Search

Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.

The kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), HePTP, PTPSL and STEP, are involved in the negative regulation of mitogen-activated protein kinase (MAPK) signalling pathways and are important therapeutic targets for a number of diseases. We have used VSpipe, a virtual screening pipeline, to identify a ligand cluster distribution that is unique to this subfamily of PTPs. Several clusters map onto KIM-PTP specific sequence motifs in contrast to the cluster distribution obtained for PTP1B, a classic PTP that mapped to general PTP motifs. Importantly, the ligand clusters coincide with previously reported functional and substrate binding sites in KIM-PTPs. Assessment of the KIM-PTP specific clusters, using ligand efficiency index (LEI) plots generated by the VSpipe, ascertained that the binders in these clusters reside in a more drug-like chemical–biological space than those at the active site. LEI analysis showed differences between clusters across all KIM-PTPs, highlighting a distinct and specific profile for each phosphatase. The most druggable cluster sites are unexplored allosteric functional sites unique to each target. Exploiting these sites may facilitate the delivery of inhibitors with improved drug-like properties, with selectivity amongst the KIM-PTPs and over other classical PTPs.

Orientador: Paulo Cesar Muniz de Lacerda Miranda Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Este trabalho investigou o planejamento, síntese e avaliação biológica de diferentes ligantes da proteína tirosina fosfatases (PTPs). Essas enzimas são sobre-expressas em muitos tipos de Câncer: proteína tirosina fosfatase 1B (PTP1B) e fosfatase baixo peso molecular (LMW-PTP). PTP1B e LMW-PTP estão envolvidas em várias importantes vias de sinalização celular. Estas enzimas têm um sitio ativo conservado, o que sugere uma abordagem geral para investigar as interações moleculares em todos eles. Mesmo assim, é um desafio encontrar inibidores seletivos de PTP, devido às homologias estruturais em muitas enzimas nesta família. Baseado nas informações disponíveis na literatura de PTPs e estudos de docking, propusemos 10 séries de ligantes a serem avaliados neste estudo. Os estudos de modelação foram utilizados para planejar cinco diferentes séries de compostos, tais como, o ácidos (N-fenilamino)oxoacético (série 1), ácidos N-fenilo sulfúrico (série 2), ácidos isoxazole carboxílicos (série 3), ácidos tioacético (série 4) e ácidos N- (2- (1H-benzo [d] imidazol-2-iltio) acetamido) benzóicos (série 5). As cinco séries restantes de compostos foram planejadas utilizando uma abordagem de síntese orientada pela biologia (do inglês biology-orinted synthesis, BIOS) para os derivados de ácidos fosfónicos (série 6), ácidos sulfónicos (séries de 7), ácidos borónicos (série 8), ácidos carboxílicos (série 9) e ácidos cinâmico (série 10). No total, 97 compostos foram planejados e sintetizados. 61 compostos adicionais foram obtidos a partir de fornecedores comerciais. No total, 154 compostos foram avaliados contra PTP1B e LMW-PTP em uma concentração de 5 mM e 1 mM de inibidor. Além disso, os valores de IC50 foram calculados para os compostos mais ativos desta triagem. Estes compostos exibiram valores de IC50 de milimolar (mM) a micromolar (µ M). Os melhores compostos foram selecionados com base nos valores de IC50 (MKR-18a (IC50 = 70.2 µM), MK-35 (IC50 = 0.18 mM), MK-38 (IC50 = 2.33 µM), KSR-6ab (IC50 = 5.0 µM) e KSR-6f (IC50 = 89.5 µM)) e foram submetidos a ensaios de cocristalização contra PTP1B e LMW-PTP. Os dados cocristal foram coletados em Diamond Light Source-UK e difractómetro Bruker Kappa Apex II Duo. Depois de o refinamento de dados, as análises estruturais mostraram que os ligantes não apareceram nos mapas de densidade de elétrons. Além disso, 69 compostos foram selecionados de estudos de relação estrutura e atividade quantitativa (QSAR) contra PTP1B e sua análise do coeficiente de correlação (R2) = 0,89. Mas, as análises preliminares de estudos de relação estrutura e atividade (SAR) mostraram que os derivados de ácido (N-fenilamino)oxoacético são os melhores inibidores de PTP1B e LMW-PTP Abstract: This work investigated the design, synthesis and biological evaluation of ligands to different protein tyrosine phosphatase (PTP) enzymes which are overexpressed in many cancer diseases: protein tyrosine phosphatase 1B (PTP1B) and low molecular weight phosphatase (LMW-PTP). PTP1B and LMW-PTP are involved in several important cell signaling pathways. These enzymes have a conserved active site, which suggests a general approach to investigate molecular interactions in all of them. Nevertheless, it is a challenging task to find selective PTP inhibitor, because of the structural homologies in many enzymes in this family. Based on available literature information of PTPs and docking studies, we proposed 10 new chemical series of ligands to be assessed in this study. Modeling studies were used to design five different series of compounds such as (N-phenylamino)oxoacetic acids (series 1), N-phenylsulfamic acids (series 2), isooxazole carboxylic acids (series 3), thioacetic acids (series 4) and N-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)benzoic acids (series 5). The remaining five series of compounds were designed using a Biology-Oriented Synthesis (BIOS) approach for the compounds of phosphonic acids (series 6), sulfonic acids (series 7), boronic acids (series 8), carboxylic acids (series 9) and cinnamic acids (series 10). In total, 97 compounds were designed and synthesized. Additional 61 compounds were obtained from commercial suppliers. In total, 154 compounds were assayed against PTP1B and LMW-PTP at a 5 mM and 1 mM inhibitor concentration. Further, IC50 values were calculated for the most active compounds in this trial setup. These compounds showed IC50 values in range millimolar (mM) to micromolar (µM) range. The best compounds were selected based on the IC50 values, five compounds (such as: MKR-18a (IC50 = 70.2 µM), MK-35 (IC50 = 0.18 mM), MK-38 (IC50 = 2.33 µM), KSR-6ab (IC50 = 5.0 µM) and KSR-6f (IC50 = 89.5 µM)) selected for cocrystallization against PTP1B and LMW-PTP. The cocrystal data was collected in Diamond Light Source ¿UK and diffractometer Bruker Kappa Apex II Duo. After refinement of data, structure analysis showed that ligand did not appear in the electron density maps. Furthermore, 69 compounds were selected for Quantitative Structure¿Activity Relationship (QSAR) studies against PTP1B and their analysis of Correlation coefficient (R2) = 0.89. But, preliminary analysis of Structure-Activity Relationship (SAR) studies showed that (N-phenylamino)oxoacetic acid derivatives are the best inhibitors for PTP1B and LMW-PTP Doutorado Química Orgânica Doutor em Ciências CNPQ 190655/2011-9

Orientador: Ronaldo Aloise Pilli Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica Resumo: Fostriecina (10) é um fosfato orgânico natural citotóxico isolado de Streptomyces pulveraceu e apresenta potente atividade anticâncer contra células leucêmicas (IC50=0,46 mM) e outras linhagens de células tumorais. As propriedades citotóxicas desse composto são atribuídas à inibição seletiva da proteína fosfatase 2A (IC50=3,2 mM). A fostriecina havia avançado para estudos clínicos, no entanto os estudos foram suspensos devido à instabilidade do composto, que foi atribuída à porção triênica da sua estrutura. Com base nisso, foi decidido estudar análogos de fostriecina (105a e 105b) que apresentem estabilidade química atrelada a potente atividade biológica. A estratégia de síntese proposta para a obtenção dos análogos 105a e 105b foi orientada por algumas idéias centrais que visavam preservar a integridade do anel di-hidropirano, controlar adequadamente a geometria das ligações duplas presentes na estrutura e a estereoquímica absoluta dos centros estereogênicos, sem comprometer a eficiência da rota sintética. As etapas chave na preparação dos análogos de fostriecina foram: a reação de alilação catalítica e assimétrica de Yamamoto para instalação do centro estereogênico em C5; a reação de di-hidroxilação assimétrica de Sharpless para construção do centro estereogênico em C8 e a reação aldólica para construção da ligação C10-C11. Os produtos finais da síntese, bem como os intermediários avançados da síntese, foram submetidos a três ensaios: de atividade antiproliferativa in vitro contra 10 linhagens de células tumorais, de inibição da enzima LMWPTP isolada e de viabilidade celular em linhagens que superexpressam a LMWPTP. Três compostos se mostraram bastante promissores nos ensaios, apresentando inibição superior aos controles positivos e com baixa toxicidade para células sadias. Os resultados obtidos sugerem que os compostos induzem morte celular via perturbação das vias de sinalização da enzima LMWPTP Abstract: Fostriecin (10) is a natural cytotoxic organic phosphate isolated from Streptomyces pulveraceus which exhibits potent antitumor activity against leukemic cells (IC50=0,46 mM), among others. The cytotoxic properties of this compound are presumably linked to the selective inhibition of the phosphatase protein 2A (IC50=3,2 mM). Although fostriecin has advanced to clinical studies, these were suspended due to its chemical instability, which was assigned to the trienic moiety of the structure. Therefore it was decided to prepare fostriecin analogues (105a and 105b), which should present chemical stability and potent biological activity.The synthetic strategy proposed towards the achievement of analogues 105a and 105b was guided by a main idea that aimed to preserve the integrity of the dihydropyran ring and to properly control the geometry of the double bonds, as well as the absolute stereochemistry of the stereogenic centers, without compromising the efficiency of the synthetic route. The key steps in the preparation of the fostriecin analogues were: a Yamamoto catalytic and asymmetric allylation reaction to install the stereogenic center in C5; a Sharpless asymmetric dihydroxilation reaction to access the stereogenic center in C8; and an aldol reaction to form the bond between C10-C11. The target products, as well as advanced synthetic intermediates were submitted to three biological assays: antiproliferative activity in vitro against 10 cancer cell lines; LMWPTP isolated enzyme inhibition; and cell viability within cell lines that overexpress the LMWPTP. Three of those compounds showed promising results, exhibiting higher inhibition than the positive controls and with low cytotoxicity to healthy cells. These results suggest that these compounds induct apoptosis via disruption of the LMWPTP enzyme signaling pathways Doutorado Química Orgânica Doutor em Ciências

Protein phosphatases 1 and 2A (PP1 and PP2A) are the most ubiquitous and abundant serine/threonine phosphatases in eukaryotic cells. They play fundamental roles in the regulation of various cellular functions. This review focuses on recent advances in the functional studies of these enzymes in the field of smooth muscle physiology. Many naturally occurring protein phosphatase inhibitors with different relative PP1/PP2A affinities have been discovered and are widely used as powerful research tools. Current topics in the chemical biology of PP1/PP2A inhibitors are introduced and discussed, highlighting the identification of the gene cluster responsible for the biosynthesis of calyculin A in a symbiont microorganism of a marine sponge.

The protein tyrosine phosphatase (PTP) family is involved in multiple cellular functions and plays an important role in various pathological and physiological processes. In many chronic diseases, for example cancer, PTP is a potential therapeutic target for cancer treatment. In the last two decades, dozens of PTP inhibitors which specifically target individual PTP molecules were developed as therapeutic agents. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and is the second most lethal cancer worldwide due to a lack of effective therapies. Recent studies have unveiled both oncogenic and tumor suppressive functions of PTP in HCC. Here, we review the current knowledge on the involvement of PTP in HCC and further discuss the possibility of targeting PTP in HCC.

Key message PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used. Abstract Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).

Patients with inherited dilated cardiomyopathy (DCM) often suffer from severe heart failure based on impaired cardiac contractility leading to increased morbidity and mortality. Integrin-linked kinase (ILK) as a part of the cardiac mechanical stretch sensor was found to be an essential genetic regulator of cardiac contractility. Integrin-linked kinase localizes to z-disks and costameres in vertebrate hearts and regulates the activity of the signaling molecule protein kinase B (PKB/Akt) by controlling its phosphorylation. Despite identification of several potential drug targets in the ILK signaling pathway, pharmacological treatment strategies to restore contractile function in ILK-dependent cardiomyopathies have not been established yet. In recent years, the zebrafish has emerged as a valuable experimental system to model human cardiomyopathies as well as a powerful tool for the straightforward high-throughput in vivo small compound screening of therapeutically active substances. Using the ILK deficient zebrafish heart failure mutant main squeeze (msq), which shows reduced PKB phosphorylation and thereby impaired cardiac contractile force, we identified here, in an automated small compound screen, the protein phosphatase inhibitors calyculin A and okadaic acid significantly restoring myocardial contractile function by reconstituting PKB phosphorylation in msq ILK-deficient zebrafish embryos.

Seven PNA-encoded combinatorial libraries targeting proteases and phosphatases with covalent reversible and irreversible mechanism-based inhibitors were prepared. The libraries were synthesized using modified PNA monomers, which dramatically increase the water solubility of the libraries in biologically relevant buffers. The libraries were shown to selectively inhibit targeted enzymes.

Contains fulltext : 136010.pdf (Publisher’s version ) (Open Access) Background:Plasma exposure of sunitinib shows large inter-individual variation. Therefore, a pharmacokinetic (PK) study was performed to determine safety and feasibility of sunitinib dosing based on PK levels.Methods:Patients were treated with sunitinib 37.5 mg once daily. At days 15 and 29 of treatment, plasma trough levels of sunitinib and N-desethyl sunitinib were measured. If the total trough level (TTL) was

The chicken erythroblast cell line, HD3, has high glucose transport activity which is lost upon differentiation to the red cell phenotype. HD3 cells, when incubated under conditions where maturation occurs, show substantial loss of GLUT1 and GLUT3 mRNAs. To assess whether cAMP or cellular protein phosphorylation affected GLUT mRNA and protein, the HD3 cells were incubated in the presence of different phosphatase inhibitors. Treatment of HD3 cells with the phosphatase inhibitors okadaic acid, vanadate or with 3-isobutyl-1-methyl-xanthine induced glucose transport and GLUT mRNAs. This suggests that phosphorylation events enhance glucose transport and that their reduction may be involved in the decrease in glucose transport that occurs upon HD3 cells differentiation.

A short enantioselective synthesis of (+)-eleutherin, (+)-allo-eleutherin and the formal synthesis of (+)-nocardione B is described. The synthesis is completed in six steps in overall yields of 8% for eleutherin and 14% for allo-eleutherin. The synthetic strategy features an efficient combination of the Dotz annulation reaction with a chiral alkyne and an oxa-Pictet Spengler reaction as the keys steps in the stereodivergent synthesis of (+)-eleutherin and (+)-allo-eleutherin. The synthesis of (S)-(+)-2-(2′-hydroxypropyl)-5-methoxy-1,4-naphthoquinone entails the formal synthesis of (+)-nocardione B.

The first documented case of diabetes mellitus occurred earlier than 4000 BC. Since then, many of the brightest minds in medicinehave dedicated their time and effort toward developing treatments that can reverse the course of this deadly disease. As our understandingof the pathogenesis of diabetes increases, so does the availability of treatment options. The fight against diabetes once onlyhad metformin and sulfonylureas as the cornerstone of oral treatment, but now, multiple classes have been added to this armamentariumincluding thiazolidenediones (TZDs) and dipeptidyl peptidase IV (DDP IV) inhibitors. These therapies provide reasonable durable glycemic control but are unable to arrest the natural progression of diabetes or the eventual need for insulin. By utilizing our growingknowledge on the pathogenesis of diabetes, a number of new therapeutic agents are in development to overcome the shortcomingsof current therapies. Promising options on the horizon include sodium-coupled glucose cotransport 2 (SGLT2) inhibitors, Ranolazine,Salicylates, second generation Peroxisome proliferator ac-tivator receptor agonists (PPARs), and 11-beta hydroxysteroid dehydrogenasetype 1 inhibitors (11-beta HSD1 inhibitors). Various molecules, including some enzymes, are also in development particularly to addressbeta cell preservation and its sensitivity to glucose, while minimizing hypoglycemia. Most of these new classes of drugs consistof daily administration, simplifying the regimen for patients and likely increasing medication compliance.This article reviews the new agents that are advancing through clinical trials, their mechanism of actions, glucose lowering effect andpossible side effects and limitations

Calyculins, highly cytotoxic polyketides, originally isolated from the marine sponge Discodermia calyx by Fusetani and co-workers, belong to the lithistid sponges group. These molecules have become interesting targets for cell biologists and synthetic organic chemists. The serine/threonine protein phosphatases play an essential role in the cellular signalling, metabolism, and cell cycle control. Calyculins express potent protein phosphatase 1 and 2A inhibitory activity, and have therefore become valuable tools for cellular biologists studying intracellular processes and their control by reversible phosphorylation. Calyculins might also play an important role in the development of several diseases such as cancer, neurodegenerative diseases, and type 2-diabetes mellitus. The fascinating structures of calyculins have inspired various groups of synthetic organic chemists to develop total syntheses of the most abundant calyculins A and C. However, with fifteen chiral centres, a cyano-capped tetraene unit, a phosphate-bearing spiroketal, an anti, anti, anti dipropionate segment, an α-chiral oxazole, and a trihydroxylated γ-amino acid, calyculins reach versatility that only few natural products can surpass, and truly challenge modern chemists’ asymmetric synthesis skills.

Bis(maltolato)oxovanadium(IV) (BMOV), a protein phosphotyrosine phosphatase inhibitor, selectively induced apoptosis (as quantitated by TUNEL staining) in a B-cell line (Ramos) but not in a T-cell line (Jurkat). The pattern of BMOV-induced protein tyrosine phosphorylation was different in B-cells versus T-cells. Further, BMOV induced a 2-fold increase in ceramide levels in B-cells but not in T-cells and this resembled the ceramide increase following activation of the B-cell antigen receptor. A 2-fold increase in the ratio of ceramide to sphingomyelin in B-cells treated with BMOV suggested that sphingomyelinase activation was the result of the sustained tyrosine phosphorylation of specific proteins and activated the cell death pathway.

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine - 186%, L-leucine - 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ss-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ss-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ss-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.