Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp b ) and native wild-type (wt) empty capsids (MVMp e ), determined and refined to 3.25 and 3.75 Å resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 Å resolution in this study. A comparison of the MVMp b and MVMp e capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the "shoulder" of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.
Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.
Legumes and the polyphenolic compounds present in them have gained a lot of interest due to their beneficial health implications. Dietary polyphenolic compounds, especially flavonoids, exert antioxidant properties and are potent inhibitors of xanthine oxidase (XO) activity. XO is the main contributor of free radicals during exercise but it is also involved in pathogenesis of several diseases such as vascular disorders, cancer and gout. In order to discover new natural, dietary XO inhibitors, some polyphenolic fractions and pure compounds isolated from two legume plant extracts were tested for their effects on XO activity. The fractions isolated from both Vicia faba and Lotus edulis plant extracts were potent inhibitors of XO with IC50 values range from 40–135 µg/mL and 55–260 µg/mL, respectively. All the pure polyphenolic compounds inhibited XO and their Ki values ranged from 13–767 µM. Ten of the compounds followed the non competitive inhibitory model whereas one of them was a competitive inhibitor. These findings indicate that flavonoid isolates from legume plant extracts are novel, natural XO inhibitors. Their mode of action is under investigation in order to examine their potential in drug design for diseases related to overwhelming XO action.
Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to a-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of P-D-glucopyranosylamine, N-acetyl-P-D-glucopyranosylamine (I-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K, = 32 pM) and the a ( K , = 35 PM) forms of the enzyme with respect to glucose I-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-I-GlcNAc-IMP complex were grown in space group P432,2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1 %, for data between 8 and 2.3 A resolution. I-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of a-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-I-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-a-D-glucose complex structure. The structure of the I-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.
Sialic acid precursors are mediators of the sialic acid pathway. In this manuscript we present evidence that the application of sialic acid a precursor modulates gene expression and cell differentiation. The concept that sugars are involved in cellular transcription was first proposed by Jacob and Monod nearly 40 years ago studying the regulation of the lac-operon in prokaryotes. Surprisingly, these findings have never been transferred to eukaryotic systems. For our studies we have chosen PC12 cells. PC12-cells differentiate after application of NGF into a neuron-like phenotype. It is shown that treatment of PC12 cells with two different sialic acid precursors N-acetyl- or N-propanoylmannosamine, without application of NGF also induces neurite outgrowth. Moreover, the PC12 cells show the same morphology as the NGF-treated cells. Surprisingly, after application of both sialic acid precursors the phosphorylation and translocation of erk1/2 into the nucleus are activated, thus influencing the expression of genes involved in the differentiation of cells, such as the transcription factor c-Jun or TOAD-64/Ulip/CRMP (Turned ON After Division, 64 kd/ unc-33-like phosphoprotein/Collapsin Response Mediator Protein). These are the first experimental data showing that the sialic acid metabolism is closely associated with signal transduction and regulation of neuronal differentiation.
Sialic acids usually represent the terminal monosaccharide of glycoconjugates and are directly involved in many biological processes. The cellular concentration of their nucleotide-activated form is one pacemaker for the highly variable sialylation of glycoconjugates. Hence, the determination of CMP-sialic acid levels is an important factor to understand the complex glycosylation machinery of cells and to standardize the production of glycotherapeutics. We have established a highly sensitive strategy to quantify the concentration of nucleotide-activated sialic acid by a combination of reduction and fluorescent labeling using the fluorophore 1,2-diamino-4,5-methylenedioxybenzene (DMB). The labeling with DMB requires free keto as well as carboxyl groups of the sialic acid molecule. Reduction of the keto group prior to the labeling process precludes the labeling of nonactivated sialic acids. Since the keto group is protected against reduction by the CMP-substitution, labeling of nucleotide-activated sialic acids is still feasible after reduction. Subsequent combination of the DMB-high-performance liquid chromatography (HPLC) application with mass spectrometric approaches, such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and electrospray-ionization (ESI)-MS, allows the unambiguous identification of both natural and modified CMP-sialic acids and localization of potential substituents. Thus, the described strategy offers a sensitive detection, identification, and quantification of nucleotide-activated sialic acid derivatives in the femtomole range without the need for nucleotide-activated standards.
C5 halogen substituted glucopyranosyl nucleosides (1-(β-D-glucopyranosyl)-5-X-uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective K(i) values of 1.02, 3.27, and 1.94 μM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ-hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1-(β-D-glucopyranosyl)uracil (K(i) =12.39 μM), as revealed by X-ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM-PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell.
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