Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with monoaryl P4 motifs

Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-one-based factor Xa inhibitors, incorporating neutral and basic monoaryl P4 groups, ultimately producing potent inhibitors with effective levels of anticoagulant activity and extended oral pharmacokinetic profiles. However, time dependant inhibition of Cytochrome P450 3A4 was a particular issue with this series.     

ADP competes with FAD binding in putrescine oxidase

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.     

Mass spectrometry of ribosomes from Saccharomyces cerevisiae: implications for assembly of the stalk complex

The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes. In yeast this structure is composed of two heterodimers (P1alpha-P2beta and P1beta-P2alpha) attached to the ribosome via P0. Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes. We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae. The resulting mass spectra reveal a noncovalent complex of mass 77,291 +/- 7 Da assigned to the pentameric stalk. Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0. No other oligomers are observed, confirming the specificity of the pentameric complex. At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks. To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1alpha or P2beta are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2alpha molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1alpha or P2beta destabilizes binding of the partner protein on the ribosome. This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.     

Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P <  0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T₀ plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.     

High Prevalence of Shared International Type 53 among Mycobacterium tuberculosis Complex Strains in Retreated Patients from C?te d’Ivoire

Background

Genotyping methods are useful tools to provide information on tuberculosis epidemic. They can allow a better response from health authorities and the implementation of measures for tuberculosis control. This study aimed to identify the main lineages and clades of Mycobacterium tuberculosis complex strains circulating in Côte d’Ivoire.

Methods/Main Findings

Strains isolated from sputum samples of patients ongoing retreatment from all the country were characterized by spoligotyping and by MIRU-VNTR. Profiles obtained by spoligotyping were first compared to the SITVIT/SpolDB4 database for family assignment. Of 194 strains analysed, 146 (75.3%) belonged to the T lineage. The most predominant spoligotype was the shared international type 53 with 135 strains (69.6%). In contrast with neighbouring countries, LAM (11 strains, 5.7%) and H (9 strains 4.6%) lineages were slightly represented. Only 3 Beijing strains (1.5%) and 4 strains of Mycobacterium africanum (2%) were found. Analysis of the results obtained with MIRU-VNTR revealed also a high level of clustering.

Conclusion/Significance

The population of Mycobacterium tuberculosis complex strains among retreatment cases in Côte d’Ivoire exhibits a low diversity, allowing to assume recent transmission and locally based infection.