with TP1L at indicated concentrations for 16 hours and stimulated with 2.5 μg ml⁻¹ anti-CD3 antibody for 10 min. Reproduced from Ref. [53] with permission from the Royal Society of Chemistry.

More recently, Wang et al. identified a subtype selective TC-PTP degrader PVD-06 based on a thiadiazolidinone dioxide–naphthalene scaffold, which is related to AC484 with a VHL ligand (Table 1).^[54] PVD-06 shows a DC₅₀ of 217 nM for TC-PTP, while the DC₅₀ for PTP1B is greater than 13 μM in Jurkat cells. Proteomic analysis affirmed that PVD-06 exhibited broad degradation selectivity across the proteome and excellent subtype specificity over other PTPs. Furthermore, the subtype-selective TC-PTP degradation induced by PVD-06 enhanced T cell activation and augmented IFN-γ-mediated inhibition of B16F10 cell growth, underscoring the therapeutic potential of selective TC-PTP degradation.

Summary and Outlook

The generation of potent, selective, and bioavailable PTP inhibitors suitable for therapeutic applications is experiencing a renaissance as a promising approach in drug discovery. Small molecule degraders of PTPs provide unprecedented opportunities for targeting these challenging enzymes. Many reported PROTACs exhibit superior cellular activities and improved selectivities compared to their parent inhibitors as a result of the distinct targeted protein degradation mechanism. These observations will likely motivate the exploration of new PTP inhibitors, as there are supplementary opportunities to improve the potency and selectivities. On the other hand, the discovery and leverage of non-inhibitory binders of PTPs for degrader development presents a new direction, given that they often provide starting points with preferable specificity and druglike properties. While several PROTAC drugs have progressed to clinical trials,^[55] the clinical translation of PROTAC degraders faces the challenge of limited bioavailability due to their relatively high molecular weight and the presence of multiple hydrogen bond donors and acceptors. To address this issue, structure-based modification of the effective PTP PROTACs for optimal drug-like properties might be necessary. Furthermore, other approaches that induce protein degradation based on proximity, such as lysosome-targeting chimera (LYTAC),^[56,57] autophagy-targeting chimera (AUTAC),^[58] autophagosome-tethering compound (ATTEC),^[59] and molecular glue,^[60] may open up additional possibilities for targeted PTP degradation. In conclusion, we anticipate continued interest in the advancement of PTP degraders for use as both chemical probes and therapeutic applications.

Figure 5.

PVD-06 enhanced T cell activation and augmented IFN-γ-mediated inhibition of B16F10 cell growth. (A) PVD-06 promoted T cell proliferation. Normalized cell proliferation presented as the percentage of control cells (DMSO as 1, 100%) and as the mean ± SD, n = 3, *p < 0.05, one-way ANOVA. (B) PVD-06 amplified IFN-γ-induced B16F10 melanoma cell growth inhibition. B16F10 cell growth curve, 0–96 h, presented as mean ± SD and as the percentage of control cells (DMSO, 100%). Adapted with permission from Ref. [54]. Copyright 2023 American Chemical Society.

Biographies

Jinmin Miao received his BS degree from the college of Chemistry at Nankai University, China. He obtained his Ph.D. in 2016 from the Indiana University Purdue University Indianapolis, where he studied organic chemistry. After his graduation, he worked as a postdoc fellow in the research group of Zhong-Yin Zhang at Purdue University. He is currently a research associate in the Department of Medicinal Chemistry and Molecular Pharmacology at Purdue University. His current research interest lies in the development of potent and druglike small molecule inhibitors of protein tyrosine phosphatases.

Zhong-Yin Zhang obtained his Ph.D. in Chemistry from Purdue University in 1990. He completed his postdoctoral training at the University of Michigan from 1991-1994 in the laboratory of Dr. Jack Dixon. He established his laboratory in 1994 at Albert Einstein College of Medicine and moved to Indiana University School of Medicine in 2005. In 2016, he moved back to Purdue as Distinguished Professor of Medicinal Chemistry and Head of the Borch Department of Medicinal Chemistry and Molecular Pharmacology. His research focuses on PTP structure and function, signaling mechanism, and development of PTP inhibitors as novel therapeutics.