Activation of MAPK by potassium bisperoxo(1,10-phenanthroline)oxovanadate (V).

Potassium bisperoxo(1,10-phenantroline)oxovanadate (V) [bpV(phen)] is a potent protein tyrocine phosphatase inhibitor which mediates a variety of biological effects. The aim of these studies was to examine the role(s) of mitogen activated protein kinase (MAPK) pathways in PC12 cell proliferation and toxicity by bpV(phen). BpV(phen) exerts a bimodal effect in PC12 cells: proliferation at low and cell death at higher micromolar concentrations. Activation of MAPK by bpV(phen) depends on time and concentration. The phosphorylation pattern of extracellular regulated kinases (ERK 1/2), c-jun N-terminal activated kinases (JNK) and p38 in PC12 cells is strikingly different. Activation of JNK is sustained in PC12 cells. In contrast, ERK 1/2 activation is transient and treatment with PD98059 indicates that ERK activation by bpV(phen) is partly independent from the ras-MEK pathway. Stability studies of bpV(phen) in DMEM and PBS showed linear relationship with T1/2 about 6 h and 10 days in DMEM and PBS, respectively. Comparison between the time courses of MAPK activation and kinetics of bpV(phen) decomposition as assessed by 51V-NMR analysis show that the initial and maximal phosphorylation signals are produced in the presence of the complex bpV(phen) and not caused by the decomposition products of bpV(phen).     

Molecular mechanism of T-cell protein tyrosine phosphatase (TCPTP) activation by mitoxantrone

T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α₁β₁ directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α₁-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α₁-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α₁-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics.     

Molecular modeling of protein tyrosine phosphatase 1B (PTP 1B) inhibitors

Binding modes of a series of aryloxymethylphosphonates and monoanionic biosteres of phosphate group from a series of benzylic alpha,alpha-diflluoro phosphate and its biosteres as protein tyrosine phosphatase 1B (PTP 1B) inhibitors have been identified by molecular modeling techniques. We have performed docking and molecular dynamics simulations of these inhibitors with PTP 1B enzyme. The initial conformation of the inhibitors for docking was obtained from simulated annealing technique. Solvent accessible surface area calculations suggested that active site of PTP 1B is highly hydrophobic. The results indicate that for aryloxymethylphosphonates, in addition to hydrogen bonding interactions, Tyr46, Arg47, Asp48, Val49, Glu115, Lys116, Lys120 amino acid residues of PTP 1B are responsible for governing inhibitor potency of the compounds. The sulfonate and tetrazole functional groups have been identified as effective monoanionic biosteres of phosphate group and biphenyl ring system due to its favorable interactions with Glu115, Lys116, Lys120 residues of PTP 1B found to be more suitable aromatic functionality than naphthalene ring system for benzylic alpha,alpha-diflluoro phosphate and its biosteres. The information generated from the present study should be useful in the design of more potent PTP 1B inhibitors as anti diabetic agents.     

DNA binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline

Absorption spectroscopy and circular dichroism (CD) have been used to characterize the DNA binding of [Fe(phen)3]2+, [Fe(phen)2(DIP)]2+ and [Fe(phen)(DIP)2]2+ where phen and DIP stand for 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline, respectively. Both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ bind weakly to calf thymus DNA (CT-DNA) in an electrostatic mode, while [Fe(phen)(DIP)2]2+ binds more strongly to CT-DNA, possibly in an intercalation mode. The hypochromicity, red shift and Kb increase in the order [Fe(phen)3]2+ < [Fe(phen)2(DIP)]2+ < [Fe(phen)(DIP)2]2+ in accordance with the increase in size and hydrophobicity of the iron(II) complexes. The thermodynamic parameters obtained suggest that the DNA binding of both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ is entropically driven, while that of [Fe(phen)(DIP)2]2+ is enthalpically driven. A strong CD spectrum in the UV and visible region develops upon addition of CT-DNA into the racemate solution of each iron(II) complex (Pfeiffer effect). This has revealed that a shift in diastereomeric inversion equilibrium takes place in the solution to yield an excess of one of the DNA-complex diastereomers. The striking resemblance of the CD spectral profiles to those of the pure delta-enantiomer indicates that the delta-enantiomer of the iron(II) complexes is preferentially bound to CT-DNA. The mechanism of the development of Pfeiffer CD is proposed on the basis of kinetic studies on the DNA binding of the racemic iron(II) complexes.     

Engineering non-natural inhibitor sensitivity in protein tyrosine phosphatase H1

Protein tyrosine phosphatase H1, a member of the ubiquitous protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important signaling molecule, mutant forms of which have been found in human colorectal cancers. Selective PTPH1 inhibitors would be valuable tools for investigating PTPH1's roles in cellular regulation. However, no PTPH1-specific inhibitors are known. To identify target-selective inhibitors of human PTPH1, we have redesigned a PTPH1/inhibitor interface. Structure-based protein design was used to identify two amino-acid residues, isoleucine 846 and methionine 883, that control PTPH1's sensitivity to oxalylaminoindole PTP inhibitors. Mutation of residues 846 and 883 to alanine and glycine, respectively, conferred novel inhibitor sensitivity onto PTPH1. From a small panel of putative inhibitors, compounds that potently and selectively target the inhibitor-sensitized PTPH1 mutants were identified.     

Engineered inhibitor sensitivity in the WPD loop of a protein tyrosine phosphatase

Small molecules that can be used to turn off the activities of specific cellular proteins are essential tools for chemical biology. Few such compounds are known, however, and they are particularly difficult to identify for members of large protein families. Here, we present a method for insertion of a chemical "off switch" into a catalytically essential loop region (the "WPD loop") of a protein tyrosine phosphatase (PTP). Using a combination of point mutations and amino acid insertions, we have engineered variants of T-cell PTP (TCPTP) that possess cysteine-rich WPD loops. The engineered WPD loops, which contain sequences that appear in no wild-type PTP, confer upon TCPTP the ability to bind a cell-permeable small molecule (the biarsenical fluorescein derivative, FlAsH) that is not an inhibitor of wild-type PTPs. We have identified sites in TCPTP's WPD loop that can be modified to display FlAsH-binding cysteine residues without disrupting TCPTP's inherent PTP activity, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. Upon addition of the FlAsH ligand, however, the activities of the mutants drop dramatically. Inhibition of the FlAsH-sensitized TCPTP mutants is rapid and specific; and strong FlAsH sensitivity was observed in mutants that contain as few as two cysteine point mutations in their engineered WPD loops. Our results show that relatively conservative substitutions can be used to engineer precise small-molecule control of PTP activity. Moreover, since all known classical PTPs utilize the WPD-loop mechanism targeted in this study, it is likely that a substantial fraction of the PTP superfamily can be sensitized through an analogous approach.     

Mechanism of action of pyridazine analogues on protein tyrosine phosphatase 1B (PTP1B)

The inhibitory effect on PTP1B caused by the addition of pyridazine analogues has been investigated. Biophysical techniques, that is, mass spectrometry (MS), nuclear magnetic resonance (NMR), and isothermal titration calorimetry (ITC) were used for the characterization. Pyridazine analogues cause catalytic oxidation of the reducing agent, generating hydrogen peroxide that oxidizes the active site cysteine on the enzyme, leading to enzyme inactivation. Two additional compound classes show the same effect. We found one common structural feature in these molecules that allows the reaction with triplet molecular oxygen to be less endothermic. A proposed mechanism for the catalytic redox cycle is described.     

Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion

Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive interactions, whereas the intracellular catalytic regions can modulate the phosphorylation state of cadherin/catenin complexes. We describe a systematic investigation of the cell-adhesive properties of the extracellular region of RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at 2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals extensive interactions between the two domains, which form a rigid structural unit. Structure-based site-directed mutagenesis, serial domain deletions and cell-adhesion assays allowed us to identify the four N-terminal domains (MAM, Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit. Biophysical characterization revealed at least two independent types of homophilic interaction which, taken together, suggest that there is the potential for formation of a complex and possibly ordered array of receptor molecules at cell contact sites.     

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

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

A new polymorph of dichlorido(1,10-phenanthroline)platinum(II)

A yet unreported polymorph of [PtCl₂(1,10-phenanthroline)] was obtained by slow decomposition, in CH₂Cl₂ solution, of [Pt{CH₂CH₂N(CH₂CH₃)₂-κC,κN}(1,10-phenanthroline)](ClO₄). The structure of the new orthorhombic form, III, (space group Pna2₁), is described and compared to those of the two already reported forms, I and II, which are monoclinic (space group P2₁/c) and orthorhombic (space group Pca2₁), respectively [21]. Polymorph III appears to be the least stable of the three.     

Potassium Bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) Induces Apoptosis and Pyroptosis and Disrupts the P62-HDAC6 Protein Interaction to Suppress the Acetylated Microtubule-dependent Degradation of Autophagosomes

Autophagy is a cellular process that controls and executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activates the initiation of autophagy. Autophagosomes migrate along acetylated microtubules to fuse with lysosomes to execute the degradation of the engulfed substrates that usually bind with sequestosome 1 (SQSTM1, p62). Microtubule-associated protein 1 light chain 3 (LC3) traces the autophagy process by converting from the LC3-I to the LC3-II isoform and serves as a major marker of autophagy flux. Potassium bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) is an insulin mimic and a PTEN inhibitor and has the potential to treat different diseases. Here we show that bpV(phen) enhances the ubiquitination of p62, reduces the stability of p62, disrupts the interaction between p62 and histone deacetylase 6 (HDAC6), activates the deacetylase activity of HDAC6 on α-tubulin, and impairs stable acetylated microtubules. Microtubular destabilization leads to the blockade of autophagosome-lysosome fusion and accumulation of autophagosomes. Autophagy defects lead to oxidative stress and lysosomal rupture, which trigger different types of cell death, including apoptosis and pyroptosis. The consistent results from multiple systems, including mouse and different types of mammalian cells, are different from the predicted function of bpV(phen) as a PTEN inhibitor to activate autophagy flux. In addition, levels of p62 are reduced but not elevated when autophagosomal degradation is blocked, revealing a novel function of p62 in autophagy regulation. Therefore, it is necessary to pay attention to the roles of bpV(phen) in autophagy, apoptosis, and pyroptosis when it is developed as a drug.     

Crystal structures of α- and β-(1,10-phenanthroline)tetrahydroborato(triphenylphosphine)copper(I) and (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline)tetrahydroboratocopper(I)

Copper(I) is five coordinate in (1,10-phenanthroline)tetrahydroborato(triphenylphosphine)copper(I). This compound crystallizes from either toluene as the yellow, α-form, a = 16.247(8), b = 9.750(7), c = 9.322(5) Å, α = 62.92(4), β = 84.77(4), γ = 84.34(5)°, triclinic P$\text{1}$, Z = 2, or from a xylene/methylene chloride mixture as the red β-form, X-ray cell, a = 13.675(11), b = 10.115(8), c = 9.700(7) Å, α = 95.22(6), β = 96.22(6), γ = 101.02(6)°; neutron cell, as the tetradeuteroborate, a = 13.703(1), b = 10.096(8), c = 9.74(1) Å, α = 95.23(9), β = 96.51(8), γ = 101.04(2)°, triclinic, P$\text{1}$, Z = 2. For both forms, unidentate triphenylphosphine, bidentate 1,10-phenanthroline and unsymmetrical bidentate BH₄^? completes the copper(I) coordination but there are subtle differences between the two. When the ligand 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, dmdp, replaces 1,10-phenanthroline, the compound obtained is four coordinate with no tpp in the crystal. [C(dmdp)BH₄] is monoclinic, Cc, a = 14.522(4), b = 20.07(2), c = 7.718(2) Å, β = 106.17(2)°, Z = 4.     

Facile fabrication of promising protein tyrosine phosphatase (PTP) inhibitor entities based on 'clicked' serine/threonine-monosaccharide hybrids

Protein tyrosine phosphatases (PTPs) are well-validated therapeutic targets for many human major diseases. The development of their potent inhibitors has therefore become a main focus of both academia and the pharmaceutical industry. We report herein a facile strategy toward the fabrication of new and competent PTP inhibitor entities by simply 'clicking' alkynyl amino acids onto diverse azido sugar templates. Triazolyl glucosyl, galactosyl, and mannosyl serine and threonine derivatives were efficiently synthesized via click reaction, which were then identified as potent CDC25B and PTP1B inhibitors selective over a panel of homologous PTPs tested. Their inhibitory activity and selectivity were found to largely lie on the structurally and configurationally diversified monosaccharide moieties whereon serinyl and threoninyl residues were introduced. In addition, MTT assay revealed the triazole-connected sugar-amino acid hybrids may also inhibit the growth of several human cancer cell lines including A549, Hela, and especially HCT-116. On the basis of such compelling evidence, we consider that this compound series could furnish promising chemical entities serving as new CDC25B and PTP1B inhibitors with potential cellular activity. Furthermore, the 'click' strategy starting from easily accessible and biocompatible amino acids and sugar templates would allow the modular fabrication of a rich library of new PTP inhibitors efficaciously and productively.     

Molecular docking and virtual screening for novel protein tyrosine phosphatase 1B (PTP1B) inhibitors

Protein tyrosine phosphatase 1B (PTP1B) functions as major negative regulator of insulin and leptin signaling pathways. In view of this, PTP1B is an significant target for drug development against cancer, diabetes and obesity. The aim of the current study is to identify PTP1B inhibitors by means of virtual screening with docking. 523,366 molecules from ZINC database have been screened and based on DOCK grid scores and hydrogen bonding interactions five new potential inhibitors were identified. ZINC12502589, ZINC13213457, ZINC25721858, ZINC31392733 and ZINC04096400 were identified as potential lead molecules for inhibition of PTP1B. The identified molecules were subjected to Lipinski''s rule of five parameters and found that they did not violate any rule. More specific analysis of pharmacological parameters may be scrutinized through a complete ADME/Tox evaluation. Pharma algorithm was used to Calculate ADME–Tox profiles for such molecules. In general, all the molecules presented advantages and as well as disadvantages when compared to each other. No marked difference in health effects and toxicity profiles were observed among these molecules.     

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV)

Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST-PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST-PTP1B was reversibly inhibited by solutions of BMOV with an IC₅₀ value of 0.86 ± 0.02 μM. Steady state kinetic studies showed that inhibition of GST-PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST-PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC₅₀ value of 32.1 ± 0.6 μM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST-PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST-PTP1B and ALP are very different.     

Oxidative DNA damage of mixed copper(II) complexes with sulfonamides and 1,10-phenanthroline. Crystal structure of [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)2(1,10-phenanthroline)

Mixed coordination compounds of Cu(II) with sulfonamides and 1,10-phenanthroline as ligands have been prepared and characterised. Single crystal structural determination of the complex [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)(2)(phen)] shows Cu(II) ions are located in a highly distorted octahedral environment, probably as a consequence of the Jahn-Teller effect. The FT-IR and electronic paramagnetic resonance (EPR) spectra are also discussed. The mixed complexes prepared undergo an extensive DNA cleavage in the presence of ascorbate and hydrogen peroxide. Two of the complexes have higher nucleolytic efficiency than the bis(o-phenanthroline)copper(II) complex.     

The protein tyrosine phosphatase PTP-BL associates with the midbody and is involved in the regulation of cytokinesis

PTP-BL is a highly modular protein tyrosine phosphatase of unknown function. It consists of an N-terminal FERM domain, five PDZ domains, and a C-terminally located tyrosine phosphatase domain. Here we show that PTP-BL is involved in the regulation of cytokinesis. We demonstrate localization of endogenous PTP-BL at the centrosomes during inter- and metaphase and at the spindle midzone during anaphase. Finally PTP-BL is concentrated at the midbody in cytokinesis. We show that PTP-BL is targeted to the midbody and centrosome by a specific splicing variant of the N-terminus characterized by an insertion of 182 amino acids. Moreover, we demonstrate that the FERM domain of PTP-BL is associated with the contractile ring and can be cosedimented with filamentous actin, whereas the N-terminus can be cosedimented with microtubules. We demonstrate that elevating the expression level of wild-type PTP-BL or expression of PTP-BL with an inactive tyrosine phosphatase domain leads to defects in cytokinesis and to the generation of multinucleate cells. We suggest that PTP-BL plays a role in the regulation of cytokinesis.     

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I)

The bis(1,10-phenanthroline)copper(I) complex is a relatively simple molecule previously shown to cause DNA cleavage with a strong preference for gene control regions such as the Pribnow box. Sequence level mapping of sites of [(Phen)2CuI]+ cleavage in greater than 2000 bases in histone genes and the plasmid pUC9 showed that the specificity for control regions is related to a predominant preference for minor groove binding at TAT triplets, which were cleaved most strongly at the adenosine sugar ring. The related sequences TGT, TAAT, TAGPy, and CAGT (Py = pyrimidine) were moderately preferred, while CAT and TAC triplets, PyPuPuPu quartets, PuPuPuPy quartets, and CG-rich PyPuPuPy quartets were cleaved with low to average frequency. Polypurine and polypyrimidine sequences were cleaved with low frequency. The sequence preferences of [(Phen)2CuI]+ can be ascribed predominantly to (i) a requirement for binding in the minor groove at a pyrimidine 3'----5' step and (ii) stereoelectronic effects of the 2-amino group of guanine in the minor groove, which inhibit binding. Although the reagent appears primarily to recognize sequence features at the triplet or quartet level, lower than expected cleavage was observed for two TAT sequences adjacent to several other preferred sequences and higher than expected cleavage was observed at CAAGC sequences, suggesting that longer range sequence-dependent DNA conformational effects influence specificity in certain cases.     

Molecular mechanisms of the action of miraculin,a taste-modifying protein

Miraculin (MCL) is a homodimeric protein isolated from the fruits of Richadella dulcifica, a shrub native to West Africa. Although it is flat in taste at neutral pH, MCL has taste-modifying activity in which sour stimuli produce a sweet perception. Once MCL enters the mouth, strong sweetness can be detected for more than 1 h each time we taste a sour solution. While the human sweet taste receptor (hT1R2–hT1R3) has been identified, the molecular mechanisms underlying the taste-modifying activity of MCL remain unclear. Recently, experimental evidence has been published demonstrating the successful quantitative evaluation of the acid-induced sweetness of MCL using a cell-based assay system. The results strongly suggested that MCL binds hT1R2–hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH. Since sweet-tasting proteins may be used as low-calorie sweeteners because they contain almost no calories, it is expected that MCL will be used in the near future as a new low-calorie sweetener or to modify the taste of sour fruits.