Aza-bicyclic amino acid sulfonamides as alpha(4)beta(1)/alpha(4)beta(7) integrin antagonists

The design, synthesis, and biological activity of novel alpha(4)beta(1) and alpha(4)beta(7) integrin antagonists, containing a bridged azabicyclic nucleus, are reported. Conformational analysis of targets containing an azabicyclo[2.2.2]octane carboxylic acid and known integrin antagonists indicated that this azabicycle would be a suitable molecular scaffold. Variation of substituents on the pendant arylsulfonamide and phenylalanine groups resulted in potent alpha(4)beta(1)-selective and dual alpha(4)beta(1)/alpha(4)beta(7) antagonists. Potent compounds 11i, 11h, and 14 were effective in the antigen-sensitized sheep model of asthma.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

Crystal structure of the YffB protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> suggests a glutathione-dependent thiol reductase function

Background  

The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein.     

Combinatorial synthesis of substituted 3-(2-indolyl)piperidines and 2-phenyl indoles as inhibitors of ZipA-FtsZ interaction

The ZipA-FtsZ protein-protein interaction is a potential target for antibacterial therapy. The design and parallel synthesis of a combinatorial library of small molecules, which target the FtsZ binding area on ZipA are described. Compounds were demonstrated to bind to the FtsZ binding domain of ZipA by HSQC NMR and to inhibit cell division in a cell elongation assay.     

Proton affinity changes driving unidirectional proton transport in the bacteriorhodopsin photocycle

Bacteriorhodopsin is the smallest autonomous light-driven proton pump. Proposals as to how it achieves the directionality of its trans-membrane proton transport fall into two categories: accessibility-switch models in which proton transfer pathways in different parts of the molecule are opened and closed during the photocycle, and affinity-switch models, which focus on changes in proton affinity of groups along the transport chain during the photocycle. Using newly available structural data, and adapting current methods of protein protonation-state prediction to the non-equilibrium case, we have calculated the relative free energies of protonation microstates of groups on the transport chain during key conformational states of the photocycle. Proton flow is modeled using accessibility limitations that do not change during the photocycle. The results show that changes in affinity (microstate energy) calculable from the structural models are sufficient to drive unidirectional proton transport without invoking an accessibility switch. Modeling studies for the N state relative to late M suggest that small structural re-arrangements in the cytoplasmic side may be enough to produce the crucial affinity change of Asp96 during N that allows it to participate in the reprotonation of the Schiff base from the cytoplasmic side. Methodologically, the work represents a conceptual advance compared to the usual calculations of pK(a) using macroscopic electrostatic models. We operate with collective states of protonation involving all key groups, rather than the individual-group pK(a) values traditionally used. When combined with state-to-state transition rules based on accessibility considerations, a model for non-equilibrium proton flow is obtained. Such methods should also be applicable to other active proton-transport systems.     

Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes

Using comparative analysis of genes, operons, and regulatory elements, we describe the cobalamin (vitamin B12) biosynthetic pathway in available prokaryotic genomes. Here we found a highly conserved RNA secondary structure, the regulatory B12 element, which is widely distributed in the upstream regions of cobalamin biosynthetic/transport genes in eubacteria. In addition, the binding signal (CBL-box) for a hypothetical B12 regulator was identified in some archaea. A search for B12 elements and CBL-boxes and positional analysis identified a large number of new candidate B12-regulated genes in various prokaryotes. Among newly assigned functions associated with the cobalamin biosynthesis, there are several new types of cobalt transporters, ChlI and ChlD subunits of the CobN-dependent cobaltochelatase complex, cobalt reductase BluB, adenosyltransferase PduO, several new proteins linked to the lower ligand assembly pathway, l-threonine kinase PduX, and a large number of other hypothetical proteins. Most missing genes detected within the cobalamin biosynthetic pathways of various bacteria were identified as nonorthologous substitutes. The variable parts of the cobalamin metabolism appear to be the cobalt transport and insertion, the CobG/CbiG- and CobF/CbiD-catalyzed reactions, and the lower ligand synthesis pathway. The most interesting result of analysis of B12 elements is that B12-independent isozymes of the methionine synthase and ribonucleotide reductase are regulated by B12 elements in bacteria that have both B12-dependent and B12-independent isozymes. Moreover, B12 regulons of various bacteria are thought to include enzymes from known B12-dependent or alternative pathways.     

Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase: identification of a new activity on the tautomerase superfamily

Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro1-(X)8 -Gly-Arg11-X-Asp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro1 and Arg75 as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a beta-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro1. These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro1 and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechanistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro1 and Arg75 play prominent roles.     

Canonical inhibitor-like interactions explain reactivity of alpha1-proteinase inhibitor Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, alpha1-proteinase inhibitor (alpha1PI) Pittsburgh (P1 Met --> Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound alpha1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2'. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with alpha1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for alpha1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.     

Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion

Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the initial vertex ring, independent of t-SNAREs or actin; (b) F-actin disassembly and GTP-bound Ypt7p direct the localization of other fusion factors; (c) The t-SNAREs Vam3p and Vam7p regulate each other's vertex enrichment, but do not affect Ypt7p localization. The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked. Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS). In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy. Our findings provide a unique view of the functional relationships between GTPases, SNAREs, and actin in membrane fusion.