Protein Tyrosine Phosphatase Enzyme Research Articles

Abstract 1183: A first-in-class and SHP1 allosteric inhibitor, SB8091, shows a good efficacy and safety profile in preclinical models

Abstract Introduction: Src homology 2 domain-containing phosphatase 1 (SHP1) is a cytoplasmic protein tyrosine phosphatase (PTP) that is primarily expressed in hematopoietic cells and is commonly recognized as a negative regulator of multiple signaling pathways1. Previously, we showed that inhibition of the phosphatase activity of SHP1 by a novel SHP1 allosteric inhibitor, SB6299, results in the activation of innate and adaptive immune cells and enhanced anti-tumor immune responses2. Here, we report SB8091, an advanced SHP1 allosteric inhibitor with a promising initial safety profile and improved pharmacokinetic properties. Methods: SHP1 inhibitors were developed through a structure-based drug design approach, and optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. The lead series was assessed in in-vitro studies with mouse or human primary immune cells. In-vivo efficacy and pharmacodynamic data were generated using mouse syngeneic tumor models. Results: Structurally, SB8091 seemed to stabilize the autoinhibitory form of SHP1 as an allosteric inhibitor, in a nanomolar range and which can explain the outstanding selectivity compared to other PTP enzymes, including SHP2. In several human primary immune cell types, SB8091 significantly increased cytokine responses and demonstrated significantly enhanced activity in NK cells and macrophages against various cancer target cells. As a single agent, this compound exhibited inhibitory effects on tumor growth when administered orally, and the efficacy was further enhanced when co-administered with anti-PD1 antibodies in various syngeneic tumor models. Notably, this effect was associated with a pharmacodynamic response marked by increased levels of phosphorylated substrates regulated by SHP1. SB8091 exhibited favorable pharmacokinetic properties for oral administration, and SafetyScreen data showed a very low potential for off-target activity and drug-drug interactions. Additionally, during a 2-week repeat pilot toxicity study in mice, SB8091 demonstrated excellent tolerability even at high doses, indicating a high therapeutic index (TI > 100). Conclusion: Our advanced compound, SB8091, is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP1. It has demonstrated excellent efficacy as a monotherapy or in combination with immune checkpoint blockers, with favorable drug properties and safety profiles. Research to advance to the clinical stage is being conducted.

DUSP15 expression is reduced in the hippocampus of Myrf knock-out mice but attention and object recognition memory remain intact.

The atypical protein tyrosine phosphatase enzyme, dual-specificity phosphate 15 (DUSP15) is thought to be activated by myelin regulatory factor (MyRF) and to have a role in oligodendrocyte differentiation. Here, we assess whether Dusp15 is reduced in the hippocampus of mice with conditional knock-out of Myrf in oligodendrocyte precursor cells. Using quantitative polymerase chain reaction (qPCR) we found that Dusp15 expression was indeed lower in these mice. Alterations in myelin have been associated with Alzheimer's disease (AD), autism spectrum disorder (ASD) and attention deficit/hyperactivity disorder (ADHD). Symptoms of these disorders can include impairments of object recognition and attention. We, therefore tested the mice in the object recognition task (ORT) and 5-choice serial reaction time task (5CSRTT). However, we did not find behavioural impairments indicating that attentional abilities and object recognition are not impacted by reduced oligodendrogenesis and hippocampal Dusp15 expression. Gaining insight into the role of newly formed oligodendrocytes and Dusp15 expression is helpful for the development of well targeted treatments for myelin dysregulation.

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.

Investigation of antidiabetic properties of shikonin by targeting aldose reductase enzyme: In silico and in vitro studies.

Diabetes is a complicated multifactorial disorder in which the patient generally observes polyphagia, polydipsia, and polyuria due to uncontrolled growth in blood sugar levels. For its management, the pharmaceutical industry is working day and night to find a better drug with no or least toxicity. That's why nowadays a more focused branch is to use herbal phytoconstituents for its prevention. Shikonin is a naphthoquinone natural dye that is isolated from the plants of the Boraginaceae family and has proven its role as an anti-cancer, anti-inflammatory, and anti-gonadotrophic agent. In our previous study, we have published its anti-diabetic action by inhibiting the enzyme protein tyrosine phosphatase 1B. In this study, we were more focused on finding out the role of Shikonin and its pharmacophores by inhibiting the action of aldose reductase (AR) enzyme. The study was conducted using pharmacophore modeling, molecular docking, and molecular dynamics simulation studies. The absorption, distribution, metabolism, excretion (ADME), and toxicity profile were also evaluated in this study. Along with all the computational biology parameters we also focused on the in vitro activity and kinetic study of inhibitory activity of Shikonin against aldose reductase.

Ribes nigrum and Juglans regia, Potential Functional Foods as a Source of Protein Tyrosine Phosphatase Enzyme: Biochemical and Kinetics Studies

Functional foods have gained substantial attention in the healthcare industry for their less explored bioactive ingredients with significant therapeutic potential. Extensive research in the fields of plants is required to identify the sources of PTPase, a key enzyme in regulating proteins and involved in various ailments. Ribes nigrum (Munakka; M), Juglans regia (Walnut; W); dry fruit and nut, have been purified through salt fractionation. In M, 0-45% fraction exhibited a 3.51-fold increase in purification with specific activity of 10.39 U.mg-1 (M), whereas in W, 45-90% fraction exhibited values of 7.20-fold and 31.35 U.mg-1 (W). PTPase from W and M exhibited thermostability up to 700C and 600C, respectively, with 75% activity retention. Km values were determined to be 4.67 mM (W) and 14.29 mM (M), respectively whereas, the activation energy (Ea) values were 30.59 KJ.mol-1 (W) and 51.84 KJ.mol-1 (M). Fe2+, Mn2+, and Zn2+ ions showed prominent inhibitory effects on PTPase; Ki value showed Mn2+ ions exhibit strong binding affinity differentially in PTPase from two sources of 0.075 mM(W), 0.103 mM(M) may have clinical importance in the future. The findings demonstrate that dry fruit and nuts are sources of PTPases as potential functional foods that may serve significant multifunctional eatery for holistic development.

Structural characterization of a pathogenic mutant of human protein tyrosine phosphatase PTPN2 (Cys216Gly) that causes very early onset autoimmune enteropathy.

PTPN2 is an important protein tyrosine phosphatase (PTP) that plays a key role in cell signaling. Deletions or inactivating mutations of PTPN2 have been described in different pathologies and underline its critical role in hematopoiesis, autoimmunity, and inflammation. Surprisingly, despite the major pathophysiological implications of PTPN2, the structural analysis of this PTP and notably of its pathogenic mutants remains poorly documented. Contrary to other human PTP enzymes, to date, only one structure of PTPN2 (wild-type form) has been reported. Here, we report the first crystal structure of a pathogenic mutant of PTPN2 (Cys216Gly) that causes an autoimmune enteropathy. We show in particular that this mutant adopts a classical PTP fold. More importantly, albeit inactive, the mutant retains its ability to bind substrates and to adopt the characteristic catalytically competent closed form of PTP enzymes. This novel PTPN2 structure may serve as a new tool to better understand PTP structures and the structural impacts of pathogenic mutations. Moreover, the C216G PTPN2 structure could also be helpful to design specific ligands/inhibitors.

Flavonoids and Terpenoids with PTP-1B Inhibitory Properties from the Infusion of Salvia amarissima Ortega

An infusion prepared from the aerial parts of Salvia amarissima Ortega inhibited the enzyme protein tyrosine phosphatase 1B (PTP-1B) (IC50~88 and 33 μg/mL, respectively). Phytochemical analysis of the infusion yielded amarisolide (1), 5,6,4′-trihydroxy-7,3′-dimethoxyflavone (2), 6-hydroxyluteolin (3), rutin (4), rosmarinic acid (5), isoquercitrin (6), pedalitin (7) and a new neo-clerodane type diterpenoid glucoside, named amarisolide G (8a,b). Compound 8a,b is a new natural product, and 2–6 are reported for the first time for the species. All compounds were tested for their inhibitory activity against PTP-1B; their IC50 values ranged from 62.0 to 514.2 μM. The activity was compared to that of ursolic acid (IC50 = 29.14 μM). The most active compound was pedalitin (7). Docking analysis predicted that compound 7 has higher affinity for the allosteric site of the enzyme. Gas chromatography coupled to mass spectrometry analyses of the essential oils prepared from dried and fresh materials revealed that germacrene D (15) and β-selinene (16), followed by β-caryophyllene (13) and spathulenol (17) were their major components. An ultra-high performance liquid chromatography coupled to mass spectrometry method was developed and validated to quantify amarisolide (1) in the ethyl acetate soluble fraction of the infusion of S. amarissima.

Does Fermentation Really Increase the Phenolic Content in Cereals? A Study on Millet.

Millet is underutilized in Europe, despite its advantages compared to other common cereals. In Asia and Africa, millet is mainly eaten in fermented form; its consumption has beneficial properties on human health. Three millet batches were compared in terms of free and bound phenols by High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS). The richest one in terms of bound phenols was selected for testing via a basic (0.1 M NaOH) and an acidic (1.2 M H2SO4) hydrolysis, in which 149.3 and 193.6 mg/100 g of phenols were recovered, respectively. The ability of fermentation, with yeast and Lactobacilli, to increase the content of phenolic compounds was verified. Five withdrawalswere performed to verify the influence of fermentation time on the total phenolic content. The greatest phenolic content was observed after 72 h. Fermentation increased the cinnamic acids and flavonoids contents by approximately 30%. Vitexin and vitexin 2″-O-rhamnoside contents were significantly higher in the fermented millet; these compounds partially inhibit the protein tyrosine phosphatase enzyme, which is overexpressed in type-2 diabetes. A molecular dynamic simulation showed the two flavonoids to be allosteric inhibitors. The phenolic extract from fermented millet demonstrated a higher level of antioxidant protection on human erythrocytes by ex vivo cellular antioxidant activity in red blood cells. In this context, functional foods based on fermented millet could represent a new trend in European markets.

Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells.

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation-during which the PTP enzymes act on the peptide substrate-the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.

Uncovering the Molecular Interactions in the Catalytic Loop That Modulate the Conformational Dynamics in Protein Tyrosine Phosphatase 1B.

Active-site loops are integral to the function of numerous enzymes. They enable substrate and product binding and release, sequester reaction intermediates, and recruit catalytic groups. Here, we examine the catalytic loop in the enzyme protein tyrosine phosphatase 1B (PTP1B). PTP1B has a mobile so-called WPD loop (named for its three N-terminal residues) that initiates the dephosphorylation of phosphor-tyrosine substrates upon loop closure. We have combined X-ray crystallography, solution NMR, and pre-steady-state kinetics experiments on wild-type and five WPD loop mutants to identify the relationships between the loop structure, dynamics, and function. The motions of the WPD loop are modulated by the formation of weak molecular interactions, where perturbations of these interactions modulate the conformational equilibrium landscape. The point mutants in the WPD loop alter the loop equilibrium position from a predominantly open state (P185A) to 50:50 (F182A), 35:65 (P188A), and predominantly closed states (T177A and P188A). Surprisingly, there is no correlation between the observed catalytic rates in the loop mutants and changes to the WPD loop equilibrium position. Rather, we observe a strong correlation between the rate of dephosphorylation of the phosphocysteine enzyme intermediate and uniform millisecond motions, not only within the loop but also in the adjacent α-helical domain of PTP1B. Thus, the control of loop motion and thereby catalytic activity is dispersed and resides within not only the loop sequence but also the surrounding protein architecture. This has broad implications for the general mechanistic understanding of enzyme reactions and the role that flexible loops play in the catalytic cycle.

A REVIEW ON PROTEIN TYROSINE PHOSPHATASES - AN IMPORTANT TARGET FOR VARIOUS DISEASES

The enzyme protein tyrosine phosphatase (PTP) is responsible for the regulation of cellular functions including cell growth, replication, and signal transduction. Dysregulation of this enzyme leads to various diseases including Type II diabetes and cancers as well. The PTP enzyme functions as a promising drug target for these diseases, leading to new innovations in developing new drug targets in the field of clinical studies and pharmacology. The PTP is considered as the next generation drug targets. The protein tyrosine drug targets can be targeted to cure the diseases caused due to its dysregulations. However, due to its complex structure and highly conserved active sites are the major challenges which block this strategy. Moreover, two enzyme proteins PTP A and PTP B of PTP enzyme family are essential for the survival of Mycobacterium in host macrophages and cause infection resulting in chronic tuberculosis. The novel drug-like properties of L335-M34 and L01Z08 compounds are selective inhibitors of this enzyme responsible for the tuberculosis virulence in mammals. Some of the commercial inhibitors such as ertiprotafib, arylbenzonaphthofurans, and arylbenzonaphthothiophenes have also proven to inhibit the enzyme’s virulence. This review summarizes the latest innovations to lead a map for developing new innovative drugs against the various classes of target enzymes of tyrosine phosphatase.

Vanadium in Biosphere and Its Role in Biological Processes.

Ultra-trace elements or occasionally beneficial elements (OBE) are the new categories of minerals including vanadium (V). The importance of V is attributed due to its multifaceted biological roles, i.e., glucose and lipid metabolism as an insulin-mimetic, antilipemic and a potent stress alleviating agent in diabetes when vanadium is administered at lower doses. It competes with iron for transferrin (binding site for transportation) and with lactoferrin as it is secreted in milk also. The intracellular enzyme protein tyrosine phosphatase, causing the dephosphorylation at beta subunit of the insulin receptor, is inhibited by vanadium, thus facilitating the uptake of glucose inside the cell but only in the presence of insulin. Vanadium could be useful as a potential immune-stimulating agent and also as an antiinflammatory therapeutic metallodrug targeting various diseases. Physiological state and dose of vanadium compounds hold importance in causing toxicity also. Research has been carried out mostly on laboratory animals but evidence for vanadium importance as a therapeutic agent are available in humans and large animals also. This review examines the potential biochemical and molecular role, possible kinetics and distribution, essentiality, immunity, and toxicity-related study of vanadium in a biological system.

Revisiting the roles of VHR/DUSP3 phosphatase in human diseases

Protein tyrosine phosphatases have long been considered key regulators of biological processes and are therefore implicated in the origins of various human diseases. Heterozygosity, mutations, deletions, and the complete loss of some of these enzymes have been reported to cause neurodegenerative diseases, autoimmune syndromes, genetic disorders, metabolic diseases, cancers, and many other physiological imbalances. Vaccinia H1-related phosphatase, also known as dual-specificity phosphatase 3, is a protein tyrosine phosphatase enzyme that regulates the phosphorylation of the mitogen-activated protein kinase signaling pathway, a central mediator of a diversity of biological responses. It has been suggested that vaccinia H1-related phosphatase can act as a tumor suppressor or tumor-promoting phosphatase in different cancers. Furthermore, emerging evidence suggests that this enzyme has many other biological functions, such as roles in immune responses, thrombosis, hemostasis, angiogenesis, and genomic stability, and this broad spectrum of vaccinia H1-related phosphatase activity is likely the result of its diversity of substrates. Hence, fully identifying and characterizing these substrate-phosphatase interactions will facilitate the identification of pharmacological inhibitors of vaccinia H1-related phosphatase that can be evaluated in clinical trials. In this review, we describe the biological processes mediated by vaccinia H1-related phosphatase, especially those related to genomic stability. We also focus on validated substrates and signaling circuitry with clinical relevance in human diseases, particularly oncogenesis.

High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy: Proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC–HRMS–SPE–NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0±5.9μM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

Structure Revision of Four Acylphloroglucinols Isolated from the Leaves of Syzygium polyanthum

The structures of four 1-acyl-2,3,5-trihydroxybenzene derivatives (1–4) that inhibit the enzyme protein tyrosine phosphatase 1B, previously reported from the leaves of Syzygium polyanthum, have been revised based upon reinterpretation of NMR spectroscopic data. The corrected structures are all 1-acylphloroglucinol derivatives (5–8).

Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.

A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases.

Effect of Natural Product Extracts on Lipoxygenase, Cyclooxygenase, and Protein Tyrosine Phosphatase 1β

The purpose of this research project is to examine the inhibition of enzymes linked to inflammation and associated diseases by plants traditionally as anti-inflammatory medicines: Tussilago farfara, Grindelia squarrosa, and Uritca dioica. Arachidonic acid is metabolized in the body through two main metabolic pathways with the enzymes: cyclooxygenases (COX) and lipoxygenases (LOX). Elevated levels of prostanoids and leukotrienes, products of the two respective pathways, have been linked to inflammatory diseases. Finding a dual inhibitor of COX and LOX is promising in preventing the inflammation and diseases that are linked to the overproduction of both pathways while minimizing the side effects associated with inhibition of individual pathways. Furthermore, the enzyme protein tyrosine phosphatase 1β (PTP1β) is linked between inflammation and metabolic disease through the leptin receptor-associated Janus Kinase (JAK). PTP1β is a negative regulator of insulin and leptin receptors thus being explored as a possible therapeutic for type II diabetes and obesity. Crude methanolic extracts of Tussilago farfara, Grindelia squarrosa, and Urtica dioica were taken to approximate the plants' components released in the body upon consumption. The bioactivities of the standardized extracts were then determined using enzymatic assay kits for COX I and II, LOX, and PTP1β. T. farfara is the strongest LOX inhibitor at 60% inhibition of 15-LOX, followed by U. dioica and G. squarrosa at 45% and 39%, respectively. Studies for COX I and II as well as PTP1β inhibition are ongoing, and the results will be presented.

Real-Time Monitoring of the Dephosphorylating Activity of Protein Tyrosine Phosphatases Using Microarrays with 3-Nitrophosphotyrosine Substrates.

Phosphatases and kinases regulate the crucial phosphorylation post-translational modification. In spite of their similarly important role in many diseases and therapeutic potential, phosphatases have received arguably less attention. One reason for this is a scarcity of high-throughput phosphatase assays. Herein, a new real-time, dynamic protein tyrosine phosphatase (PTP) substrate microarray assay measuring product formation is described. PTP substrates comprising a novel 3-nitrophosphotyrosine residue are immobilized in discrete spots. After reaction catalyzed by a PTP a 3-nitrotyrosine residue is formed that can be detected by specific, sequence-independent antibodies. The resulting microarray was successfully evaluated with a panel of recombinant PTPs and cell lysates, which afforded results comparable to data from other assays. Its parallel nature, convenience, and low sample requirements facilitate investigation of the therapeutically relevant PTP enzyme family.

Design, Synthesis, and Evaluation of 2‐(arylsulfonyl)oxiranes as Cell‐permeable Covalent Inhibitors of Protein Tyrosine Phosphatases

A structure-based design approach has been applied to develop 2-(arylsulfonyl)oxiranes as potential covalent inhibitors of protein tyrosine phosphatases. A detailed kinetic analysis of inactivation by these covalent inhibitors reveals that this class of compounds inhibits a panel of protein tyrosine phosphatases in a time- and dose-dependent manner, consistent with the covalent modification of the enzyme active site. An inactivation experiment in the presence of sodium arsenate, a known competitive inhibitor of protein tyrosine phosphatase, indicated that these inhibitors were active site bound. This finding is consistent with the mass spectrometric analysis of the covalently modified protein tyrosine phosphatase enzyme. Additional experiments indicated that these compounds remained inert toward other classes of arylphosphate-hydrolyzing enzymes, and alkaline and acid phosphatases. Cell-based experiments with human A549 lung cancer cell lines indicated that 2-(phenylsulfonyl)oxirane (1) caused an increase in intracellular pTyr levels in a dose-dependent manner thereby suggesting its cell-permeable nature. Taken together, the newly identified 2-(arylsulfonyl)oxiranyl moiety could serve as a novel chemotype for the development of activity-based probes and therapeutic agents against protein tyrosine phosphatase superfamily of enzymes.

Chimeric design, synthesis, and biological assays of a new nonpeptide insulin-mimetic vanadium compound to inhibit protein tyrosine phosphatase 1B

Prior to its total synthesis, a new vanadium coordination compound, called TSAG0101, was computationally designed to inhibit the enzyme protein tyrosine phosphatase 1B (PTP1B). The PTP1B acts as a negative regulator of insulin signaling by blocking the active site where phosphate hydrolysis of the insulin receptor takes place. TSAG001, [V(V)O(2)(OH)(picolinamide)], was characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy; IR: ν/cm(-1) 3,570 (NH), 1,627 (C=O, coordinated), 1,417 (C-N), 970/842 (O=V=O), 727 δ̣ (pyridine ring); (13)C NMR: 5 bands between 122 and 151 ppm and carbonyl C shifted to 180 ppm; and (1)H NMR: 4 broad bands from 7.6 to 8.2 ppm and NH(2) shifted to 8.8 ppm. In aqueous solution, in presence or absence of sodium citrate as a biologically relevant and ubiquitous chelator, TSAG0101 undergoes neither ligand exchange nor reduction of its central vanadium atom during 24 hours. TSAG0101 shows blood glucose lowering effects in rats but it produced no alteration of basal- or glucose-induced insulin secretion on β cells during in vitro tests, all of which excludes a direct mechanism evidencing the extrapancreatic nature of its activity. The lethal dose (LD(50)) of TSAG0101 was determined in Wistar mice yielding a value of 412 mg/kg. This value is one of the highest among vanadium compounds and classifies it as a mild toxicity agent when compared with literature data. Due to its nonsubstituted, small-sized scaffold design, its remarkable complex stability, and low toxicity; TSAG0101 should be considered as an innovative insulin-mimetic principle with promising properties and, therefore, could become a new lead compound for potential nonpeptide PTP1B inhibitors in antidiabetic drug research. In view of the present work, the inhibitory concentration (IC(50)) and extended solution stability will be tested.