A novel neurotrophic drug for cognitive enhancement and Alzheimer's disease

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer''s disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer''s disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.     

Hexokinase and glucose-6-phosphatase activity in woodchuck model of hepatitis virus-induced hepatocellular carcinoma

2-Deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) is used for PET imaging of woodchuck (Marmota monax) model of hepatocellular carcinoma (HCC). The usefulness of FDG on this animal model needs to be validated according to the hypothesized mechanisms. In this study, two key enzymes involved in glucose or [(18)F] FDG metabolism, hexokinase (HK) and glucose-6-phophatase (G6Pase), were examined for their enzymatic activities in the woodchuck models of HCC, which has not been studied before. After dynamic PET scans, woodchuck liver tissue samples were harvested and the homogenate was centrifuged. The supernatant was used for HK activity assay and the microsomal pellet was used for G6Pase assay. HK and G6Pase activities were measured by means of colorimetric reactions via kinetic and end-point assays, respectively. Total protein content was measured by the Bradford method and used to normalize all enzyme activities. HK and G6Pase activities in woodchuck HCC will be used to correlate with in vivo PET imaging data. The woodchuck model of HCC had significantly increased levels of HK in the livers compared to the age-matching healthy woodchuck (7.96 +/- 1.27 vs. 2.74 +/- 0.66 mU/mg protein, P < 0.01) and significantly decreased levels of G6Pase compared to healthy woodchuck (40.35 +/- 19.28 vs. 237.01 +/- 17.32 mU/mg protein, P < 0.01), reflecting an increase in glycolysis. In addition, significant differences were found in HK and G6Pase activities between HCC liver region (HK: 7.96 +/- 1.27 mU/mg protein; G6Pase: 40.35 +/- 19.28 mU/mg protein) and surrounding normal liver region (HK: 2.98 +/- 0.92 mU/mg protein; G6Pase: 140.87 +/- 30.62 mU/mg protein) in the same woodchuck model of HCC (P < 0.01). Our study demonstrated an increased HK activity and a decreased G6Pase activity in liver of the woodchuck models of HCC as compared to normal woodchuck liver.     

Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-Å crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.Dengue fever (DF) affects tens of millions of people each year, with an average mortality rate of 5%, comprising mostly young children. The increasing spread and frequency of global epidemics of this disease have heightened the urgency for developing effective strategies for prevention, diagnosis, and treatment. Though several groups are involved in vaccine development (17, 23, 25), dengue presents a unique, complex challenge. Four antigenically distinct serotypes of the causative dengue virus (DENV) (DEN1 to -4) occur in nature, with differing pathogenicities in partially overlapping geographic locations. In individuals previously exposed to a certain serotype, repeat assault by a different serotype can lead to potentially fatal complications of the disease, dengue hemorrhagic fever and dengue shock syndrome. The presence of subneutralizing levels of antibodies against the first serotype, resulting in antibody-dependent enhancement (ADE), is believed to be a causative mechanism underlying this complication. Forty percent of the world population lives in dengue risk areas, mainly in tropical and subtropical regions, which necessitates the development of vaccines and therapeutics that can simultaneously protect against all four serotypes (24).DEN1 to -4 belong to the family Flaviviridae (genus Flavivirus) of positive-stranded RNA viruses transmitted by Aedes aegypti mosquitoes. Upon infection, the genomic RNA is translated by the host cell machinery into a 370-kDa polyprotein, which is subsequently cleaved and processed into 10 distinct structural (C, E, and prM) and nonstructural (NS1, 2A, 2B, 3, 4A, 4B, and 5) proteins. A majority of this processing (junctions of NS2A/2B, 2B/3, 3/4A, and 4B/5, as well as internal sites within C, 2A, 3, and 4A) is carried out by a virus-encoded two-component protease, NS2B-NS3. Additional processing, especially of sites toward the N terminus (junctions of C/prM, prM/E, E/NS1, NS1/NS2A, and NS4A/4B), employs cellular proteases, such as signalase. The NS2B-NS3-mediated cleavage forms an essential step in the viral replicative cycle, as evidenced by the lack of production of infectious virions in mutants carrying inactivating substitutions in the protease. Of the duo, the N terminus of NS3 encodes the enzymatic core, while a hydrophilic core within NS2B provides an essential cofactor function (9).Owing to its essential nature in viral replication and the promise and success of drugs targeting the proteases in the treatment of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections (4), several recent studies have concentrated on identifying inhibitors of the flaviviral protease (5, 10, 21, 26-28). An added advantage of targeting the protease is that it is highly conserved between the serotypes (63 to 74%). For such structure-based drug design efforts, the availability of three-dimensional (3D) structures is an essential prerequisite, and given the differing pathogenicities and immune complexity generated by the multiple serotypes, it is a prudent first step to determine the structures of all four DENV proteases. The crystal structures of the DEN2 and DEN4 proteases in complex with various lengths of NS2B are available (7, 15). In this paper, we present the structures of an optimal construct of DEN1pro in complex with the essential hydrophilic core of NS2B (NS2Bc), as well as its active-site mutant. These structures reveal a novel and unexpected serotype-specific structural element embedded in the DENV genome, with implications for the discovery of broad-spectrum drugs. We also report the identification of a 10-residue stretch within the NS3 sequence that allows the separation of the substrate-binding function of the enzyme from its catalytic efficiency.     

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apoferredoxin in an ATP-dependent reaction

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.     

Inhibition of calmodulin-dependent cyclic AMP phosphodiesterase by phenoxazines

Phenoxazine derivatives were examined for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase, which is based on the hydrolysis of cAMP to AMP by phosphodiesterase in the presence or absence of inhibitor, followed by quantitative analysis by HPLC method. Anticalmodulin activity of phenoxazines with respect to substitution at C-2 position follows the order: 2-trifluoromethyl>2-chloro>unsubstituted phenoxazines. The interaction of phenoxazines with calmodulin using fluorescence spectroscopy has been performed. Binding study showed that calmodulin has two types of binding sites for phenoxazines. One is high affinity binding site (Kd value 0.07-0.46 microM) and the other, a low affinity binding site (Kd value 0.7-34.5 microM). The change in secondary structure of calmodulin upon binding to phenoxazines was studied by circular dichroism (CD) method, which showed that the percentage of helicity decreased with an extensive change in tertiary structure of calmodulin. Kinetic analysis of the phenoxazine-calmodulin interaction showed that phenoxazines competitively inhibited the activation of phosphodiesterase without affecting Vmax. Thus, these studies showed a good correlation between the ability of phenoxazines to block the activation of phosphodiesterase and their ability to bind to the activator.     

Effects of poly-ether B on proteome and phosphoproteome expression in biofouling Balanus amphitrite cyprids

Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds.     

Notch receptor-ligand binding and activation: insights from molecular studies

The Notch receptor is part of a core signalling pathway which is highly conserved in all metazoan species. It is required for various cell fate decisions at multiple stages of development and in the adult organism, with dysregulation of the pathway associated with genetic and acquired diseases including cancer. Although cellular and in vivo studies have provided considerable insight into the downstream consequences of Notch signalling, relatively little is known about the molecular basis of the receptor/ligand interaction and initial stages of activation. Recent advances in structure determination of the extracellular regions of human Notch-1 and one of its ligands Jagged-1 have given new insights into docking events occurring at the cell surface which may facilitate the development of new highly specific therapies. We review the structural data available for receptor and ligands and identify the challenges ahead.     

Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling

The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.     

Determinants in the pathways followed by the carbons of acetone in their conversion to glucose

[2-14C]Acetone was infused into rats that were fed or fasted. Each was infused with either a trace quantity of acetone or a large quantity that resulted in a blood concentration of acetone of at least 4 mM. The distribution of 14C in the carbons of glucose from each rat was determined. Two of the rats were given acetone in their drinking water and one was diabetic. Whether a rat was chronically exposed to acetone, fed or fasted, normal or diabetic, if given the trace dose, over 80% of the 14C in the glucose it formed was in carbons 1, 2, 5, and 6 of the glucose. If a rat was given the large dose, about 50% was in carbons 3 and 4. Thus, the major determinant of the pathways followed by acetone when it is metabolized is its concentration and not the prior dietary state of the animal or its previous exposure to acetone. Incorporation into carbons 1, 2, 5, and 6 occurs in the conversion of the carbons of [2-14C]lactate into glucose, whereas incorporation into carbons 3 and 4 occurs in the conversion of the carbons of [1-14C]acetate into glucose. Therefore, at high acetone concentration, the pathway that has been proposed for acetone's metabolism via acetate predominates, and via acetate there can be no net synthesis of glucose from acetone. When rats were given cyanamide and then the large dose of acetone, 74% of the 14C in the glucose they formed was in carbons 3 and 4 of the glucoses. Thus, the relative contribution of the pathway to lactate, or its metabolic equivalent, that has been proposed appears to be lessened by the administration of an aldehyde dehydrogenase inhibitor.