Protein Tyrosine Phosphatase 1B Activity Research Articles

A fast and efficient method for screening and evaluation of hypoglycemic ingredients of Traditional Chinese Medicine acting on PTP1B by capillary electrophoresis

As a pivotal enzyme that regulates dephosphorylation in cell activities and participates in the insulin signaling pathway, protein tyrosine phosphatase 1B (PTP1B) is considered to be an important target for the therapy of diabetes. In this work, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE), and used for screening and evaluating the inhibition effect of Traditional Chinese Medicine on PTP1B. Response Surface Methodology was used for optimizing the conditions of analysis. After method validation, the enzyme kinetic study and inhibition test were performed. As a result, the IC50 of PTP1B inhibitors Ⅳ and ⅩⅧ were consistent with reported values measured by a conventional method. It was found that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. showed prominent inhibition on the activity of PTP1B, which were stronger than the positive controls. Meanwhile, on top of the excellent advantages of CE, the whole analysis time is less than 2 min. Thus, the results demonstrated that a fast and efficient screening method was successfully developed. This method could be a powerful tool for screening inhibitors from complex systems. It can also provide an effective basis for lead compound development in drug discovery.

Switching photocurrent polarity and mimicking enzyme functions of MIL-53(Fe)@hemin for homogeneous dual-modal ultrasensitive detection of PTP1B

Protein tyrosine phosphatase 1B (PTP1B) as a regulatory enzyme is an important target for the early diagnosis and drug discovery of diabetes, obesity, and cancer. Herein, a homogeneous dual-modal biosensor integrating photoelectrochemical and colorimetric (PEC-CL) strategies was developed in light of the switching photocurrent polarity and mimicking enzyme functions of MIL-53(Fe)@hemin for the sensitive detection of PTP1B activity. Magnetic Fe3O4@Au NPs were synthesized for more sufficient homogeneous biorecognition, and modified with the phosphorylated peptides (p-Peptide), and sequentially connected with MIL-53(Fe)@hemin by the specific coordination of phosphate groups with Fe(III). After PTP1B-mediated specific recognition and dephosphorylation, the separated MIL-53(Fe)@hemin were quickly collected via magnetic separation for further PEC and CL assay. MIL-53(Fe)@hemin not only switched the photocurrent polarity of SnS2 NFs from anode to cathode, but also exhibited excellent peroxidase-like activity for oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. As a signal amplifier, MIL-53(Fe) acted as a nanocarrier to load abundant PEC signal switching factor of hemin, and synergistically enhanced its enzyme-mimic property. Consequently, ultrasensitive PEC biosensing (with linear response range of 0.1 pg mL−1 to 3000 ng mL−1 and detection limit of 0.074 pg mL−1) assisted by visual CL strategy (with linear response range of 10 pg mL−1 to 3000 ng mL−1 and detection limit of 0.24 pg mL−1) for PTP1B activity assay were achieved, which not only provided high sensitivity, but also possessed portability, perceptual intuition and reliability.

The synergistic regulatory effect of PTP1B and PTK inhibitors on the development of Oedaleus decorus asiaticus Bei-Bienko

Tyrosine phosphorylation is crucial for controlling normal cell growth, survival, intercellular communication, gene transcription, immune responses, and other processes. PTP (protein tyrosine phosphatase) and PTK (protein tyrosine kinases) can achieve this goal by regulating multiple signaling pathways. Oedaleus decorus asiaticus is an important pest that infests the Mongolian Plateau grassland. We aimed to evaluate the survival rate, growth rate, overall performance, and ovarian developmental morphology of the 4th instar nymphs of O. decorus asiaticus while inhibiting the activity of PTP1B (protein tyrosine phosphatase-1B) and PTK. In addition, the expression and protein phosphorylation levels of key genes in the MAPK signaling pathway and antioxidant enzyme activity were assessed. The results showed no significant differences in survival rate, growth rate, or overall performance between PTP1B inhibitor treatment and control. However, after PTK inhibitor treatment, these indexes were significantly lower than in the control. The ovarian size of female larvae after 15 days of treatment with PTK inhibitors showed significantly slower development, while female larvae treated with PTP1B exhibited faster ovarian growth than the control group. In comparison to controls and nymphs treated with PTK inhibitors, the expression and phosphorylation levels of key genes in the MAPK signaling pathway under PTP1B inhibitor treatments were significantly higher in 4th instar nymphs. However, ROS levels and the activities of NADPH oxidase and other antioxidant enzymes were considerably reduced, although they were significantly greater in the PTK inhibitor treatment. The results suggest that PTP1B and PTK feedback inhibition in the MAPK signal transfer can regulate the physiological metabolism of the insect as well as its developmental rate. These findings can facilitate future uses of PTP1B and PTK inhibitors in controlling insect development to help control pest populations.

Iridoids from the aerial parts of Adenosma caeruleum with their nitric oxide inhibitory effects

Three new iridoids, adenocaeroids A−C (1−3), and seven known analogs, umbellatolide B (4), pedicularis-lactone (5), iridolactone (6), artselaenin C (7), eucommiol (8), rehmaglutin D (9), and 3-deoxy-artselaenin C (10) were isolated from the aerial parts of Adenosma caeruleum. Their chemical structures were identified by the comprehensive spectroscopic methods, including 1D and 2D NMR and HRESI mass spectra. The relative configurations of 1−3 were established by analyzing their NOESY spectra as well as by DFT-GIAO NMR calculations followed by ML-J-DP4, DP4+, and DU8 methods. The absolute configurations of 1−3 were determined by comparing experimental and calculated ECD spectra and specific optical rotation values. All of the compounds, except 7 and 10, inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV2 microglial cells in a dose-dependent manner, with IC50 values ranging from 28.4−61.8 µM. However, none of the compounds showed inhibitory effect on protein-tyrosine phosphatase 1B (PTP1B) activity.

Schnurri-3 drives tumor growth and invasion in cancer cells expressing interleukin-13 receptor alpha 2

Interleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/β-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.

Oleanolic acid derivative alleviates cardiac fibrosis through inhibiting PTP1B activity and regulating AMPK/TGF-β/Smads pathway

Cardiac fibrosis (CF) in response to persistent exogenous stimuli or myocardial injury results in cardiovascular diseases (CVDs). Protein tyrosine phosphatase 1B (PTP1B) can promote collagen deposition through regulating AMPK/TGF-β/Smads signaling pathway, and PTP1B knockout improves cardiac dysfunction against overload-induced heart failure. Oleanolic acid (OA) has been proven to be an inhibitor of PTP1B, and its anti-cardiac remodeling effects have been validated in different mouse models. To improve the bioactivity of OA and to clarify whether OA derivatives with stronger inhibition of PTP1B activity have greater prevention of cardiac remodeling than OA, four new OA derivatives were synthesized and among them, we found that compound B had better effects than OA in inhibiting cardiac fibrosis both in vivo in the isoproterenol (ISO)-induced mouse cardiac fibrosis and in vitro in the TGF-β/ISO-induced 3T3 cells. Combining with the results of molecular docking, surface plasmon resonance and PTP1B activity assay, we reported that OA and compound B directly bound to PTP1B and inhibited its activity, and that compound B showed comparable binding capability but stronger inhibitory effect on PTP1B activity than OA. Moreover, compound B presented much greater effects on AMPK activation and TGF-β/Smads inhibition than OA. Taken together, OA derivative compound B more significantly alleviated cardiac fibrosis than OA through much greater inhibition of PTP1B activity and thus much stronger regulation of AMPK/TGF-β/Smads signaling pathway.

Vinyl-functionalized covalent organic frameworks with photoelectric property and light-responsive oxidase-mimetic activity for photoelectrochemical and colorimetric dual-mode assay of PTP1B activity

The sensitive and precise assay of protein tyrosine phosphatase 1B (PTP1B) activity plays a considerable role in clinical diagnosis and cancer therapy. Herein, a photoelectrochemical (PEC)-colorimetric (CL) dual-mode biosensor based on magnetic Fe3O4 nanospheres and vinyl-functionalized covalent organic framework (COF-V) with photoelectric property and light-responsive oxidase-mimetic activity was developed for PTP1B activity assay. Fe3O4 nanospheres modified with polydopamine (PDA) and chelated Ti4+ (Fe3O4@PDA-Ti4+) were synthesized by the self-polymerization of dopamine and the chelation reaction between hydroxyl groups in the PDA shell and Ti4+. Subsequently, the PTP1B-specific peptide (P-peptide) was modified on the surface of Fe3O4@PDA-Ti4+ nanosphere via the formation of Ti-O-P bonds between Ti4+ and phosphate group on the P-peptide. Before PTP1B activity assay, COF-V was introduced to the Fe3O4@PDA-Ti4+/P-peptide complex by thiol-ene click reaction of the vinyl group on COF-V with the thiol group of P-peptide, and the Fe3O4@PDA-Ti4+/P-peptide/COF-V complex was obtained. With addition of PTP1B, it can specifically identify and dephosphorylate the P-peptide, resulting in the release of some P-peptide and COF-V from the Fe3O4@PDA-Ti4+/P-peptide/COF-V complex. Then, the undamaged Fe3O4@PDA-Ti4+/P-peptide/COF-V complex was obtained by magnetic separation and used for PEC and CL analysis. Based on the excellent photoelectric property and light-responsive oxidase-mimetic activity of COF-V, PTP1B activity was sensitively assayed, and the linear ranges of 0.01 ng mL−1–1000 ng mL−1 (detection limit, 0.006 ng mL−1) for PEC technique and 0.1 ng mL−1–1000 ng mL−1 (detection limit, 0.03 ng mL−1) for CL method were obtained. Such a design opens a new avenue to detect protein phosphatases activity in biomedical study, disease diagnosis, and cancer treatment.

Ginsenoside F4 Alleviates Skeletal Muscle Insulin Resistance by Regulating PTP1B in Type II Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with increasing morbidity. Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin signaling cascade and has attracted intensive investigation in the T2DM study. Ginseng is widely used to treat metabolic diseases, while the effects of ginsenoside F4 (F4) on T2DM have remained unknown. Here, we identify F4 as an inhibitor of skeletal muscle insulin resistance. The results showed that F4 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the inhibition of the PTP1B activity. On the one hand, the inhibition of PTP1B activity by F4 resulted in increased insulin receptor (INSR) and insulin receptor substrate 1 tyrosine phosphorylation and enhanced insulin sensitivity. On the other hand, F4 as a PTP1B inhibitor inhibited the inositol-requiring enzyme 1 (IRE-1)/recombinant TNF receptor associated factor 2 (TRAF2)/c-Jun N-terminal kinase signaling pathway and alleviated skeletal muscle endoplasmic reticulum (ER) stress, thereby reducing IRS-1 serine phosphorylation. Both finally activated the PI3K/AKT signaling pathway and promoted glucose transporter protein 4 translocation to the cell membrane for glucose uptake. Taken together, our experiments demonstrate that F4 activates the insulin signaling pathway by inhibiting the activity of PTP1B while inhibiting the IRE-1/TRAF2/JNK signaling pathway, enhancing insulin sensitivity, and alleviating ER stress in the skeletal muscle of db/db mice. Our results indicate that F4 can be used as a PTP1B inhibitor for the treatment of T2DM.

Analysis of neutral mutational drift in an allosteric enzyme.

Neutral mutational drift is an important source of biological diversity that remains underexploited in fundamental studies of protein biophysics. This study uses a synthetic transcriptional circuit to study neutral drift in protein tyrosine phosphatase 1B (PTP1B), a mammalian signaling enzyme for which conformational changes are rate limiting. Kinetic assays of purified mutants indicate that catalytic activity, rather than thermodynamic stability, guides enrichment under neutral drift, where neutral or mildly activating mutations can mitigate the effects of deleterious ones. In general, mutants show a moderate activity-stability tradeoff, an indication that minor improvements in the activity of PTP1B do not require concomitant losses in its stability. Multiplexed sequencing of large mutant pools suggests that substitutions at allosterically influential sites are purged under biological selection, which enriches for mutations located outside of the active site. Findings indicate that the positional dependence of neutral mutations within drifting populations can reveal the presence ofallosteric networks and illustrate an approach for using synthetic transcriptional systems to explore these mutations in regulatory enzymes.

Medicinal parts of mulberry (leaf, twig, root bark, and fruit) and compounds thereof are excellent traditional Chinese medicines and foods for diabetes mellitus

Diabetes mellitus (DM) is one of the main chronic diseases severely threatening human health, of which type 2 DM (T2DM) is the most common type. Traditional Chinese medicine has unique advantages in the treatment of T2DM. Mulberry (Morus alba L.) is a medicinal and edible plant that is well known for its rich nutrition and medicinal values. The medicinal parts of mulberry include leaf, twig, root bark, and fruit, all of which have remarkable hypoglycemic effects. In this paper, the extracts and compounds with hypoglycemic effects from four medicinal parts of mulberry are reviewed. It is found that the hypoglycemic compounds in mulberry are mainly flavonoids, polysaccharides, alkaloids, stilbenes, and Diels-Alder compounds. Their mechanisms of action are mainly related to the regulation of lipid profiles and glucose metabolism, protection of pancreatic β-cells, promotion of insulin secretion, antioxidation, inhibition of protein tyrosine phosphatase 1B and α-glycosidase activities. In addition, mulberry treatment could change intestinal microbiota of mice, which are associated with insulin resistance and diabetes. This review provides reference for further research and development of different medicinal parts of mulberry for the treatment of DM.

Physical inactivity induces insulin resistance in plantaris muscle through protein tyrosine phosphatase 1B activation in mice.

Inactivity causes insulin resistance in skeletal muscle and exacerbates various lifestyle-related diseases. We previously found that 24-h hindlimb cast immobilization (HCI) of the predominantly slow-twitch soleus muscle increased intramyocellular diacylglycerol (IMDG) and insulin resistance by activation of lipin1, and HCI after a high-fat diet (HFD) further aggravated insulin resistance. Here, we investigated the effects of HCI on the fast-twitch-predominant plantaris muscle. HCI reduced the insulin sensitivity of plantaris muscle by approximately 30%, and HCI following HFD dramatically reduced insulin sensitivity by approximately 70% without significant changes in the amount of IMDG. Insulin-stimulated phosphorylation levels of insulin receptor (IR), IR substrate-1, and Akt were reduced in parallel with the decrease in insulin sensitivity. Furthermore, tyrosine phosphatase 1B (PTP1B), a protein known to inhibit insulin action by dephosphorylating IR, was activated, and PTP1B inhibition canceled HCI-induced insulin resistance. In conclusion, HCI causes insulin resistance in the fast-twitch-predominant plantaris muscle as well as in the slow-twitch-predominant soleus muscle, and HFD potentiates these effects in both muscle types. However, the mechanism differed between soleus and plantaris muscles, since insulin resistance was mediated by the PTP1B inhibition at IR in plantaris muscle.

Ursonic acid from Artemisia montana exerts anti-diabetic effects through anti-glycating properties, and by inhibiting PTP1B and activating the PI3K/Akt signaling pathway in insulin-resistant C2C12 cells

Artemisia is one of the largest genera in the plant family Asteraceae and has long been used in traditional medicine for its antitussive, analgesic, antihypertensive, antitoxic, antiviral, antimalarial, and anti-inflammatory properties. However, the anti-diabetic activity of Artemisia montana has not been broadly studied. The goal of this study was to determine whether extracts of the aerial parts of A. montana and its main constituents inhibit protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase activities. We isolated nine compounds from A. montana including ursonic acid (UNA) and ursolic acid (ULA), which significantly inhibited PTP1B with IC50 values of 11.68 and 8.73 μM, respectively. In addition, UNA showed potent inhibitory activity against α-glucosidase (IC50 = 61.85 μM). Kinetic analysis of PTP1B and α-glucosidase inhibition revealed that UNA was a non-competitive inhibitor of both enzymes. Docking simulations of UNA demonstrated negative binding energies and close proximity to residues in the binding pockets of PTP1B and α-glucosidase. Molecular docking simulations between UNA and human serum albumin (HSA) revealed that UNA binds tightly to all three domains of HSA. Furthermore, UNA significantly inhibited fluorescent AGE formation (IC50 = 4.16 μM) in a glucose-fructose-induced HSA glycation model over the course of four weeks. Additionally, we investigated the molecular mechanisms underlying the anti-diabetic effects of UNA in insulin-resistant C2C12 skeletal muscle cells and discovered that UNA significantly increased glucose uptake and decreased PTP1B expression. Further, UNA increased GLUT-4 expression level by activating the IRS-1/PI3K/Akt/GSK-3 signaling pathway. These findings clearly demonstrate that UNA from A. montana shows great potential for treatment of diabetes and its complications.

Modulation of Insulin Resistance by Silybum marianum Leaves, and its Synergistic Efficacy with Gymnema sylvestre, Momordica charantia, Trigonella-foenum graecum Against Protein Tyrosine Phosphatase 1B

ABSTRACT Prolonged insulin resistance is considered one of the reasons for Type 2 Diabetes Mellitus. Upregulation of Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signalling, has been well studied as a key regulator in prognosis to insulin resistance. It has been widely studied as a desirable molecular therapeutic target. The study aimed to evaluate the efficacy of leaf extract of the medicinal plants Silybum marianum on the inhibition of PTP1B activity. It also explored the synergistic effect with extracts of Gymnema sylvestre (leaves), Momordica charantia (seeds), and Trigonella foenum graecum (seeds). The S. marianum leaves showed dose-dependent inhibition of PTP1B ranging from 9.48–47.95% (25–1000 μg mL−1). Assay with individual plant extracts showed comparatively lesser inhibition of PTP1B as compared to metformin as a control (38% inhibition). However, a synergistic effect showed nearly 45% PTP1B inhibition (higher than metformin) after the assay was done with selected four plant extracts in combination. The effect of leaf extracts of S. marianum was studied for glucose uptake efficiency in yeast cell lines which was found to be increased by 23% as compared to the control (without extract). Metformin improves glucose upake by yeast cells by ~15–31%. GC-MS analysis revealed 23 phytochemicals, some of which possessed anti-diabetic properties. A dose-dependent increase in antioxidant activity of S. marianum leaves extracts was observed (40–53%). The findings of the study highlighted the presence of various phytochemicals in leaves extracts that are effective against PTP1B inhibition and may help in reinvigorating drug development.

Activating Pyruvate Kinase Improves Red Blood Cell Integrity By Reducing Band3 Tyrosine Phosphorylation

Background: The scientific rationale for mitapivat (AG-348) as an anti-sickling therapy is that it enhances red cell pyruvate kinase (PKR) activity and glycolytic flux, leading to a reduction of intracellular 2,3-diphosphoglycerate (2,3-DPG) and a concomitant increase in adenosine triphosphate (ATP). Reducing 2,3-DPG destabilizes hemoglobin S (HbS) fibers, while increasing ATP improves red blood cell (RBC) integrity and cellular hydration. Mitapivat increased ATP and decreased 2,3-DPG levels in a dose-dependent manner in this Phase 1 study (Xu et al, 2022). The Hb increase was sustained after stopping mitapivat until end of study (EOS) at 4 weeks, suggesting an improvement in RBC survival. However, pathways modulated by mitapivat that may contribute to sickle RBC survival remain largely unknown. Mitapivat has been shown to improve oxidative stress by increasing GSH/GSSG ratio (Matte et al 2021). In SCD, elevated oxidative stress inhibits RBC tyrosine (Tyr) phosphatases that normally maintain the RBC Band3 (Bd3) tyrosines in an unphosphorylated state. Tyr-phosphorylation of Bd3 destabilizes membrane integrity and reduces RBC deformability (Noomuna et al, 2020). We hypothesize that PKR activators, such as mitapivat and AG-946 (a next-generation activator of PKR), improve the membrane integrity via regulating Tyr-phosphorylation of Bd3. Methods: Of the 16 patients in the Phase1 study, frozen whole blood samples were evaluable in 15, from which RBC ghosts (membranes) at the different timepoints (baseline and following 2 weeks of treatment at each dose level) were isolated. Tyr-phosphorylation of Bd3 was analyzed by Western blotting using a specific phospho-Tyr Bd3 antibody. Ex-vivo experiments were conducted by treating RBCs from different HbSS patients with different concentrations (1 µM, 3 µM, 10 µM, and 30 µM) of mitapivat or AG-946, for 4 hours at 37°C. To investigate the effect of mitapivat on kinases and phosphatases, HbSS RBCs were treated with different concentrations of mitapivat in a combination or without Syk kinase inhibitor-fostamatinib (MedChemExpress). We measured the effect of mitapivat on ATP production in-vitro by treating HbSS RBCs with 30 µM mitapivat for 2h, 4h, 6h, and 8h. The ATP level was detected using CellTiter-Glo® 2.0 Cell Viability Assay (Promega). Meanwhile, the same samples were used to explore the effect of mitapivat on the expression of intact protein tyrosine phosphatase 1B (PTP1B) on the RBC membrane. Results: A dose-dependent decrease in mean Bd3 Tyr-phosphorylation of 20%, 34%, 42%, and 42%, from baseline was observed in the 15 subjects, corresponding to ascending BID dose levels of 2 weeks mitapivat 5 mg, 20 mg, 50 mg, 100 mg, respectively; followed by a return to near baseline by EOS (Figure 1A). Consistent with the in-vivo data, ex-vivo treated HbSS RBCs also showed a dose-dependent decrease in Bd3 Tyr-phosphorylation after either mitapivat or AG-946 treatment (Figure 1B, C). Interestingly, combination of mitapivat with fostamatinib showed an enhanced effect on the reduction of Bd3 Tyr-phosphorylation compared with single drug treatment, which indicated that mitapivat may affect the activity of PTP1B (Figure 1D). Since the activity of PTP1B could be regulated by ATP, we then investigated the effect of mitapivat on ATP level. The ATP level was elevated after 6 hours' mitapivat treatment, even after correction for cell number (Figure 1E). Concomitantly, the intact PTP1B associated with cell membrane also increased after 6 hours of mitapivat treatment (Figure 1F), which might explain the de-phosphorylation of Bd3. Conclusion: A recent study showed that PTP1B is the major phosphatase that maintains Bd3 tyrosine in an unphosphorylated state (Noomuna et al, 2022). PTP1B could be cleaved into an inactive form by calpain, which needs to be activated by calcium. The balance of calcium in RBC relies on the activity of the plasma membrane Ca2+ ATPase (PCMA) pump, which is fueled by ATP. Our results suggest that in enhancing PKR activity, mitapivat leads to an increase in intracellular ATP which increases activity of the PCMA pump, and an increased efflux of intracellular calcium. A reduction in intracellular Ca2+ reduces the activity of calpain, and thereby suppresses calpain's cleavage of phosphatase-PTP1B, resulting in reduction of Bd3 Tyr-phosphorylation. ATP increase is thus a key mechanism of mitapivat's beneficial effect on the hemoglobin increase in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of p-Hydroxybenzaldehyde, Vanillin, and Syringaldehyde on Protein Tyrosine Phosphatase 1B Activity

Background: The PTP 1B is a negative regulator of insulin signal transduction and hence, serves as a therapeutic target in the treatment of diabetes. Objective: The present study investigated the inhibitory effects of p-hydroxybenzaldehyde, vanillin, and syringaldehyde on the activity of protein tyrosine phosphatases phosphatase 1B (PTP 1B) in vitro. Method: The PTP 1B inhibitory assay and mode of inhibition of the three compounds were determined using p-nitrophenyl phosphate (p-NPP) in a 96 well microplate. Molecular docking was used to predict the binding affinities of the compounds with the PTP 1B. Results: The results showed that syringaldehyde exhibited significantly (p< 0.05) higher PTP 1B inhibitory activity (IC50: 12.75 µM) compared to p-hydroxybenzaldehyde (IC50: 33.79 µM) and vanillin (IC50: 42.82 µM) as well as the standards suramin (IC50: 28.35 µM) and ursolic acid (IC50: 19.45 µM). Syringaldehyde and vanillin showed uncompetitive inhibition whereas, p-hydroxybenzaldehyde showed a mixed inhibition type. The molecular docking simulation predicted negative binding energies of -5.0 kcal/mol, -5.5 kcal/mol, and -5.5 kcal/mol for p-hydroxybenzaldehyde, vanillin, and syringaldehyde respectively. Conclusion: Syringaldehyde showed higher inhibition of PTP 1B compared to other phenolic aldehydes and could be the mechanism of its antidiabetic activity. Hence, further studies are warranted to confirm the efficacy and toxicity of the compound

Bifunctional Magnetic Fe3O4@Cu2O@TiO2 Nanosphere-Mediated Dual-Mode Assay of PTP1B Activity Based on Photocurrent Polarity Switching and Nanozyme-Engineered Biocatalytic Precipitation Strategies.

Dysregulation of protein phosphatases is associated with the progression of various human diseases and cancers. Herein, a photoelectrochemical (PEC)-quartz crystal microbalance (QCM) dual-mode sensing platform was developed for protein tyrosine phosphatase 1B (PTP1B) activity assay based on bifunctional magnetic Fe3O4@Cu2O@TiO2 nanosphere-mediated PEC photocurrent polarity switching and QCM signal amplification strategies. The PTP1B-specific phosphopeptide (P-peptide) with a cysteine end was designed and immobilized onto the QCM Au chip via the Au-S bond. Subsequently, the Fe3O4@Cu2O@TiO2 nanosphere was connected to the P-peptide via the specific interaction between the phosphate group on the P-peptide and TiO2. After incubation with PTP1B, the dephosphorylation of the P-peptide occurred, causing some Fe3O4@Cu2O@TiO2 nanospheres to be released from the chip surface. The released magnetic Fe3O4@Cu2O@TiO2 nanospheres (labeled as R-Fe3O4@Cu2O@TiO2) were quickly separated via magnetic separation technology and attached to the Bi2S3-decorated magnetic indium-tin oxide (Bi2S3/MITO) electrode by magnetic force, inducing the switch of the photocurrent polarity of the electrode from anodic current (the Bi2S3/MITO electrode) to cathodic current (the R-Fe3O4@Cu2O@TiO2/Bi2S3/MITO electrode). Also, the nondephosphorylated P-peptide linked Fe3O4@Cu2O@TiO2 nanospheres as nanozymes with horseradish peroxidase activity to catalyze the formation of precipitation on the surface of the Au chip, leading to a frequency change of the QCM. Thus, the proposed PEC-QCM dual-mode sensing platform achieved accurate and reliable assay of PTP1B activity because of the different mechanisms and independent signal transductions. In addition, this dual-mode sensing platform can be easily extended for other protein phosphatase activity analysis and shows great potential in the early diagnosis of the protein phosphatase-related diseases and the protein phosphatase-targeted drug discovery.

Inhibitors of recombinant protein-tyrosine phosphatase 1B (PTP1B) from Khaya senegalensis: Towards a strategic target for therapeutic intervention in trypanosomiasis

Background: Tyrosine dephosphorylation, catalyzed by protein-tyrosine phosphatase (PTP), prevents trypanosome differentiation to a procyclic form that lacks diverse mechanisms for survival within the mammalian host's bloodstream. Thus, differentiation to a procyclic form in the mammalian host's bloodstream would be potentially lethal to the parasite. This demonstrates that PTP is a critical regulator of trypanosome differentiation, making it a strategic therapeutic target for trypanosomiasis.Purpose: The in vitro inhibitory effect of seven compounds from the active fraction of Khaya senegalensis stem bark (4‑hydroxy-2-butanone, 1,3-butanediol, 2-methylpropyl butanoate, n-tridecanoic acid (CH3-(CH2)11-COOH), n-tetratriacontane, n-hexadecanoic (CH3-(CH2)14-COOH), and 14-pentadecenoic acid (CH2=CH-(CH2)12-COOH)) on the enzymatic activity of recombinant human PTP1B (MBL-SE332–0050) was investigated in this study.Method: The inhibition study was performed according to the standard procedure for analytical experiment and was monitored spectrophotometrically. PTP1B was assayed with 10 mM of p-nitrophenylphosphate. Bioassay-guided fractionation of methanol stem bark extract from K. senegalensis yielded the potent PTP1B inhibitors. Sodium orthovanadate was used as the positive control for inhibition. The kinetic parameters were determined by Lineweaver-Burk plots and calculated using Sigma Plot (SPCC Inc., Chicago, IL, USA)Results: The active fraction that contains the seven compounds inhibited PTP1B dose-dependently with an IC50 of 1.32 µM. Lineweaver-Burk plots revealed that PTP1B was inhibited non-competitively with a Ki of 0.46 ± 0.03 mg/ml, while sodium orthovanadate inhibited the enzyme competitively with an IC50 of 1.02 µM and Ki of 0.26 ± 0.01 mg/ml. Cleavage of the substrate para-Nitrophenylphosphate (pNPP) by PTP1B showed a KM of 4.99 mM and Vmax of 0.053 µmol/min.Conclusion: The mixture of compounds tested is potent PTP1B inhibitors that could serve as scaffolds for new trypanocidal drug candidates that target protein-tyrosine phosphatase activity.

Zinc-Air Battery-Assisted Self-Powered PEC Sensors for Sensitive Assay of PTP1B Activity Based on Perovskite Quantum Dots Encapsulated in Vinyl-Functionalized Covalent Organic Frameworks.

The self-powered sensors have attracted widespread attention in the analysis field due to a huge demand of point-of-care testing (POCT) in the early diagnosis of diseases. However, the output voltage of the reported self-powered sensors is always small, resulting in a narrow linear detection range and low assay sensitivity. Herein, a self-powered photoelectrochemical (PEC) sensor with zinc-air batteries as a power source was developed for activity assay of protein tyrosine phosphatase 1B (PTP1B) based on perovskite quantum dots encapsulated in the vinyl-functionalized covalent organic framework (COF-V). CsPbBr3 nanocrystals were stabilized by the confinement effect of the COF-V cage without aggregation, and the resulting CsPbBr3@COF-V composite was used as the cathodic photoelectric material to construct the zinc-air battery with a large open-circuit voltage (OCV, 1.556 V). Before PTP1B activity assay, an auxiliary peptide-polyamidoamine-phosphopeptide (P2-PAMAM-P1) hybrid was introduced into the photocathode via thiol-ene click reaction between the thiol group on the P1 and the vinyl group on the COF-V. The steric hindrance effect of the P1-PAMAM-P2 hybrid inhibited the PEC performance of the photocathode, resulting in a small OCV of the zinc-air battery. When the PTP1B existed, PTP1B-catalyzed dephosphorylation of tyrosine on P1 facilitated the cleavage process of P1 by chymotrypsin, leading to the removal of the P2-PAMAM-P1 hybrid from the photocathode and consequently the enhancement of the OCV. Therefore, the activity of PTP1B was sensitively detected. The developed self-powered PEC sensor showed superior performance for PTP1B activity assay (broad linear response range, 0.1 pM to 10 nM and low detection limit, 0.032 pM) due to the large output voltage of the constructed zinc-air battery and has great potential in POCT of protein phosphatase-related diseases and the discovery of protein phosphatase-targeted drugs.

Novel fluorescence biosensor custom-made for protein tyrosine phosphatase 1B detection based on titanium dioxide-decorated single-walled carbon nanohorn nanocomposite

This paper presents a new fluorescent approach for the detection of protein tyrosine phosphatase 1B (PTP1B) based on titanium dioxide-decorated single-wall carbon nanohorns (TiO2-SWCNHs). The novel TiO2-SWCNHs nanocomposite was synthesized and characterized for the first time and the phosphorylated peptide as the substrate of PTP1B was designed. Properties of SWCNHs and TiO2 were combined by growing nano-sized TiO2 particles on SWCNHs, resulting in TiO2-SWCNHs. TiO2 provides SWCNHs a large adsorption surface area and can specifically bind to phosphopeptide substrate. TiO2-SWCNHs effectively quenched the fluorescence of the phosphorylated peptide substrate labeled by the fluorophore, and the system had a low fluorescence background. In the presence of PTP1B, dephosphorylation of the peptide occurred owing to the reaction between PTP1B and the peptide, causing the separation of the dye-labeled peptide from TiO2-SWCNHs, which resulted in fluorescence enhancement of the reaction system. Thus, a simple and rapid strategy for the detection of PTP1B activity was developed, with a detection limit of 0.01 ng/mL and linear range of 0–10 ng/mL. The system can be used to detect PTP1B in serum using the standard addition method. This system provides a new approach for screening PTP1B inhibitors.

The Emerging Target Protein Tyrosine Phosphatase 1B (PTP1B) for Type 2 Diabetes Mellitus Management

Type 2 diabetes mellitus is one of the greatest health burdens of our society. Despite the extensive options of current drug treatments, novel therapies are under development to manage this chronic disorder and its subsequent complications. The inhibition of the enzyme protein tyrosine phosphatase 1B (PTP1B) is a promising therapeutic intervention in diabetes and obesity. However, finding selective PTP1B inhibitors remains a challenge. The pyrazole scaffold is considered of high pharmaceutical interest due to the high range of pharmacological activities. Nonetheless, the study of their effect against PTP1B activity have not yet been fully explored, opening a new field of research to find a new potential approach for type 2 diabetes mellitus management. This commentary presents and discusses PTP1B as an emerging target for type 2 diabetes mellitus management.