Synthesis and biological evaluation of novel irreversible serine protease inhibitors using amino acid based sulfonyl fluorides as an electrophilic trap

We have designed and synthesized novel irreversible serine protease inhibitors containing aliphatic sulfonyl fluorides as an electrophilic trap. These substituted taurine sulfonyl fluorides derived from taurine or protected amino acids were conveniently synthesized from β-aminoethanesulfonyl chlorides using KF/18-crown-6 or from β-aminoethanesulfonates using DAST. Their potency of irreversible inhibition of serine proteases is described in different enzyme assays using chymotrypsin leading to binding affinities up to 22 μM.     

Synthesis and biological activity of polygalloyl-dendrimers as stable tannic acid mimics

Inspired by the structure of tannic acid, first- to third-generation dendrimers containing two, four, and eight galloyl moieties were synthesized. Stability, antioxidant activity and collagen cross-linking activity of the natural product and its dendrimer analogues were compared. The experimental results indicate that polygalloyl dendrimers might be used as new lead compounds to improve the long-term healing characteristics of burn wounds.     

Role of solution conformation and flexibility of short peptide ligands that bind to the p56(lck) SH2 domain

A general approach in drug design is making ligands more rigid in order to avoid loss in conformational entropy (deltaS(conf)) upon receptor binding. We hypothesized that in the high affinity binding of pYEEI peptide ligands to the p56(lck) SH2 domain this loss in deltaS(conf) might be diminished due to preorganization of the fourfold negatively charged pYEEI peptide in the bound, extended, conformation. A thermodynamic analysis was performed on the peptides Ac-pYEEI-NH(2), Ac-pYAAI-NH(2) and Ac-pYGGI-NH(2) using surface plasmon resonance (SPR) competition experiments to assay affinity constants at different temperatures. To study the effect of solution conformation and flexibility a computational conformation analysis was performed from which low energy conformations in solution were calculated, and S(conf) estimated. It was found that the calculated low energy conformations for especially the pYE moiety in solution resemble that in the bound state. In the calculated minimum energy conformation in solution isoleucine is bent towards the pY aromatic ring, the occurrence of such conformation is experimentally confirmed by NMR. The estimated values for S(conf) of the EE- and AA-peptide were similar, suggesting no predominant role of preorganization of the solution conformation due to electrostatic repulsion. Apparently the thermodynamics obey the same entropy-enthalpy compensation relationship, which also was found to hold for other peptides and peptidomimetics binding to p60(src) family SH2 domains. The implications of the results for drug design are discussed.     

Backbone-modified amylin derivatives: implications for amyloid inhibitor design and as template for self-assembling bionanomaterials.

This report reviews our approach to the design, synthesis and structural/morphological analysis of backbone-modified amylin(20-29) derivatives. Depending on the position in the peptide backbone and the type of amide bond isostere/modification, the amylin(20-29) peptides behave either as inhibitors of amyloid fibril formation, which are able to retard amyloid formation of native amylin(20-29), or as templates for the formation of self-assembled supramolecular structures. Molecular fine-tuning of the hydrogen-bond accepting/donating properties allows the control over the morphology of the supramolecular aggregation motifs such as helical ribbons and tapes, ribbons progressing to closed peptide nanotubes, (twisted) lamellar sheets or amyloid fibrils.     

beta-Sheet structured beta-amyloid(1-40) perturbs phosphatidylcholine model membranes

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Abeta40 assemblies.     

Large scale production of phage antibody libraries using a bioreactor

One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.     

The Yo-Yo intermittent recovery test level 1 is reliable in young high-level soccer players

The aim of the study was to investigate test reliability of the Yo-Yo intermittent recovery test level 1 (YYIR1) in 36 high-level youth soccer players, aged between 13 and 18 years. Players were divided into three age groups (U15, U17 and U19) and completed three YYIR1 in three consecutive weeks. Pairwise comparisons were used to investigate test reliability (for distances and heart rate responses) using technical error (TE), coefficient of variation (CV), intra-class correlation (ICC) and limits of agreement (LOA) with Bland-Altman plots. The mean YYIR1 distances for the U15, U17 and U19 groups were 2024 ± 470 m, 2404 ± 347 m and 2547 ± 337 m, respectively. The results revealed that the TEs varied between 74 and 172 m, CVs between 3.0 and 7.5%, and ICCs between 0.87 and 0.95 across all age groups for the YYIR1 distance. For heart rate responses, the TEs varied between 1 and 6 bpm, CVs between 0.7 and 4.8%, and ICCs between 0.73 and 0.97. The small ratio LOA revealed that any two YYIR1 performances in one week will not differ by more than 9 to 28% due to measurement error. In summary, the YYIR1 performance and the physiological responses have proven to be highly reliable in a sample of Belgian high-level youth soccer players, aged between 13 and 18 years. The demonstrated high level of intermittent endurance capacity in all age groups may be used for comparison of other prospective young soccer players.     

Current versus historical population sizes in vertebrate species with high gene flow: a comparison based on mitochondrial DNA lineages and inbreeding theory for neutral mutations

Using inbreeding theory as applied to neutral alleles inherited maternally, we generate expected probability distributions of times to identity by descent for random pairs of mitochondrial genotypes within a population or within an entire species characterized by high gene flow. For comparisons with these expectations, empirical distributions of times to most recent common ancestry were calculated (by conventional mtDNA clock calibrations) from mtDNA haplotype distances observed within each of three vertebrate species--American eels, hardhead catfish, and redwinged blackbirds. These species were chosen for analysis because census population size in each is currently large and because both genetic and life-history data are consistent with the postulate that historical gene flow within these species has been high. The observed molecular distances among mtDNA lineages were two to three orders of magnitude lower than predicted from census sizes of breeding females, suggesting that rate of mtDNA evolution is decelerated in these species and/or that long-term effective population size is vastly smaller than present-day population size. Several considerations point to the latter possibility as most likely. The genetic structure of any species is greatly influenced by historical demography; even for species that are currently abundant, mtDNA gene lineages appear to have been channeled through fairly small numbers of ancestors.      

Developmental and wound-, cold-, desiccation-, ultraviolet-B-stress-induced modulations in the expression of the petunia zinc finger transcription factor gene ZPT2-2

The ZPT2-2 gene belongs to the EPF gene family in petunia (Petunia hybrida), which encodes proteins with TFIIIA-type zinc-finger DNA-binding motifs. To elucidate a possible function for ZPT2-2, we analyzed its pattern of expression in relation to different developmental and physiological stress signals. The activity of the ZPT2-2 promoter was analyzed using a firefly luciferase (LUC) reporter gene, allowing for continuous measurements of transgene activity in planta. We show that ZPT2-2::LUC is active in all plant tissues, but is strongly modulated in cotyledons upon germination, in leaves in response to desiccation, cold treatment, wounding, or ultraviolet-B light, and in petal tissue in response to pollination of the stigma. Analysis of mRNA levels indicated that the modulations in ZPT2-2::LUC expression reflect modulations in endogenous ZPT2-2 gene expression. The change in ZPT2-2::LUC activity by cold treatment, wounding, desiccation, and ultraviolet-B light suggest that the phytohormones ethylene and jasmonic acid are involved in regulating the expression of ZPT2-2. Although up-regulation of expression of ZPT2-2 can be blocked by inhibitors of ethylene perception, expression in plants is not induced by exogenously applied ethylene. The application of jasmonic acid does result in an up-regulation of gene activity and, thus, ZPT2-2 may play a role in the realization of the jasmonic acid hormonal responses in petunia.     

Synthesis and characterization of biodegradable peptide-based polymers prepared by microwave-assisted click chemistry

In this study, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction was used to synthesize peptide triazole-based polymers from two novel peptide-based monomers: azido-phenylalanyl-alanyl-lysyl-propargyl amide (1) and azido-phenylalanyl-alanyl-glycolyl-lysyl-propargyl amide (2). The selected monomers have sites for enzymatic degradation as well as for chemical hydrolysis to render the resulting polymer biodegradable. Depending on the monomer concentration in DMF, the molecular mass of the polymers could be tailored between 4.5 and 13.9 kDa (corresponding with 33-100 amino acid residues per polymer chain). As anticipated, both polymers can be enzymatically degraded by trypsin and chymotrypsin, whereas the ester bond in the polymer of 2 undergoes chemical hydrolysis under physiological conditions, as was shown by a ninhydrin-based colorimetric assay and MALDI-TOF analysis. In conclusion, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction is an effective tool for synthesizing biodegradable peptide polymers, and it opens up new approaches toward the synthesis of (novel) designed biomedical materials.