Beata Wilenska

Enantioseparation of β2-amino acids on cinchona alkaloid-based zwitterionic chiral stationary phases. Structural and temperature effects

The enantiomers of sixteen unusual β(2)-amino acids were directly separated on chiral stationary phases containing quinine- or quinidine-based zwitterionic selectors. The effects of the mobile phase composition, the structure of the analyte and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range -5 to 55°C in order to study the effects of temperature, and thermodynamic parameters were estimated from plots of lnk or lnα vs. 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantiomeric separations were in most cases enthalpically driven, but entropically driven separation was also observed. The sequence of elution of the enantiomers was determined in some cases.

Design, synthesis and in vitro biological evaluation of a small cyclic peptide as inhibitor of vascular endothelial growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR (vascular endothelial growth factor receptor), but it is also suggested that NRP-1 in tumour cells may serve as a separate receptor for VEGF165. Therefore molecules interfering with VEGF165 binding to NRP-1 seem to be promising candidates as new anti-angiogenic and anti-tumour drugs. Here, we report the design, synthesis, biological evaluation and molecular modelling of the small cyclic peptide, which shows a good inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18μM). The reported compound could be considered as one of the smallest cyclic peptides (MW=510) interfering with VEGF165/NRP-1 binding presented up to now.

Structure-activity relationship study of a small cyclic peptide H-c[Lys-Pro-Glu]-Arg-OH: a potent inhibitor of Vascular Endothelial Growth Factor interaction with Neuropilin-1

Inhibition of angiogenesis is one of the most promising approaches in anticancer therapy. It was recently suggested that Neuropilin-1 (NRP-1) in tumour cells may serve as a separate receptor for Vascular Endothelial Growth Factor-165 (VEGF165) which is one of the main pro-angiogenic agents in the organism. Therefore molecules inhibiting VEGF165 binding to NRP-1 could be potential candidates for new antiangiogenic and anticancer drugs. Here we present a structure-activity relationship study of the peptide H-c[Lys-Pro-Glu]-Arg-OH which showed high inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18μM) in our previous study. We report the design, synthesis, in vitro assays and docking analysis of four small cyclic peptides (14-,15-membered ring) and one bigger cyclic compound (30-membered ring). Our study shows that both the ring size and configuration of amino acid residues present in the structure are crucial for high inhibitory effect.

Conformational latitude – activity relationship of KPPR tetrapeptide analogues toward their ability to inhibit binding of vascular endothelial growth factor 165 to neuropilin-1

Neuropilin‐1 (NRP‐1) has been found to be overexpressed in several kinds of malignant tumors, and it is postulated that in pathological angiogenesis, its interaction with the vascular endothelial growth factor 165 (VEGF165) leads to progression of tumor vascularization and growth. Herein, we present synthesis and in vitro evaluation of tetrapeptides with the general sequence KxxR, where xx represents two canonical amino acid residues or a single amino acid possessing hydrocarbon backbone. All synthesized compounds were found to be inhibitors of VEGF165/NRP‐1 interactions. The rationale behind their design was to elucidate the relationship between the xx flexibility and their inhibitory activity. Detailed molecular dynamics simulations for all synthesized compounds have been performed. A clear and quantitative relationship was found in the peptide analogues: the more flexible is the xx, the less active the analogue is. But surprisingly, highly flexible peptidomimetics with non‐natural amino acids (NH2‐(CH2)n‐COOH; n = 4,5) residues used possessed higher inhibitory activity than it was expected. Our molecular dynamics study of inhibitor/NRP‐1 complexes provides convincing explanations of divergence in the observed inhibitory activity; thus, it might give indication for design of the next generation NRP‐1 inhibitors. Copyright

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1

HIGHLIGHTS13 tetrapeptides were synthesized, tested for inhibitory activity on VEGF165/NRP‐1 binding and modeled by molecular dynamics.Arg or Har in 1st position allow optimal interactions with NRP‐1 (length and delocalized charge of guanidinium group).Rigidity of central part of molecule should be partially preserved, changes in position 2 more favorable than in position 3.Har‐Dab‐Pro‐Arg, exhibits a submicromolar IC50, is almost 10 times stronger than a reference inhibitor A7R.the results give an important insight into structural requirements for high inhibitory activity on VEGF165/NRP‐1 interaction. ABSTRACT Neuropilin‐1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP‐1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP‐1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence. Here, we present a structure–activity relationship study of the systematic optimization of amino acid residues in positions 1–3 in the above tetrapeptides. All the 13 synthesized analogs possessed C‐terminal arginine that is a necessary element for interaction with NRP‐1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin‐1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP‐1.

Imaging and identification of endogenous peptides from rat pituitary embedded in egg yolk

RATIONALE Mass spectrometry imaging (MSI) can provide accurate data containing the spatial distribution of endogenous peptides in tissue sections without previous treatment. One of the key issues in analyzing small samples is establishing a proper technique for mounting and manipulating collected tissue in order to avoid contamination of the sample with optimal cutting temperature (OCT) resin. METHODS We present a method for embedding rat pituitary tissue in a frozen egg yolk block, which enables its further imaging in experiments on a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer with time-of-flight (TOF) analyzer. Embedding the sample in the egg yolk prevents contamination from the OCT resin, which decreases MALDI signal quality. RESULTS In the present study we detected numerous m/z peaks related to endogenous peptides. We identified fifteen peptides and their post-translational modifications by tandem mass spectrometry (MS/MS) directly on tissue sections of the hypophysis posterior and intermediate lobes; among these peptides were vasopressin, oxytocin, copeptin, melanocyte-stimulating hormones and beta-endorphin. We also showed that egg yolk itself does not affect localization of peptides in the pituitary. CONCLUSIONS Egg yolk embedding enables preparation of tissue sections from small tissue fragments to organs such as the pituitary gland, which is suitable for localization and identification of endogenous peptides by the MALDI-MSI and MALDI-MS/MS techniques.

Synthesis of rigid tryptophan mimetics by the diastereoselective Pictet–Spengler reaction of β3-homo-tryptophan derivatives with chiral α-amino aldehydes

The Pictet–Spengler (PS) cyclizations of β3‐hTrp derivatives as arylethylamine substrates were performed with L‐α‐amino and D‐α‐amino aldehydes as carbonyl components. During the PS reaction, a new stereogenic center was created, and the mixture of cis/trans 1,3‐disubstituted 1,2,3,4‐tetrahydro‐β‐carbolines was obtained. The ratio of cis/trans diastereomers depends on the stereogenic centre of used amino aldehyde and the size of substituents. It was confirmed by 1H and 2D NMR (ROESY) spectra. The conformations of cyclic products were studied by 2D NMR ROESY spectra. Products of the PS condensation after removal of protecting group(s) can be incorporated into a peptide chain as tryptophan mimetics with the possibility of the β‐turn induction. Copyright

Enkephalin degradation in serum of patients with inflammatory bowel diseases

BACKGROUND Inflammatory bowel diseases (IBD) are a group of chronic and recurrent gastrointestinal disorders that are difficult to control. Recently, a new IBD therapy based on the targeting of the endogenous opioid system has been proposed. Consequently, due to the fact that endogenous enkephalins have an anti-inflammatory effect, we aimed at investigating the degradation of serum enkephalin (Met- and Leu-enkephalin) in patients with IBD. METHODS Enkephalin degradation in serum of patients with IBD was characterized using mass spectrometry methods. Calculated half-life (T1/2) of enkephalins were compared and correlated with the disease type and gender of the patients. Additionally, statistical analysis was used to examine the dynamics of changes in terms of inhibition of enkephalins degradation within research groups. RESULTS Our research indicates that the degree of enkephalins degradation depends on the gender of the patients. The difference is most evident for the rate of Met-enkephalin degradation between men (mean T1/2 = 13.61 min) and women (mean T1/2  = 21.84 min) with Crohns disease (CD). CONCLUSIONS The most significant alternation of enkephalins degradation in serum samples of IBD patients, compared to control group, were observed in both Crohns disease and ulcerative colitis (UC) female patients. We suggest that the differences observed between the genders in IBD patients may be explained by regulation of enkephalinases activity by estradiol.