Tyrosine Phosphatase Inhibitor Research Articles

Protein tyrosine phosphatase 1B (PTP1B) function, structure, and inhibition strategies to develop antidiabetic drugs.

Tyrosine protein phosphatase non-receptor type 1 (PTP1B; also known as protein tyrosine phosphatase 1B) is a member of the protein tyrosine phosphatase (PTP) family and is a soluble enzyme that plays an essential role in different physiological processes, including the regulation of metabolism, specifically in insulin and leptin sensitivity. PTP1B is crucial in the pathogenesis of type 2 diabetes mellitus and obesity. These biological functions have made PTP1B validated as an antidiabetic and anti-obesity, and potentially anticancer, molecular target. Four main approaches aim to inhibit PTP1B: orthosteric, allosteric, bidentate inhibition, and PTPN1 gene silencing. Developing a potent and selective PTP1B inhibitor is still challenging due to the enzyme's ubiquitous expression, subcellular location, and structural properties. This article reviews the main advances in the study of PTP1B since it was first isolated in 1988, as well as recent contextual information related to the PTP family to which this protein belongs. Furthermore, we offer an overview of the role of PTP1B in diabetes and obesity, and the challenges to developing selective, effective, potent, bioavailable, and cell-permeable compounds that can inhibit the enzyme.

New phenylthiosemicarbazide-phenoxy-1,2,3-triazole-N-phenylacetamides as dual inhibitors against α-glucosidase and PTP-1B for the treatment of type 2 diabetes.

This study describes the design, synthesis, and evaluation of a novel series of phenylthiosemicarbazide-phenoxy-1,2,3-triazole-N-phenylacetamide derivatives (7a-l) as dual inhibitors of α-glucosidase and protein tyrosine phosphatase 1-B (PTB-1B). The latter enzymes are two important targets in the treatment of type 2 diabetes. The in vitro obtained data demonstrated that all title compounds 7a-l were more potent than the standard inhibitor acarbose against α-glucosidase while only four derivatives (7a, 7g, 7h, and 7h) were more potent than the standard inhibitor suramin against PTP-1B. Furthermore, these data showed that the most potent α-glucosidase inhibitor was compound 7i, with sixfold higher inhibitory activity than acarbose, and the most potent PTP-1B inhibitor was compound 7a with 3.5-fold higher inhibitory activity than suramin. Kinetic studies of compounds 7i and 7a revealed that they inhibited their target enzymes in a competitive mode. The docking study demonstrated that compounds 7i and 7a well occupied the active site pockets of α-glucosidase and PTP-1B, respectively. In silico pharmacokinetic and toxicity assays of the most potent compounds were performed, and the obtained results were compared with those of the standard inhibitors.

Citric Acid Controls the Activity of YopH Bacterial Tyrosine Phosphatase.

Citric acid (CA) is a tricarboxylic acid with antioxidant and antimicrobial properties. Based on previous studies, the small compound with its three carboxylic groups can be considered a protein tyrosine phosphatase inhibitor. YopH, a protein tyrosine phosphatase, is an essential virulence factor in Yersinia bacteria. We performed enzymatic activity assays of YopH phosphatase after treatment with citric acid in comparison with the inhibitory compound trimesic acid, which has a similar structure. We also measured the cytotoxicity of these compounds in Jurkat T E6.1 and macrophage J774.2 cell lines. We performed molecular docking analysis of the binding of citric acid molecules to YopH phosphatase. Citric acid and trimesic acid reversibly reduced the activity of YopH enzyme and decreased the viability of Jurkat and macrophage cell lines. Importantly, these two compounds showed greater inhibitory properties against bacterial YopH activity than against human CD45 phosphatase activity. Molecular docking simulations confirmed that citric acid could bind to YopH phosphatase. Citric acid, a known antioxidant, can be considered an inhibitor of bacterial phosphatases.

β-Cell Insulin Resistance Plays a Causal Role in Fat-Induced β-Cell Dysfunction In Vitro and In Vivo.

In the classical insulin target tissues of liver, muscle, and adipose tissue, chronically elevated levels of free fatty acids (FFA) impair insulin signaling. Insulin signaling molecules are also present in β-cells where they play a role in β-cell function. Therefore, inhibition of the insulin/insulin-like growth factor 1 pathway may be involved in fat-induced β-cell dysfunction. To address the role of β-cell insulin resistance in FFA-induced β-cell dysfunction we co-infused bisperoxovanadate (BPV) with oleate or olive oil for 48 hours in rats. BPV, a tyrosine phosphatase inhibitor, acts as an insulin mimetic and is devoid of any antioxidant effect that could prevent β-cell dysfunction, unlike most insulin sensitizers. Following fat infusion, rats either underwent hyperglycemic clamps for assessment of β-cell function in vivo or islets were isolated for ex vivo assessment of glucose-stimulated insulin secretion (GSIS). We also incubated islets with oleate or palmitate and BPV for in vitro assessment of GSIS and Akt (protein kinase B) phosphorylation. Next, mice with β-cell specific deletion of PTEN (phosphatase and tensin homolog; negative regulator of insulin signaling) and littermate controls were infused with oleate for 48 hours, followed by hyperglycemic clamps or ex vivo evaluation of GSIS. In rat experiments, BPV protected against fat-induced impairment of β-cell function in vivo, ex vivo, and in vitro. In mice, β-cell specific deletion of PTEN protected against oleate-induced β-cell dysfunction in vivo and ex vivo. These data support the hypothesis that β-cell insulin resistance plays a causal role in FFA-induced β-cell dysfunction.

Stereoselective Syntheses of Organophosphorus Compounds

The review is devoted to the theoretical and synthetic aspects of the stereochemistry of organophosphorus compounds. Organophosphorus compounds are not only widely exist in biologically active pharmaceuticals and agrochemicals, but also have widespread applications in material science and organic synthesis as ligands for transition metal complexes. One of the mainstreams for the development in this field is the creation of biologically active organophosphorus compounds that are searched and used as drugs or plant-protecting agents, which leads to the elaboration of advanced methods and monitoring, yielding up-to-date approaches to perform synthesis in an environmentally friendly manner. The review consists of two parts. The first part presents methods for the asymmetric synthesis of organophosphorus compounds using asymmetric organocatalysis and metal complex catalysis. In the review is described the nature of the chirality generation in the prebiotic period, the mechanisms of asymmetric induction, and double stereodifferentiation are discussed. The use of these methods for the preparation of chiral phosphorus analogs of natural compounds (phosphono-isonorstatin, phosphono-GABOB, phosphacarnitine, bis-phosphonates, and others) is described. Some data concerning of λ5-phosphanediones as metaphosphate anion analogues are also reported. The second part of the presented review shows examples of the use of these methods for the synthesis of phosphorus analogues of natural compounds—chiral phosphonoamino acids and hydroxyphosphonates: phosphonoaspartic acid, phosphonoglutamic acid, phosphonohomoproline, chiral bis-phosphonates. The reaction of dehydration aromatization with the formation of pho sphono isoindolinones, including isoindolinone bis-phosphonates, has been studied. Some of the synthesized compounds showed biological activity as protein tyrosine phosphatase inhibitors. A phosphonic analogue of iso-norstatine was synthesized. A stereoselective method for the synthesis of tetradecapentaenoic acid derivatives was developed.

Natural product-inspired molecules for covalent inhibition of SHP2 tyrosine phosphatase

Natural products have been playing indispensable roles in the development of lifesaving drug molecules. They are also valuable sources for covalent protein modifiers. However, they often are scarce in nature and have complex chemical structures, which are limiting their further biomedical development. Thus, natural product-inspired small molecules which still contain the essence of the parent natural products but are readily available and amenable for structural modification, are important and desirable in searching for lead compounds for various disease treatment. Inspired by the complex and diverse ent-kaurene diterpenoids with significant biological activities, we have created a synthetically accessible and focused covalent library by incorporating the bicyclo[3.2.1]octane α-methylene ketone, which is considered as the pharmacophore of ent-kaurene diterpenoids, as half of the structure, and replacing the other half with much less complex but more druglike scaffolds. From this library, we have identified and characterized selective covalent inhibitors of protein tyrosine phosphatase SHP2, an important anti-cancer therapeutic target. The success of this study demonstrated the importance of creating and evaluating natural product-inspired library as well as their application in targeting challenging disease targets.

Vanadium Complexes with Thioanilide Derivatives of Amino Acids: Inhibition of Human Phosphatases and Specificity in Various Cell Models of Metabolic Disturbances.

In the text, the synthesis and characteristics of the novel ONS-type vanadium (V) complexes with thioanilide derivatives of amino acids are described. They showed the inhibition of human protein tyrosine phosphatases (PTP1B, LAR, SHP1, and SHP2) in the submicromolar range, as well as the inhibition of non-tyrosine phosphatases (CDC25A and PPA2) similar to bis(maltolato)oxidovanadium(IV) (BMOV). The ONS complexes increased [14C]-deoxy-D-glucose transport into C2C12 myocytes, and one of them, VC070, also enhanced this transport in 3T3-L1 adipocytes. These complexes inhibited gluconeogenesis in hepatocytes HepG2, but none of them decreased lipid accumulation in the non-alcoholic fatty liver disease model using the same cells. Compared to the tested ONO-type vanadium complexes with 5-bromosalicylaldehyde and substituted benzhydrazides as Schiff base ligand components, the ONS complexes revealed stronger inhibition of protein tyrosine phosphatases, but the ONO complexes showed greater activity in the cell models in general. Moreover, the majority of the active complexes from both groups showed better effects than VOSO4 and BMOV. Complexes from both groups activated AKT and ERK signaling pathways in hepatocytes to a comparable extent. One of the ONO complexes, VC068, showed activity in all of the above models, including also glucose utilizatiand ONO Complexes are Inhibitors ofon in the myocytes and glucose transport in insulin-resistant hepatocytes. The discussion section explicates the results within the wider scope of the knowledge about vanadium complexes.

Effect of sodium stibogluconate in recruiting and awakening immune cells in the pleural fluid of pancreatic cancer: preparation for immunotherapy.

Ascites and pleural effusion are recognized complications of pancreatic cancer. These diseases are accompanied by ascites and pleural effusion, and drug treatment is limited by high costs, long hospital stays, and failure rates. Immunotherapy may offer new option, but in most patients with late stages of cancer, immune cells may lose the ability to recognize tumor cells, how to activate their immune cells is a major problem, sodium glucosidate (SSG) is injected into ascites as a protein tyrosine phosphatase inhibitor to wake up immune cells and prepare for immunotherapy. We used single-cell RNA sequencing (scRNA-seq) to investigate whether and how SSG injected into ascites of pancreatic cancer elicits an immune response. Our study showed that the process of SSG fusion treatment of ascites and pleural effusion, the interaction between TandNK cells, MPs cells, monocytes and neutrophils was induced, and large numbers of genes were expressed, resulting in upregulation of immune response, which also approved that SSG is not only used as a protein tyrosine phosphatase inhibitor, but also it works as a protein tyrosine phosphatase inhibitor. It can also be used to regulate immune cell function, recruiting immune cells to the right place with the help of PD-1 or PD-L1 to fight cancer cells in ascites and pleural effusions in cancer patients.

Kaempferol activates chloride secretion via the cAMP/PKA signaling pathway and expression of CFTR in T84 cells.

Kaempferol is a flavonol identified as the most potent activator of chloride (Cl-) secretion among other flavonoids in airway epithelial cells. This study aimed to investigate the cellular mechanisms by which kaempferol stimulates Cl- secretion in the T84 human colon carcinoma cell line by Ussing chambers and voltage clamp technique. Bilateral addition of kaempferol (1-100µM) increased short-circuit current (I sc ) in a concentration-dependent manner. Ion substitution of Cl- or CFTR inhibitors NPPB and glibenclamide or a Na+/K+/2Cl- cotransporter inhibitor bumetanide attenuated kaempferol-induced I sc response. In permeabilized monolayers, selective channel inhibitors CFTRinh-172 and CaCCinh-A01 inhibited kaempferol-induced apical Cl- current (I Cl ), and K+ blockers BaCl2 and clotrimazole inhibited basolateral K+ current (I Kb ). The kaempferol-induced I Cl showed no additive effects with forskolin or 8cpt-cAMP. The kaempferol-induced I Cl was mostly abolished by protein kinase A inhibitor H89, but not by tyrosine kinase inhibitors, AG490 and tyrphostin A23, or tyrosine phosphatase inhibitor vanadate. Treatment with kaempferol for 24h increased the expression of CFTR protein as determined by the Western blot analysis. These results demonstrated that kaempferol activates Cl- secretion across T84 cells by activating the apical Cl- current and basolateral K+ current. The mechanisms may involve the cAMP/PKA pathway and CFTR expression. Taken together, these findings reveal the beneficial effects of kaempferol to increase fluid secretion which can be used to treat constipation.

Mitochondrial metabolic flexibility is critical for CD8+ T cell antitumor immunity.

Mitochondria use different substrates for energy production and intermediatory metabolism according to the availability of nutrients and oxygen levels. The role of mitochondrial metabolic flexibility for CD8+ T cell immune response is poorly understood. Here, we report that the deletion or pharmacological inhibition of protein tyrosine phosphatase, mitochondrial 1 (PTPMT1) significantly decreased CD8+ effector T cell development and clonal expansion. In addition, PTPMT1 deletion impaired stem-like CD8+ T cell maintenance and accelerated CD8+ T cell exhaustion/dysfunction, leading to aggravated tumor growth. Mechanistically, the loss of PTPMT1 critically altered mitochondrial fuel selection-the utilization of pyruvate, a major mitochondrial substrate derived from glucose-was inhibited, whereas fatty acid utilization was enhanced. Persistent mitochondrial substrate shift and metabolic inflexibility induced oxidative stress, DNA damage, and apoptosis in PTPMT1 knockout cells. Collectively, this study reveals an important role of PTPMT1 in facilitating mitochondrial utilization of carbohydrates and that mitochondrial flexibility in energy source selection is critical for CD8+ T cell antitumor immunity.

Ang1/Tie2/VE-Cadherin Signaling Regulates DPSCs in Vascular Maturation

Adding dental pulp stem cells (DPSCs) to vascular endothelial cell–formed vessel-like structures can increase the longevity of these vessel networks. DPSCs display pericyte-like cell functions and closely assemble endothelial cells (ECs). However, the mechanisms of DPSC-derived pericyte-like cells in stabilizing the vessel networks are not fully understood. In this study, we investigated the functions of E-DPSCs, which were DPSCs isolated from the direct coculture of human umbilical vein endothelial cells (HUVECs) and DPSCs, and T-DPSCs, which were DPSCs treated by transforming growth factor beta 1 (TGF-β1), in stabilizing blood vessels in vitro and in vivo. A 3-dimensional coculture spheroid sprouting assay was conducted to compare the functions of E-DPSCs and T-DPSCs in vitro. Dental pulp angiogenesis in the severe combined immunodeficiency (SCID) mouse model was used to explore the roles of E-DPSCs and T-DPSCs in vascularization in vivo. The results demonstrated that both E-DPSCs and T-DPSCs possess smooth muscle cell–like cell properties, exhibiting higher expression of the mural cell–specific markers and the suppression of HUVEC sprouting. E-DPSCs and T-DPSCs inhibited HUVEC sprouting by activating TEK tyrosine kinase (Tie2) signaling, upregulating vascular endothelial (VE)–cadherin, and downregulating vascular endothelial growth factor receptor 2 (VEGFR2). In vivo study revealed more perfused and total blood vessels in the HUVEC + E-DPSC group, HUVEC + T-DPSC group, angiopoietin 1 (Ang1) pretreated group, and vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor pretreated group, compared to HUVEC + DPSC group. In conclusion, these data indicated that E-DPSCs and T-DPSCs could stabilize the newly formed blood vessels and accelerate their perfusion. The critical regulating pathways are Ang1/Tie2/VE-cadherin and VEGF/VEGFR2 signaling.

Phosphoproteomics and morphology of stored human red blood cells treated by protein tyrosine phosphatases inhibitor

AbstractThe process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology. This study investigated the relationship between pY and the storage lesions. To do so, RBCs were treated (in the absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate [OV]) to stimulate phosphorylation and with 3 selective kinase inhibitors (KIs). Erythrocyte membrane proteins were studied by western blot analyses and phosphoproteomics (data are available via ProteomeXchange with identifier PXD039914) and cell morphology by digital holographic microscopy. The increase of pY triggered by OV treatment (inducing a global downregulation of pS and pT) disappeared during the storage. Phosphoproteomic analysis identified 609 phosphoproteins containing 1752 phosphosites, of which 41 pY were upregulated and 2 downregulated by OV. After these phosphorylation processes, the shape of RBCs shifted from discocytes to spherocytes, and the addition of KIs partially inhibited this transition. The KIs modulated either pY or pS and pT via diverse mechanisms related to cell shape, thereby affecting RBC morphology. The capacity of RBCs to maintain their function is central in transfusion medicine, and the presented results contribute to a better understanding of RBC biology.

Network pharmacology and experiment indicated that medicinal food homologous components play important roles in insomnia

AbstractInsomnia refers to a subjective experience where insufficient sleep duration and quality affect daytime social functioning. This study aims to screen bioactive components with medicinal food homologous (MFH), such as quercetin, beta‐sitosterol, kaempferol, stigmasterol, and capsaicin, which can treat insomnia and demonstrate that quercetin acts as a direct inhibitor of insomnia‐related glucosylceramidase and as an insulin receptor tyrosine phosphatase inhibitor in the upstream insulin signaling pathway, affecting insomnia targets in the downstream pathway for the treatment of insomnia, using network pharmacology and cell experiment approaches. The results showed that 703 components in 44 MFH were enriched in 139 metabolic pathways and acted on 210 potential targets of insomnia. The protein–protein interaction network is analyzed by cytoNCA, according to the topological analysis results, quercetin, beta‐sitosterol, kaempferol, stigmasterol, and capsaicin of betweenness ranked in the top 10. Quercetin and glucosylceramidase were shown to directly inhibit insomnia proteins through reverse molecular docking. Quercetin was used to culture and stimulate HepG2 cells to detect tyrosine phosphorylation of key cellular proteins in the insulin signaling pathway, concluding that quercetin inhibits the activity of tyrosine phosphatase in a dose‐dependent manner, activating the insulin signaling pathway, triggering a cascade reaction, and affecting the expression of insomnia targets in the downstream pathway. The molecular docking of tyrosine phosphatase and quercetin revealed that quercetin interacted with it near the catalytic site, thereby inhibiting its interaction with the substrate. Overall, this study facilitates the development of medicinal food homologous, which exerts good pharmacological effects in the treatment of insomnia.

A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources.

The detection of superoxide anion (O2●-) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O2●- since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O2●-. In O2●- producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O2●- generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO-). To exclude the effects of altered tyrosine phosphorylation, O2●- was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O2●- in biological systems.

Manipulating PTPRD function with ectodomain antibodies.

Protein tyrosine phosphatases (PTPs) are critical regulators of signal transduction but have yet to be exploited fully for drug development. Receptor protein tyrosine phosphatase δ (RPTPδ/PTPRD) has been shown to elicit tumor-promoting functions, including elevating SRC activity and promoting metastasis in certain cell contexts. Dimerization has been implicated in the inhibition of receptor protein tyrosine phosphatases (RPTPs). We have generated antibodies targeting PTPRD ectodomains with the goal of manipulating their dimerization status ectopically, thereby regulating intracellular signaling. We have validated antibody binding to endogenous PTPRD in a metastatic breast cancer cell line, CAL51, and demonstrated that a monoclonal antibody, RD-43, inhibited phosphatase activity and induced the degradation of PTPRD. Similar effects were observed following chemically induced dimerization of its phosphatase domain. Mechanistically, RD-43 triggered the formation of PTPRD dimers in which the phosphatase activity was impaired. Subsequently, the mAb-PTPRD dimer complex was degraded through lysosomal and proteasomal pathways, independently of secretase cleavage. Consequently, treatment with RD-43 inhibited SRC signaling and suppressed PTPRD-dependent cell invasion. Together, these findings demonstrate that manipulating RPTP function via antibodies to the extracellular segments has therapeutic potential.

Discovery of benzofuran-2-carboxylic acid derivatives as lymphoid tyrosine phosphatase (LYP) inhibitors for cancer immunotherapy

Lymphoid-tyrosine phosphatase (LYP) is mainly expressed in the immune system and plays an important role in the T-cell receptor (TCR) signaling pathway and tumor immunity. Herein, we identify benzofuran-2-carboxylic acid as a potent pTyr mimic and design a new series of new LYP inhibitors. The most active compound, D34 and D14, reversibly inhibits LYP (Ki = 0.93 μM and 1.34 μM) and possess a certain degree of selectivity toward other phosphatases. Meanwhile, D34 and D14 regulate the TCR signaling by specifically inhibiting LYP. In particular, D34 and D14 significantly suppress tumor growth in an MC38 syngeneic mouse model by boosting antitumor immunity, including activation of T-cell and inhibition of M2 macrophage polarization. Moreover, treatment of D34 or D14 upregulate PD-1/PD-L1 expression, which can be leveraged with PD-1/PD-L1 inhibition to augment immunotherapy. In summary, our study demonstrates the feasibility of targeting LYP for cancer immunotherapy and provides new lead compounds for further drug development.

Genetic deletion or tyrosine phosphatase inhibition of PTPRZ1 activates c-Met to up-regulate angiogenesis and lung adenocarcinoma growth.

Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) expressed in endothelial cells and required for stimulation of cell migration by vascular endothelial growth factor A165 (VEGFA165 ) and pleiotrophin (PTN). It is also over or under-expressed in various tumor types. In this study, we used genetically engineered Ptprz1-/- and Ptprz1+/+ mice to study mechanistic aspects of PTPRZ1 involvement in angiogenesis and investigate its role in lung adenocarcinoma (LUAD) growth. Ptprz1-/- lung microvascular endothelial cells (LMVEC) have increased angiogenic features compared with Ptprz1+/+ LMVEC, in line with the increased lung angiogenesis and the enhanced chemically induced LUAD growth in Ptprz1-/- compared with Ptprz1+/+ mice. In LUAD cells isolated from the lungs of urethane-treated mice, PTPRZ1 TP inhibition also enhanced proliferation and migration. Expression of beta 3 (β3 ) integrin is decreased in Ptprz1-/- LMVEC, linked to enhanced VEGF receptor 2 (VEGFR2), c-Met tyrosine kinase (TK) and Akt kinase activities. However, only c-Met and Akt seem responsible for the enhanced endothelial cell activation in vitro and LUAD growth and angiogenesis in vivo in Ptprz1-/- mice. A selective PTPRZ1 TP inhibitor, VEGFA165 and PTN also activate c-Met and Akt in a PTPRZ1-dependent manner in endothelial cells, and their stimulatory effects are abolished by the c-Met TK inhibitor (TKI) crizotinib. Altogether, our data suggest that low PTPRZ1 expression is linked to worse LUAD prognosis and response to c-Met TKIs and uncover for the first time the role of PTPRZ1 in mediating c-Met activation by VEGFA and PTN.

Mapping the Chemical Space of Active-Site Targeted Covalent Ligands for Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.

Abstract 4038: Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells

Abstract Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) has emerged as a promising cancer immunotherapy target. Previously, we reported on the functional role of PTPN2 in the pathogenesis of colorectal carcinoma (CRC). We demonstrated increased PTPN2 phosphatase activity correlates with disease progression and decreased immune responses in tumor tissues, while loss of PTPN2 in T-cells or in dendritic cells (DCs) reduces tumor burden in several CRC models and potentiates anti-PD1 efficacy (Katkeviciute et al., 2021). Others reported that loss of PTPN2 in tumor cells sensitizes to immune-mediated tumor killing (Manguso et al., 2017; Goh et al., 2022). Thus, PTPN2 was proposed as a critical node to modulate anti-tumor immunity, and PTPN2 inhibition is expected to enhance anti-tumor immunity by sensitizing tumor cells and by activating immune cells. Recently, also the closely related phosphatase PTPN1 (PTP1B) was demonstrated to restrain T-cell mediated tumor killing (Wiede et al., 2022). Inhibition of Protein Tyrosine Phosphatases has historically been a demanding area for drug discovery. However, both allosteric PTP inhibitors (eg targeting SHP2/PTPN11) and catalytic-site inhibitors (targeting PTPN2/N1) have recently progressed to clinical studies. We now identified a chemical series of novel dual PTPN2/N1 inhibitors. These inhibitors bind to the catalytic site of PTPN2, as confirmed by crystallography. The best molecules of the series are low nM inhibitors of PTPN2 and PTPN1 enzymes with excellent selectivity across other phosphatases. As an immediate consequence of PTPN2/N1 inhibition, these compounds augment phosphorylation of STAT1 and/or STAT5 isoforms in myeloid and T-cell lines, and in primary T-cells, macrophages and DCs. This results in immune-cell activation as evidenced by increased production of effector cytokines (e.g. IFNγ), immune-activation, levels of cytotoxicity markers in T-cells (e.g. granzyme B) as well as upregulation of antigen presentation and co-stimulatory markers in macrophages and DCs. Furthermore, inhibition of PTPN2/N1 sensitizes murine and human cancer cell lines to IFNγ, and enhances immune-mediated tumor killing in co-culture assays in vitro. In vivo evaluation of selected examples is in progress and results will be presented at the conference. Our data indicate that inhibition of PTPN2/N1 phosphatase activity is a powerful pharmacologic strategy to promote anti-tumor immunity, with strong potential to be exploited for cancer immunotherapy both as a single agent treatment in anti-PD1 refractory cancers, and in combination with anti-PD1 therapy. Citation Format: Kalliopi Pervolaraki, Egle Katkeviciute, Dominique Lambin, Sandro Boland, Amuri Kilonda, Vincent Pericolle, Marnik Nijs, Wanda Haeck, Kristine Metzger, Hugo Klaassen, Arnaud Marchand, Patrick Chaltin, Matthias Versele, Marianne Spalinger, Michael Scharl. Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4038.

Virtual Screening and Biological Evaluation of Novel Low Molecular Weight Protein Tyrosine Phosphatase Inhibitor for the Treatment of Insulin Resistance.

Protein tyrosine phosphatases (PTPs) play an essential way in diseases including cancer, obesity, diabetes and autoimmune disorders. As a member of PTPs, low molecular weight PTP (LMPTP) has been a well-recognized anti-insulin resistance target in obesity. However, the number of reported LMPTP inhibitors is limited. Our research aims to discover a novel LMPTP inhibitor and evaluate its biological activity against insulin resistance. A virtual screening pipeline based on the X-ray co-crystal complex of LMPTP was constructed. Enzyme inhibition assay and cellular bioassay were used to evaluate the activity of screened compounds. The screening pipeline rendered 15 potential hits from Specs chemical library. Enzyme inhibition assay identified compound F9 (AN-465/41163730) as a potential LMPTP inhibitor with a K i value of 21.5 ± 7.3 μM. Cellular bioassay showed F9 could effectively increase the glucose consumption of HepG2 cells as a result of releasing insulin resistance by regulating PI3K-Akt pathway. In summary, this study presents a versatile virtual screening pipeline for potential LMPTP inhibitor discovery and provides a novel-scaffold lead compound that is worthy of further modification to get more potent LMPTP inhibitors.