Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.

Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 µM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.

Design, synthesis and biological evaluation of imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as lymphoid-specific tyrosine phosphatase inhibitors.

Lymphoid-specific tyrosine phosphatase (LYP), which exclusively exists in immune cells and down-regulates T cell receptor signaling (TCR), has becoming a potent target for various autoimmune diseases. Herein, we designed and synthesized imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as new LYP inhibitors. Among them, the cinnamic acids-based inhibitors (9p and 9r) displayed good LYP inhibitory activities (IC50 = 2.85-6.95 µM). Especially, the most potent inhibitor 9r was identified as competitive inhibitor (Ki = 1.09 µM) and bind LYP reversibly. Meanwhile, 9r exhibited better selectivity over other phosphatases than known LYP inhibitor A15. Furthermore, compound 9r could regulate TCR associated signaling pathway in Jurkat T cell.

Identification and structure-function analyses of an allosteric inhibitor of the tyrosine phosphatase PTPN22.

Protein-tyrosine phosphatase nonreceptor type 22 (PTPN22) is a lymphoid-specific tyrosine phosphatase (LYP), and mutations in the PTPN22 gene are highly correlated with a spectrum of autoimmune diseases. However, compounds and mechanisms that specifically inhibit LYP enzymes to address therapeutic needs to manage these diseases remain to be discovered. Here, we conducted a similarity search of a commercial database for PTPN22 inhibitors and identified several LYP inhibitor scaffolds, which helped identify one highly active inhibitor, NC1. Using noncompetitive inhibition curve and phosphatase assays, we determined NC1's inhibition mode toward PTPN22 and its selectivity toward a panel of phosphatases. We found that NC1 is a noncompetitive LYP inhibitor and observed that it exhibits selectivity against other protein phosphatases and effectively inhibits LYP activity in lymphoid T cells and modulates T-cell receptor signaling. Results from site-directed mutagenesis, fragment-centric topographic mapping, and molecular dynamics simulation experiments suggested that NC1, unlike other known LYP inhibitors, concurrently binds to a "WPD" pocket and a second pocket surrounded by an LYP-specific insert, which contributes to its selectivity against other phosphatases. Moreover, using a newly developed method to incorporate the unnatural amino acid 2-fluorine-tyrosine and 19F NMR spectroscopy, we provide direct evidence that NC1 allosterically regulates LYP activity by restricting WPD-loop movement. In conclusion, our approach has identified a new allosteric binding site in LYP useful for selective LYP inhibitor development; we propose that the 19F NMR probe developed here may also be useful for characterizing allosteric inhibitors of other tyrosine phosphatases.

Protective Effect of Pinitol Against Inflammatory Mediators of Rheumatoid Arthritis via Inhibition of Protein Tyrosine Phosphatase Non-Receptor Type 22 (PTPN22).

BACKGROUND The aim of the current study was to explore the anti-arthritic effect of pinitol via assessing its effect on various inflammatory mediators and its possible mechanism of action. MATERIAL AND METHODS We assessed the anti-arthritic effect of pinitol in a formaldehyde- and CFA-induced arthritic model in Wistar Swiss albino strain rats divided into 6 groups. The rats received different doses of pinitol and indomethacin for 28 days. The arthritic index and body weight were determined at regular intervals, together with hepatic, hematological, and antioxidant parameters. The expression of proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-1ß) and inflammatory mediators (e.g., COX-2 and VEGF) were also estimated with histopathological evaluation of the joint tissue of rats. A docking study of pinitol with PTPN22 was also carried out. RESULTS The CFA-induced model rats developed redness and nodules in the tail and front paws, and the arthritic control (AC) group rats showed similar symptoms, which were decreased by pinitol administration. The body weight of AC group rats was decreased, while pinitol-treated rats showed considerably increased body weight. Hematological, hepatic, and antioxidant parameters were altered by pinitol in a dose-dependent manner. Pinitol significantly decreased the elevated concentration of proinflammatory cytokines and inflammatory mediators, with improvement in histopathological condition. The docking study suggested that pinitol efficiently interacted with PTPN22 via Arg59, Tyr60, Leu106, and Lys138 by creating close interatomic hydrogen bonds and hydrophobic contacts. CONCLUSIONS Pinitol showed anti-arthritic effects via reduction of proinflammatory cytokines and inflammatory mediators via inhibition of PTPN22.

C1858T Polymorphism of Protein Tyrosine Phosphatase Non-receptor Type 22 (PTPN22): an eligible target for prevention of type 1 diabetes?

INTRODUCTION: In type 1 diabetes (T1D), several genetic factors are associated to ß-cell autoimmunity onset and clinical progression. HLA-genes play a major role in susceptibility and initiation of ß-cell autoimmunity, whereas non-HLA genes may influence the destruction rate. Areas covered: Our review focuses on the possible role of the PTPN22 C1858 T variant as a prognostic factor, given its influence on disease variability. Moreover, we present the potential role of C1858 T as a target for tertiary prevention trials and new therapeutic strategies, such as the LYP inhibitors. We used PubMed for literature research; key words were 'PTPN22', 'C1858 T polymorphism', 'lymphoid-specific tyrosine phosphatase' and 'type 1 diabetes'. We selected publications between 2000 and 2016. Expert commentary: Current data suggest that PTPN22 can be a promising target for therapeutic interventions and identification of at-risk subjects in autoimmune diseases such as T1D.

Suppression of protein tyrosine phosphatase PTPN22 gene induces apoptosis in T-cell leukemia cell line (Jurkat) through the AKT and ERK pathways.

The aim of this study was to investigate the effect of specific PTPN22 small interfering RNAs (siRNAs) on the viability and induction of apoptosis in Jurkat cells and to evaluate apoptosis signaling pathways. In this study, Jurkat cells were transfected with specific PTPN22 siRNA. Relative PTPN22 mRNA expression was measured by Quantitative Real-time PCR. Western blotting was performed to determine the protein levels of PTPN22, AKT, P-AKT, ERK, and P-ERK. The cytotoxic effects of PTPN22 siRNA were determined using the MTT assay. Apoptosis was quantified using TUNEL assay and flow cytometry. Results showed that in Jurkat cells after transfection with PTPN22 siRNA, the expression of PTPN22 in both mRNA and protein levels was effectively reduced. Moreover, siRNA transfection induced apoptosis on the viability of T-cell acute leukemia cells. More importantly, PTPN22 positively regulated the anti-apoptotic AKT kinase, which provides a powerful survival signal to T-ALL cells as well as the suppression of PTPN22 down regulated ERK activity. Our results suggest that the PTPN22 specific siRNA effectively decreases the viability of T-cell acute leukemia cells, induces apoptosis in this cell line, and therefore could be considered as a potent adjuvant in T-ALL therapy.

Protein Flexibility in Docking-Based Virtual Screening: Discovery of Novel Lymphoid-Specific Tyrosine Phosphatase Inhibitors Using Multiple Crystal Structures.

Incorporating protein flexibility is a major challenge for docking-based virtual screening. With an increasing number of available crystal structures, ensemble docking with multiple protein structures is an efficient approach to deal with protein flexibility. Herein, we report the successful application of a docking-based virtual screen using multiple crystal structures to discover novel inhibitors of lymphoid-specific tyrosine phosphatase (LYP), a potential drug target for autoimmune diseases. The appropriate use of multiple protein structures allowed a better enrichment than a single structure in the recovery of known inhibitors. Subsequently, an optimal ensemble of LYP structures was selected and used in docking-based virtual screening. Eight novel LYP inhibitors (IC50 ranging from 7.95 to 56.6 µM) were identified among 23 hit compounds. Further studies demonstrated that the most active compound B15 possessed some selectivity over other protein phosphatases and could effectively up-regulate TCR (T cell receptor)-mediated signaling in Jurkat T cells. These novel hits not only provided good starting points for the development of therapeutic agents useful in autoimmune diseases but also demonstrated the advantages of choosing an appropriate ensemble of protein structures in docking-based virtual screening over using a single protein conformation.

Fast identification of novel lymphoid tyrosine phosphatase inhibitors using target-ligand interaction-based virtual screening.

Lymphoid-specific tyrosine phosphatase (Lyp), a critical signaling regulator of immune cells, is associated with various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Recent research suggests that Lyp is a potential drug target for autoimmune diseases. Herein, we applied a target-ligand interaction-based virtual screening method to identify novel Lyp inhibitors. Nine Lyp inhibitors with novel scaffolds were identified with eight reversible inhibitors (Ki values ranged from 2.87 to 28.03 µM) and one covalent inhibitor (Ki = 40.98 ± 13.19 µM). The top four compounds (A2, A15, A19, and A26) displayed selectivity over other phosphatases in preliminary experiments, and kinetic analysis indicated that these compounds are competitive inhibitors of Lyp. Compounds A15 and A19 up-regulated TCR (T cell receptor) mediated signaling and transcriptional activation through inhibition of Lyp activity in T cells. The new chemotypes of Lyp selective inhibitors identified through the target-ligand interaction-based virtual screening may provide new leads for Lyp targeted therapeutic development.

Covalent inhibition of the lymphoid tyrosine phosphatase.

Covalent inhibitors of lymphoid tyrosine phosphatase (LYP) were identified from a screen of the NIH Molecular Libraries Small Molecules Repository (MLSMR). Both of the two lead compounds identified have phosphotyrosine-mimetic benzoic acid moieties as well as electrophilic acrylonitrile groups. Inhibition kinetics of both compounds are consistent with covalent modification of the enzyme, with nanomolar KI and reciprocal millisecond kinact values, representing the best efficiency ratios (kinact /KI ) among currently reported covalent LYP inhibitors. Covalent inhibitors can provide longer efficacy and better selectivity than more conventional noncovalent inhibitors, and these lead compounds are an important step toward the development of protein tyrosine phosphatase (PTP)-targeted covalent therapeutic compounds.

Lymphoid-specific tyrosine phosphatase (Lyp): a potential drug target for treatment of autoimmune diseases.

Lymphoid-tyrosine phosphatase (Lyp), encoded by the PTPN22 gene, is a member of the protein tyrosine phosphatase family enzymes. Human genetics studies have shown that a single-nucleotide polymorphism in PTPN22 is often mutated in patients suffering from autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosis. Because of its critical role in the regulation of T-cell Receptor (TCR) signaling pathways, Lyp recently emerged as a candidate target for therapy of autoimmune diseases. Herein, we review the structure and splice isoforms of Lyp, the biochemistry of the disease-predisposing allele, discuss the function of the phosphatase in TCR signaling and the association with human autoimmune diseases. Especially, we summarized recent progress in the development of Lyp inhibitors, intending to provide a basis for the Lyp-based treatment of autoimmunity. Moreover, the emphasis and direction for future study of Lyp in autoimmune diseases were prospected.

A three-dimensional chemical phase pharmacophore mapping, QSAR modelling and electronic feature analysis of benzofuran salicylic acid derivatives as LYP inhibitors.

Lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has a critical negative regulatory role in T-cell antigen receptor (TCR) and emerged as a promising drug target for human autoimmune diseases. A five-point pharmacophore with two hydrogen bond acceptors, one hydrogen bond donor and two aromatic ring features was generated for a series of benzofuran salicylic acid derivatives as LYP inhibitors in order to elucidate their anti-autoimmune activity. The generated pharmacophore yielded a significant 3D-QSAR model with r(2) of 0.9146 for a training set of 27 compounds. The model also showed excellent predictive power with Q(2) of 0.7068 for a test set of eight compounds. The investigation of the 3D-QSAR model has revealed the structural insights which could lead to more potent analogues. The most active and inactive compounds were further subjected to electronic structure analysis using density functional theory (DFT) at B3LYP/3-21(∗)G level to support the 3D-QSAR predictions. The results obtained from this study are expected to be useful in the proficient design and development of benzofuran salicylic acid derivatives as inhibitors of LYP.

Thiuram disulfides as pseudo-irreversible inhibitors of lymphoid tyrosine phosphatase.

We screened a small library of thiuram disulfides for inhibition of lymphoid tyrosine phosphatase (LYP) activity. The parent thiuram disulfide, disulfiram, inhibited LYP activity in vitro and in Jurkat T cells, whereas diethyldithiocarbamate failed to inhibit LYP at the concentrations tested. Compound 13, an N-(2-thioxothiazolidin-4-one) analogue, was found to be the most potent LYP inhibitor in this series, with an IC50 value of 3 µM. Compound 13 inhibits LYP pseudo-irreversibly, as evidenced by the time-dependence of inhibition, with a K(i) value of 1.1 µM and a k(inact) value of 0.004 s⁻¹. The inhibition of LYP by compound 13 could not be reversed significantly by incubation with glutathione or by prolonged dialysis, but could be partially reversed by incubation with dithiothreitol. Compound 13 also inhibited LYP activity in Jurkat T cells.

PTPN22 silencing in the NOD model indicates the type 1 diabetes-associated allele is not a loss-of-function variant.

PTPN22 encodes the lymphoid tyrosine phosphatase (LYP) and is the second strongest non-HLA genetic risk factor for type 1 diabetes. The PTPN22 susceptibility allele generates an LYP variant with an arginine-to-tryptophan substitution at position 620 (R620W) that has been reported by several studies to impart a gain of function. However, a recent report investigating both human cells and a knockin mouse model containing the R620W homolog suggested that this variation causes faster protein degradation. Whether LYP R620W is a gain- or loss-of-function variant, therefore, remains controversial. To address this issue, we generated transgenic NOD mice (nonobese diabetic) in which Ptpn22 can be inducibly silenced by RNA interference. We found that Ptpn22 silencing in the NOD model replicated many of the phenotypes observed in C57BL/6 Ptpn22 knockout mice, including an increase in regulatory T cells. Notably, loss of Ptpn22 led to phenotypic changes in B cells opposite to those reported for the human susceptibility allele. Furthermore, Ptpn22 knockdown did not increase the risk of autoimmune diabetes but, rather, conferred protection from disease. Overall, to our knowledge, this is the first functional study of Ptpn22 within a model of type 1 diabetes, and the data do not support a loss of function for the PTPN22 disease variant.

Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT.

A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors.

Distinct functional and conformational states of the human lymphoid tyrosine phosphatase catalytic domain can be targeted by choice of the inhibitor chemotype.

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has recently been identified as a promising drug target for human autoimmunity diseases. Like the majority of protein-tyrosine phosphatases LYP can adopt two functionally distinct forms determined by the conformation of the WPD-loop. The WPD-loop plays an important role in the catalytic dephosphorylation by protein-tyrosine phosphatases. Here we investigate the binding modes of two chemotypes of small molecule LYP inhibitors with respect to both protein conformations using computational modeling. To evaluate binding in the active form, we built a LYP protein structure model of high quality. Our results suggest that the two different compound classes investigated, bind to different conformations of the LYP phosphatase domain. Binding to the closed form is facilitated by an interaction with Asp195 in the WPD-loop, presumably stabilizing the active conformation. The analysis presented here is relevant for the design of inhibitors that specifically target either the closed or the open conformation of LYP in order to achieve better selectivity over phosphatases with similar binding sites.

Discovery of a novel series of inhibitors of lymphoid tyrosine phosphatase with activity in human T cells.

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor. The mechanism of action of the lead inhibitor compound 4e was studied by a combination of hydrogen/deuterium-exchange mass spectrometry and molecular modeling. The results suggest that the inhibitor interacts critically with a hydrophobic patch located outside the active site of the phosphatase. Targeting of secondary allosteric sites is viewed as a promising yet unexplored approach to develop pharmacological inhibitors of protein tyrosine phosphatases. Our novel scaffold could be a starting point to attempt development of "nonactive site" anti-LYP pharmacological agents.

Inhibition of lymphoid tyrosine phosphatase by benzofuran salicylic acids.

The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 µM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds.

Target-specific control of lymphoid-specific protein tyrosine phosphatase (Lyp) activity.

Lymphoid-specific protein tyrosine phosphatase (Lyp), a member of the protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important mediator of human-leukocyte signaling. Lyp has also emerged as a potential anti-autoimmune therapeutic target, owing to the association of a Lyp-activating mutation with an array of autoimmune disorders. Toward the goal of generating a selective inhibitor of Lyp activity that could be used for investigating Lyp's roles in cell signaling and autoimmune-disease progression, here we report that Lyp's PTP domain can be readily sensitized to target-specific inhibition by a cell-permeable small molecule. Insertion of a tetracysteine-motif-containing peptide at a conserved position in Lyp's catalytic domain generated a mutant enzyme (Lyp-CCPGCC) that retains activity comparable to that of wild-type Lyp in the absence of added ligand. Upon addition of a tetracysteine-targeting biarsenical compound (FlAsH), however, the activity of the Lyp-CCPGCC drops dramatically, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that FlAsH-induced Lyp-CCPGCC inhibition is potent, specific, rapid, and independent of the nature of the PTP substrate used in the inhibition assay. Moreover, we show that FlAsH can be used to specifically target overexpressed Lyp-CCPGCC in a complex proteomic mixture. Since the mammalian-cell permeability of FlAsH is well established, it is likely that FlAsH-mediated inhibition of Lyp-CCPGCC will be useful for specifically targeting Lyp activity in engineered leukocytes and autoimmune-disease models.

Gold(I) phosphine mediated selective inhibition of lymphoid tyrosine phosphatase.

Selective protein tyrosine phosphatase (PTP) inhibition is often difficult to achieve owing to the high degree of similarity of the catalytic domains of this family of enzymes. Selective inhibitors of the lymphoid specific tyrosine phosphatase, LYP, are of great interest due to the involvement of LYP in several autoimmune disorders. This manuscript describes a study into the mechanistic details of selective LYP inhibition by a Au(I)-phosphine complex. The complex, [Au((CH(2)CH(2)CN)(2)PPh)Cl], selectively inhibits LYP activity both in vitro and in cells, but does not inhibit other T-cell derived PTPs including the highly homologous PTP-PEST. The mode of inhibition was probed by investigating inhibition of LYP, the LYP mutant C129/231S, and PTP-PEST. Inhibition of LYP and PTP-PEST was competitive, while the LYP double mutant appeared mixed. Wild-type LYP was inhibited more potently than LYP C129/231S, indicating an important role for at least one of these residues in Au(I) binding. Coordination of Au(I) by both the active site cysteine residue as well as either Cys129 or 231 is suggested as a potential mechanism for LYP selective inhibition.

Identifying potent, selective protein tyrosine phosphatase inhibitors from a library of Au(I) complexes.

Therapeutic inhibition of protein tyrosine phosphatase activity is a compelling yet challenging approach to the treatment of human disease. Toward this end, a library of 40 gold complexes with the general formula R(3)P-Au-Cl was screened to identify novel inhibitors of PTP activity. The most promising inhibitor obtained for the lymphoid tyrosine phosphatase LYP, (2-pyridine)(Ph(2))P-Au-Cl, is one of the most potent and selective LYP inhibitors identified to date with an IC(50) of 1.5 +/- 0.3 microM, 10-fold selectivity for LYP over PTP-PEST, HePTP, and CD45 in vitro, and activity in cellular studies as well.