Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC₅₀ value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC₅₀ of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a K_D of 0.29 μm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.     

Assessment of the 2-D Gel-Based Proteomics Application of Clinically Archived Formalin-Fixed Paraffin Embedded Tissues

Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7–10 % of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE’s maps had pl 4–6, while the percentages of proteins with pl above 6 were 3–5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67–78 % of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies.     

2-Substituted fortimicins by ring opening of 2-deoxy-1,2-epimino-2-epi-fortimicin B and by nucleophilic displacements of 2-O-(methylsulfonyl)fortimicin derivatives

Opening of the aziridine ring of 2-deoxy-1,2-epimino-2-epi-fortimicin B (10) has been effected with both chloride and azide. The reactions are both stereo- and regiospecific and give 2-chloro-2-deoxyfortimicin B (2c) and 2-azido-2-deoxy-fortimicin B (2d). The nucleophilic displacements of the methanesulfonate groups of some 1-N-benzyloxycarbonyl-2-O-(methylsulfonyl)fortimicin derivatives with chloride, azide, and cyanide in N,N-dimethylformamide are dependent both on the nature of the nucleophile and the specific 1-N-benzyloxycarbonyl-2-methanesulfonate employed as the substrate. Striking differences in the stereochemistry of the azide displacements with different 2-methanesulfonates are believed to have a conformational basis. 2-Amino-2-deoxyfortimicin A (1c) and both of the 2-epimeric 2-chloro-2-deoxyfortimicins A (1b) and (5) were prepared for antibacterial assay and the in vitro results are reported.     

Spatio-temporal pattern of the Chironomidae community: toward the use of non-biting midges in bioassessment programs

We employed the self-organizing map (SOM) method to investigate the spatio-temporal pattern of the Chironomidae community in the Southern Morava River basin (Serbia) and to examine to what extent the Chironomidae community is affected by environmental factors. Additionally, this study explores the problems of utilizing chironomids in bioassessment programs. The SOM analysis of the chironomid community data produced 3 groups of sites. The indicator species analysis presented indicator taxa for two groups. Twenty taxa (at species, species group and genus level), according to the Kruskal–Wallis test, showed the most pronounced differences among the temporal units. Out of 15 measured environmental parameters, one-way ANOVA pointed out that 10 significantly differ between the groups. Elevation had the most important influence on the chironomid community, also affecting other environmental parameters. According to our findings, the winter season and the periods with high water level are the main sources of natural variability. To avoid such variability and to successfully incorporate Chironomidae in bioassessment programs, we suggest exclusion of the arguable months from monitoring programs.     

Preparations of 2-epi-fortimicins A from 2-epi-fortimicin B by intramolecular base-catalyzed 2-O-acylation of 1,2′,6′-tri-N-benzyloxycarbonyl-2-epi-fortimicin B

Preparations of 2-epi-fortimicin A (4) from 2-epi-fortimicin B (3) are described. In contrast to the previously reported, selective 4-N-acylation of 1,2′,6′-tri-N-benzyloxycarbonylfortimicin B (8) with N-(N-benzyloxycarbonylglycyloxy)succinimide, 1,2′,6′-tri-N-benzyloxycarbonyl-2-epi-fortimicin B (5) underwent predominant 2-O,4-N-diacylation under similar conditions. Proof of the structure of the diacylated product is presented, with evidence that the diacylated product is formed by initial intramolecular, base-catalyzed 2-O-acylation. The in vitro antibacterial activities of 2-epi-fortimicin A (4), 2-O-glycyl-2-epi-fortimicin A (11), 1-N-glycyl-2-epi-fortimicin A (12), and 5-deoxy-2-epi-fortimicin A (13) are reported.