Shikimate Kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: Kinetics and Structural Dynamics of a Potential Molecular Target for Drug Development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endurance training improves responsiveness to insulin and modulates insulin signal transduction through the phosphatidylinositol 3-kinase/Akt-1 pathway

Background: Endurance training increases insulin-stimulated muscle glucose transport and leads to improved metabolic control in diabetic patients.Objective: To analyze the effects of endurance training on the early steps of insulin action in muscle of rats. Design: Male rats submitted to daily swimming for 6 weeks were compared with sedentary controls. At the end of the training period, anesthetized animals received an intravenous (i.v.) injection of insulin and had a fragment of their gastrocnemius muscle excised for the experiments.Methods: Associations between insulin receptor, insulin receptor substrates (IRS)-1 and -2 and phosphatidylinositol 3-kinase (PI3-kinase) were analyzed by immunoprecipitation and immunoblotting. Akt-1 serine phosphorylation and specific protein quantification were detected by immunoblotting of total extracts, and IRS-1/IRS-2-associated PI3-kinase activity were determined by thin-layer chromatography.Results: Insulin-induced phosphorylation of IRS-1 and IRS-2 increased respectively by 1.8-fold (P < 0.05) and 1.5-fold (P < 0.05), whereas their association with PI3-kinase increased by 2.3-fold (P < 0.05) and 1.9-fold (P < 0.05) in trained rats as compared with sedentary controls, respectively. The activity of PI3-kinase associated with IRS-1 and IRS-2 increased by 1.8-fold (P < 0.05) and 1.7-fold (P < 0.05) respectively, in trained rats as compared with their untrained counterparts. Serine phosphorylation of Akt-1/PKB increased 1.7-fold (P < 0.05) in trained rats in response to insulin. These findings were accompanied by increased responsiveness to insulin as demonstrated by a reduced area under the curve for insulin during an i.v. glucose tolerance test, by increased glucose disappearance rate during an insulin tolerance test, and by increased expression of glucose transporter-4.Conclusions: the increased responsiveness to insulin induced by chronic exercise in rat skeletal muscle may result, at least in part, from the modulation of the insulin signaling pathway at different molecular levels.

Dissociation and electrooxidation of primaquine diphosphate as an approach to the study of anti-chagas prodrugs mechanism of action

This paper describes the voltammetric behavior of primaquine as a previous support to the further understanding of the delivery and action mechanisms of its respective synthesized prodrugs. There are few papers describing the drug behavior and most of the time no correlation between oxidation process and pH is done. Our results showed that primaquine oxidation is a one-step reaction involving two electrons with the charge transfer process being strongly pH-dependent in acid medium and pH-independent in a weak basic medium, with the neutral form being easily oxidized.This leads to the conclusion that quinoline nitrogen ring neutralization is a determinant step to the formation of the oxidized primaquine form. The existence of a relationship between the primaquine dissociation equilibrium and its electrooxidation process is shown.This work points the importance of voltammetric methodology as a tool for further studies on quantitative relationship studies between chemical structure and biological activity (QSAR) for electroactive drugs. (C) 2000 Elsevier B.V. S.A. All rights reserved.

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDKI and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors. (C) 2004 Elsevier B.V. All rights reserved.

Structural Basis for Interaction of Inhibitors with Cyclin-Dependent Kinase 2

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular models of protein kinase 6 from Plasmodium falciparum

Cyclin-dependent kinases (CDKs) have been identified as potential targets for development of drugs, mainly against cancer. These studies generated a vast library of chemical inhibitors of CDKs, and some of these molecules can also inhibit kinases identified in the Plasmodium falciparum genome. Here we describe structural models for Protein Kinase 6 from P. falciparum (PfPK6) complexed with Roscovitine and Olomoucine. These models show clear structural evidence for differences observed in the inhibition, and may help designing inhibitors for PfPK6 generating new potential drugs against malaria.

Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis

Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.

Molecular model of cyclin-dependent kinase 5 complexed with roscovitine

Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data, are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives. (C) 2002 Elsevier B.V. (USA). All rights reserved.

Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase

Matrix metal loprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

Purine nucleoside phosphorylase: A potential target for the development of drugs to treat T-cell- and apicomplexan parasite-mediated diseases

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell and Apicomplexan parasites-mediated diseases.

Characterization of glycerol kinase from baker's yeast: Response surface modeling of the enzymatic reaction

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Evidence for involvement of Saccharomyces cerevisiae protein kinase C in glucose induction of HXT genes and derepression of SUC2

The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Δ mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V max of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Δ mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Δ mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.