Singlet oxygen inactivates protein tyrosine phosphatase-1B by oxidation of the active site cysteine

Singlet oxygen ((1)O(2)), an electronically excited form of molecular oxygen, is a mediator of biological effects of ultraviolet A radiation, stimulating signaling cascades in human cells. We demonstrate here that (1)O(2) generated by photosensitization or by thermodecomposition of 3,3'-(1,4-naphthylidene)dipropionate-1,4-endoperoxide inactivates isolated protein tyrosine phosphatases (PTPases). PTPase activities of PTP1B or CD45 were abolished by low concentrations of (1)O(2), but were largely restored by post-treatment with dithiothreitol. Electrospray ionization mass spectrometry analysis of tryptic digests of PTP1B exposed to (1)O(2) revealed oxidation of active-site Cys215 as the only cysteine residue oxidized. In summary, (1)O(2) may activate signaling cascades by interfering with phosphotyrosine dephosphorylation.     

Mass spectrometry-based analyses for identifying and characterizing S-nitrosylation of protein tyrosine phosphatases

All members in the protein tyrosine phosphatase (PTP) family of enzymes contain an invariant Cys residue which is absolutely indispensable for catalysis. Due to the unique microenvironment surrounding the active center of PTPs, this Cys residue exhibits an unusually low pKa characteristic, thus being highly susceptible to oxidation or S-nitrosylation. While oxidation-dependent regulation of PTP activity has been extensively examined, the molecular details and biological consequences of PTP S-nitrosylation remain unexplored. We hypothesized that the catalytic Cys residue is targeted by proximal nitric oxide (NO) and its derivatives collectively termed reactive nitrogen species (RNS), leading to nitrosothiol formation concomitant with reversible inactivation of PTPs. To test this hypothesis, we have developed novel strategies to examine the redox status of Cys residues of purified PTP1B that was exposed to NO donor S-Nitroso-N-penicillamine (SNAP). A gel-based method in conjunction with mass spectrometry (MS) analysis revealed that the catalytic Cys215 of PTP1B was reversibly modified when PTP1B was briefly treated with SNAP. In order to further identify the exact mode of NO-induced modification, we employed an online LC-ESI-MS/MS analysis incorporating a mass difference-based, data-dependent acquisition function that effectively mapped the S-nitrosylated Cys residues. Our results demonstrated that treating PTP1B with SNAP led to S-nitrosothiol formation of the catalytic Cys215. Interestingly, SNAP-induced modifications were strictly reversible as highly oxidized Cys derivatives (Cys-SO(2)H or Cys-SO(3)H) were not identified by MS analyses. Thus, the methods introduced in this study provide direct evidence to prove the direct link between S-nitrosylation of the catalytic Cys residue and reversible inactivation of PTPs.     

Benzothiazole benzimidazole (S)-isothiazolidinone derivatives as protein tyrosine phosphatase-1B inhibitors

Benzothiazole benzimidazole (S)-isothiazolidinone ((S)-IZD) derivatives 5 were discovered through a peptidomimetic modification of the tripeptide (S)-IZD protein tyrosine phosphatase 1B (PTP1B) inhibitor 1. These derivatives are potent, competitive, and reversible inhibitors of PTP1B with improved caco-2 permeability. An X-ray co-crystal structure of inhibitor 5/PTP1B at 2.2A resolution demonstrated that the benzothiazole benzimidazole forms bi-dentate H-bonds to Asp48, and the benzothiazole interacts with the surface of the protein in a solvent exposed region towards the C-site. The design, synthesis, and SAR of this novel series of benzothiazole benzimidazole containing (S)-IZD inhibitors of PTP1B are presented herein.     

Formylchromone derivatives as irreversible and selective inhibitors of human protein tyrosine phosphatase 1B. Kinetic and modeling studies

A series of formylchromone derivatives were synthesized as PTP1B inhibitors and some of them were potent against PTP1B with IC50 values as low as 1.0 microM. They exhibited remarkable selectivity for PTP1B over other human PTPases. Kinetic studies revealed that formylchromone derivatives are irreversible and active site-directed inhibitors. Molecular modeling study identified the orientation of the inhibitor bound at the active site of PTP1B.     

Docking of oxalyl aryl amino benzoic acid derivatives into PTP1B

Protein Tyrosine Phosphatases (PTPs) that function as negative regulators of the insulin signaling cascade have been identified as novel targets for the therapeutic enhancement of insulin action in insulin resistant disease states. Reducing Protein Tyrosine Phosphatase1B (PTP1B) abundance not only enhances insulin sensitivity and improves glucose metabolism but also protects against obesity induced by high fat feeding. PTP1B inhibitors such as Formylchromone derivatives, 1, 2-Naphthoquinone derivatives and Oxalyl aryl amino benzoic derivatives may eventually find an important clinical role as insulin sensitizers in the management of Type-II Diabetes and metabolic syndrome. We have carried out docking of modified oxalyl aryl amino benzoic acid derivatives into three dimensional structure of PTP1B using BioMed CAChe 6.1. These compounds exhibit good selectivity for PTP1B over most of phosphatases in selectivity panel such as SHP-2, LAR, CD45 and TCPTP found in literature. This series of compounds identified the amino acid residues such as Gly220 and Arg221 are important for achieving specificity via H-bonding interactions. Lipophilic side chain of methionine in modified oxalyl aryl amino benzoic acid derivative [1b (a2, b2, c1, d)] lies in closer vicinity of hydrophobic region of protein consisted of Meth258 and Phe52 in comparison to active ligand. Docking Score in [1b (a2, b2, c1, d)] is -131.740Kcal/mol much better than active ligand score -98.584Kcal/mol. This information can be exploited to design PTP1B specific inhibitors.     

Discovery of potent, selective and orally bioavailable triaryl-sulfonamide based PTP1B inhibitors

A novel series of pTyr mimetics containing triaryl-sulfonamide derivatives (5a-r) are reported as potent and selective PTP1B inhibitors. Some of the test compounds (5o and 5p) showed excellent selectivity towards PTP1B over various PTPs, including TCPTP (in vitro). The lead compound 5o showed potent antidiabetic activity (in vivo), along with improved pharmacokinetic profile. These preliminary results confirm discovery of highly potent and selective PTP1B inhibitors for the treatment of T2DM.     

Design, synthesis, and discovery of stilbene derivatives based on lithospermic acid B as potent protein tyrosine phosphatase 1B inhibitors

Dihydroxy stilbene derivatives were designed based on lithospermic acid B and were prepared from 4-(chloromethyl)benzoic acid. The inhibitory activities of the novel compounds against protein tyrosine phosphatase 1B (PTP1B) were evaluated. 3,4-Dihydroxy stilbene carbonyl compounds (7, 11b, 27b) inhibited PTP1B with IC50 values comparable to molybdate, while the conjugation-extended compound (15b) showed inhibition 3-fold better than preclinical RK682. The introduction of electron withdrawing groups or amides into the second phenyl ring, or extension of the conjugation into the stilbene molecule may increase stability of the generated radicals.     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

Synthesis of galactofuranose disaccharides of biological significance

Methyl beta-D-galactofuranoside was readily obtained by tin(IV) chloride-catalyzed glycosylation of penta-O-benzoyl-alpha,beta-D-galactofuranose, followed by debenzoylation with sodium methoxide. Glycosylation of 1 with 2,3,5-tri-O-benzoyl-D-galactono-1,4-lactone or with the 6-O-trityl-lactone derivative 5 gave the benzoylated beta-D-galactofuranosyl-(1----6)-D-galactono-1,4-lactone 6 in excellent yield. The structure of disaccharide 6 was confirmed by borohydride reduction to the glycosyl-alditol 7. A byproduct of the condensation reaction of 1 with 4 or 5 was identified as the benzoylated (1----1)-beta,beta'-D-galactofuranosyl disaccharide 8. Compound 8 was readily prepared (88% yield) by controlled addition of water to 1, in the presence of stannic chloride. O-Debenzoylation of 8 afforded crystalline beta'-D-galactofuranosyl-(1----1)-beta-D-galactofuranoside. The glycosyl-lactone 6 constitutes a key intermediate for the synthesis of a disaccharide derivative having both units in the furanoid form. Thus, diisoamylborane reduction of the lactone function of 6 led to the disaccharide derivative 10, from which the methyl glycoside 12 was prepared. O-Debenzoylation of 12 gave the corresponding methyl beta-D-galactofuranosyl-(1----6)-beta-D-galactofuranoside. The free disaccharide beta-D-Galf-(1----6)-D-Galp and its acetylated derivative were also synthesized from 10.     

Synthesis and PTP1B inhibition of 1,2-naphthoquinone derivatives as potent anti-diabetic agents

A new series of 1,2-naphthoquinone derivatives was synthesized by various synthetic methods and evaluated for their ability to inhibit protein tyrosine phosphatase 1B (PTP1B). 1,2-Naphthoquinone derivatives with substituent at R(4) position showed submicromolar inhibitory activity, and compound 24 demonstrated 10- to 60-fold selectivity against the tested phosphatases. Also, several 4-aryl-1,2-naphthoquinone derivatives with substituents at R(3), R(6), R(7), or/and R(8) showed submicromolar inhibitory activity and good plasma stability.     

Calix[4]arene methylenebisphosphonic acids as inhibitors of protein tyrosine phosphatase 1B

Сalix[4]arenes bearing methylenebisphosphonic or hydroxymethylenebisphosphonic acid fragments at the wide rim of the macrocycle were studied as inhibitors of PTP1B. Some of the inhibitors showed IC₅₀ values in the micromolar range and good selectivity in comparison with other protein tyrosine phosphatases such as TC-PTP, PTPβ, LAR, and CD45. Kinetic studies indicated that the calix[4]arene derivatives influence PTP1B activity as slow-binding inhibitors. Based on molecular docking results, the binding modes of the macrocyclic bisphosphonates in the active centre of PTP1B are discussed.     

Levels of protein tyrosine phosphatase 1B determine susceptibility to apoptosis in serum-deprived hepatocytes

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. To assess the importance of PTP1B in the balance between death and survival in the liver, we have developed immortalized neonatal hepatocyte cell lines lacking (PTP1B(-/-)) or overexpressing (PTP1B(+/+PTP1B)) PTP1B. Early activation of caspase-3 occurred in PTP1B(+/+PTP1B) hepatocytes but was nearly abolished in PTP1B(-/-) cells. At the molecular level, PTP1B overexpression/deficiency altered the balance of pro-(Bim) and anti-(Bcl-x(L)) apoptotic members of the Bcl-2 family upon serum withdrawal. Likewise, cytosolic cytochrome C increased rapidly in PTP1B(+/+PTP1B) hepatocytes whereas it was retained in the mitochondria of PTP1B(-/-) cells. DNA fragmentation and the increase of apoptotic cells induced by serum withdrawal in wild-type (PTP1B(+/+)) hepatocytes were absent in PTP1B(-/-) cells. Conversely, overexpression of PTP1B accelerated DNA laddering and increased the number of apoptotic cells. In serum-deprived PTP1B(+/+PTP1B) hepatocytes, a rapid entry of Foxo1 into the nucleus and an earlier activation of caspase-8 was observed. However, both events were suppressed in PTP1B(-/-) hepatocytes. Moreover, PTP1B deficiency conferred resistance to apoptosis induced by activation of Fas and constitutively active Foxo1. Rescue of PTP 1B in deficient hepatocytes recovered the phenotype of wild-type cells whereas reduction of PTP1B by siRNA suppressed apoptosis. Our results reveal a unique role for PTP1B as a mediator of the apoptotic pathways triggered by trophic factors withdrawal in hepatocytes. This novel mechanism may represent an important target in the design of therapeutic strategies for human liver regeneration after pathological damage as well as for treatment of hepatocarcinomas.     

Beta-C-glycosiduronic acids and beta-C-glycosyl compounds: new PTP1B inhibitors

Beta-C-glycosiduronic acid quinones and beta-C-glycosyl compounds have been synthesized as sugar-based PTP1B inhibitors. Benzoyl protected quinone derivatives (14 and 35) as well as aryl beta-C-glycosyl compounds (18, 22, 23 and 34) showed IC(50) values of 0.77-5.27 microM against PTP1B, with compounds 18 and 23 bearing an acidic function being the most potent.     

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.     

Discovery of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups as potential PTP1B inhibitors

Two series of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, IIIv was found to have the best in vitro inhibition activity against PTP1B (IC₅₀?=?0.67?±?0.09?µM) and the best selectivity (9-fold) between PTP1B and T-cell protein tyrosine phosphatase (TCPTP). Molecular docking studies demonstrated that compounds IIIm, IIIv and IVg could occupy simultaneously at both the catalytic site and the adjacent pTyr binding site. These results provide novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.     

trans-Beta-nitrostyrene derivatives as slow-binding inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphotyrosyl (pY) proteins to produce tyrosyl proteins and inorganic phosphate. Specific PTPs inhibitors provide useful tools for studying PTP function in signal transduction processes and potential treatment for human diseases such as diabetes, inflammation, and cancer. In this work, trans-beta-nitrostyrene (TBNS) and its derivatives are found to be slow-binding inhibitors against protein tyrosine phosphatases PTP1B, SHP-1, and Yop with moderate potencies (K(I*) = 1-10 microM). Competition experiments with a substrate (pNPP) and iodoacetate indicate that TBNS is active site-directed. The mechanism of inhibition was investigated by UV-vis absorption spectroscopy, (1)H-(13)C heteronuclear single-quantum correlation NMR spectroscopy, and site-directed mutagenesis. These studies suggested a mechanism in which TBNS acts a pY mimetic and binds to the PTP active site to form an initial noncovalent E.I complex, followed by nucleophilic attack on the TBNS nitro group by Cys-215 of PTP1B to form a reversible, covalent adduct as the tighter E.I* complex. TBNS derivatives represent a new class of neutral pY mimetic inhibitors of PTPs.     

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.     

Formylchromone derivatives as a novel class of protein tyrosine phosphatase 1B inhibitors

Formylchromone inhibits a human protein tyrosine phosphatase PTP1B with a IC(50) value of 73 microM. The chemical reactivity of formylchromone was adjusted by substitution at various positions of the formylchromone skeleton. In an initial assessment of the structure-activity relationship, the most potent inhibitor showed an IC(50) of 4.3 microM against PTP1B and strong or medium selectivity against other human PTPases, LAR and TC-PTP. This compound, however, was not selective against microbial PTPases, YPTP1 and YOP. The potency and selectivity of the formylchromone derivatives expecting further improvements provides a novel pharmacophore for the design of drugs for the treatment of type 2 diabetes and obesity.     

Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action

Previous studies implicate protein-tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related phosphatase (LAR) as negative regulators of insulin signaling. The expression and/or activity of PTP1B and LAR are increased in muscle of insulin-resistant rodents and humans. Overexpression of LAR selectively in muscle of transgenic mice causes whole body insulin resistance. To determine whether overexpression of PTP1B also causes insulin resistance, we generated transgenic mice overexpressing human PTP1B selectively in muscle at levels similar to those observed in insulin-resistant humans. Insulin-stimulated insulin receptor (IR) tyrosyl phosphorylation and phosphatidylinositol 3'-kinase activity were impaired by 35% and 40-60% in muscle of PTP1B-overexpressing mice compared with controls. Insulin stimulation of protein kinase C (PKC)lambda/zeta activity, which is required for glucose transport, was impaired in muscle of PTP1B-overexpressing mice compared with controls, showing that PTP1B overexpression impairs activation of these PKC isoforms. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed that whole body glucose disposal and muscle glucose uptake were decreased by 40-50% in PTP1B-overexpressing mice. Overexpression of PTP1B or LAR alone in muscle caused similar impairments in insulin action; however, compound overexpression achieved by crossing PTP1B- and LAR-overexpressing mice was not additive. Antibodies against specific IR phosphotyrosines indicated overlapping sites of action of PTP1B and LAR. Thus, overexpression of PTP1B in vivo impairs insulin sensitivity, suggesting that overexpression of PTP1B in muscle of obese humans and rodents may contribute to their insulin resistance. Lack of additive impairment of insulin signaling by PTP1B and LAR suggests that these PTPs have overlapping actions in causing insulin resistance in vivo.     

Synthesis and characterization of 5,7-dihydroxyflavanone derivatives as novel protein tyrosine phosphatase 1B inhibitors

A series of 5,7-dihydroxyflavanone derivatives were synthesized and identified as reversible and competitive protein tyrosine phosphatase (PTP) 1B inhibitors with IC₅₀ values in the micromolar range. Compound 4k had the most potent in vitro inhibition activity against PTP1B (IC₅₀ = 2.37?±?0.37 μM) and the greatest selectivity (3.7-fold) for PTP1B relative to T-cell protein tyrosine phosphatase. Cell-based studies revealed that 4k was membrane-permeable and enhanced insulin receptor tyrosine phosphorylation in CHO/hIR cells.