"Crystal memory" Affects the Properties of Peptide Hydrogels - The Case of the Cyclic Tyr-Tyr dipeptide.

In this work a relationship between the crystal form and morphology and rheological properties of peptide-based hydrogels is examined. We show, that under favorable circumstances a correlation between a starting solid material and a self-assembly processes in solution can exist, leading to different properties of a resulting soft matter. This observation, together with an in-depth analysis of the influence of stereochemistry of self-assembled (ll) and (dl) Tyr-Tyr cyclic dipeptides (cYY) on the observed relationship between gelation and crystallization allowed us to gain a deeper understanding of the peptide hydrogelation processes at a molecular level, using liquid state NMR, rheological studies and scanning electron microscopy. In the course of our studies, several crystal forms of (ll)-cYY has been discovered and described in details using single crystal X-ray diffraction, as well as advanced solid state NMR, X-ray diffraction of powders, thermal analysis, FTIR, circular dichroism and crystal structure prediction (CSP) calculations. Subsequently, we found that while (ll)-cYY easily assembles into hydrogels with different properties depending on the starting solid form, (dl)-cYY always precipitated as one crystal form in the tested conditions. Molecular-level justification for this observation is given.

Improved HDAC Inhibition, Stronger Cytotoxic Effect and Higher Selectivity against Leukemias and Lymphomas of Novel, Tricyclic Vorinostat Analogues.

Histone deacetylase (HDAC) inhibitors are a class of drugs used in the cancer treatment. Here, we developed a library of 19 analogues of Vorinostat, an HDAC inhibitor used in lymphomas treatment. In Vorinostat, we replaced the hydrophobic phenyl group with various tricyclic 'caps' possessing a central, eight-membered, heterocyclic ring, and investigated the HDAC activity and cytotoxic effect on the cancer and normal cell lines. We found that 3 out of the 19 compounds, based on dibenzo[b,f]azocin-6(5H)-one, 11,12-dihydrodibenzo[b,f]azocin-6(5H)-one, and benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)-dione scaffolds, showed better HDACs inhibition than the referenced Vorinostat. In leukemic cell line MV4-11 and in the lymphoma cell line Daudi, three compounds showed lower IC50 values than Vorinostat. These compounds had higher activity and selectivity against MV4-11 and Daudi cell lines than reference Vorinostat. We also observed a strong correlation between HDACs inhibition and the cytotoxic effect. Cell lines derived from solid tumours: A549 (lung carcinoma) and MCF-7 (breast adenocarcinoma) as well as reference BALB/3T3 (normal murine fibroblasts) were less susceptible to compounds tested. Developed derivatives show improved properties than Vorinostat, thus they could be considered as possible agents for leukemia and lymphoma treatment.

Isolation and structural determination of flavan-3-ol derivatives from the Polypodium vulgare L. rhizomes water extract.

Polypodium vulgare L. (Polypodiaceae) is a fern used in traditional Polish medicine as an expectorant to treat cough and pertussis. Additionally, it was used as a diuretic in renal diseases, especially in chronic nephritis and pyelonephritis. In our study, a water extract was prepared from the rhizome of common polypody and subsequently fractionated on a resin column. As a result, the mixture of flavan-3-ol derivatives was obtained after the column elution with 60% methanol. Further purification by various chromatographic techniques led us to the isolation of (+)-afzelechin (1), a new previously not reported (+)-afzelechin-7-O-α-l-arabinofuranoside (2), and three other monomer flavan-3-ol glycosides: (+)-afzelechin-7-O-ß-d-apiofuranoside (3), (+)-catechin-7-O-α-l-arabinofuranoside (4) and (+)-catechin-7-O-ß-d-apiofuranoside (5). Structures of the compounds were established by HR-ESI-MS, 1D and 2D NMR spectroscopy. The HSQC and HMBC NMR techniques were used in the structure elucidation of the position of sugar attachment.

Unsymmetrically-Substituted 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione Scaffold-A Useful Tool for Bioactive Molecules Design.

Unsymmetrically N-substituted and N,N'-disubstituted 5,12-dihydrodibenzo [b,f][1,4]diazocine-6,11-diones were synthesized in the new protocol. The desired modifications of the dibenzodiazocine scaffold were introduced at the stages of proper selection of building blocks as well as post-cyclization modifications with alkylation or acylation agents, expanding the structural diversity and possible applications of synthesized molecules. The extension of developed method resulted in the synthesis of novel: tricyclic 5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione scaffold and fused pentacyclic framework possessing two benzodiazocine rings within its structure. Additionally, the unprecedented rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones to 2-(2-aminophenyl)isoindoline-1,3-diones was observed under the basic conditions in the presence of sodium hydride for secondary dilactams. The structures of nine synthesized products have been established by single-crystal X-ray diffraction analysis. Detailed crystallographic analysis of the investigated tri- and pentacyclic systems has shed more light on their structural features. One cell line derived from non-cancerous cells (EUFA30-human fibroblasts) and three tumor cells (U87-human primary glioblastoma, HeLa-cervix adenocarcinoma, BICR18-laryngeal squamous cell carcinoma) were used to determine the cytotoxic effect of the newly synthesized compounds. Although these compounds showed a relatively weak cytotoxic effect, the framework obtained for 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione could serve as a convenient privilege structure for the design and development of novel bioactive molecules suitable for drug design, development and optimization programs.

Structural studies on radiopharmaceutical DOTA-minigastrin analogue (CP04) complexes and their interaction with CCK2 receptor.

BACKGROUND: The cholecystokinin receptor subtype 2 (CCK-2R) is an important target for diagnostic imaging and targeted radionuclide therapy (TRNT) due to its overexpression in certain cancers (e.g., medullary thyroid carcinoma (MTC)), thus matching with a theranostic principle. Several peptide conjugates suitable for the TRNT of MTC have been synthesized, including a very promising minigastrin analogue DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04). In this contribution, we wanted to see whether CP04 binding affinity for CCK-2R is sensitive to the type of the complexed radiometal, as well as to get insights into the structure of CP04-CCK2R complex by molecular modeling. RESULTS: In vitro studies demonstrated that there is no significant difference in CCK-2R binding affinity and specific cellular uptake between the CP04 conjugates complexed with [68Ga]Ga3+ or [177Lu]Lu3+. In order to investigate the background of this observation, we proposed a binding model of CP04 with CCK-2R based on homology modeling and molecular docking. In this model, the C-terminal part of the molecule enters the cavity formed between the receptor helices, while the N-terminus (including DOTA and the metal) is located at the binding site outlet, exposed in large extent to the solvent. The radiometals do not influence the conformation of the molecule except for the direct neighborhood of the chelating moiety. CONCLUSIONS: The model seems to be in agreement with much of structure-activity relationship (SAR) studies reported for cholecystokinin and for CCK-2R-targeting radiopharmaceuticals. It also explains relative insensitivity of CCK-2R affinity for the change of the metal. The proposed model partially fits the reported site-directed mutagenesis data.

Trimeric and Tetrameric A-Type Procyanidins from Peanut Skins.

Peanut skins are a rich source of oligomeric and polymeric procyanidins. The oligomeric fractions are dominated by dimers, trimers, and tetramers. A multistep chromatographic fractionation led to the isolation of four new A-type procyanidins of tri- and tetrameric structures. The structures of the new trimers were defined by NMR, electronic circular dichroism, and MS data as epicatechin-(4ß→8,2ß→O→7)-epicatechin-(4ß→8,2ß→O→7)-catechin, peanut procyanidin B (3), and epicatechin-(4ß→8,2ß→O→7)-epicatechin-(4ß→6)-catechin, peanut procyanidin C (4). The new tetramers were defined as epicatechin-(4ß→8,2ß→O→7)-epicatechin-(4ß→6)-epicatechin-(4ß→8,2ß→O→7)-catechin, peanut procyanidin E (1), and epicatechin-(4ß→8,2ß→O→7)-epicatechin-(4ß→6)-epicatechin-(4ß→8,2ß→O→7)-epicatechin, peanut procyanidin F (2). In addition, both A-type dimers A1, epicatechin-(4ß→8,2ß→O→7)-catechin, and A2, epicatechin-(4ß→8,2ß→O→7)-epicatechin, as well as two known peanut trimers, ent-epicatechin-(4ß→6)-epicatechin-(4ß→8,2ß→O→7)-catechin, peanut procyanidin A (5), and epicatechin-(4ß→8)-epicatechin-(4ß→8,2ß→O→7)-catechin, peanut procyanidin D (6), were also isolated. Dimer A1, the four trimers, and two tetramers were evaluated for anti-inflammatory activity in an in vitro assay, in which LPS-stimulated macrophages were responding with secretion of TNF-α, a pro-inflammatory cytokine. Tetramer F (2) was the most potent, suppressing TNF-α secretion to 82% at 8.7 µM (10 µg/mL), while tetramer E (1) at the same concentrations caused a 4% suppression. The results of the TNF-α secretion inhibition indicate that small structural differences, as in peanut procyanidin tetramers E and F, can be strongly differentiated in biological systems.

Effects of Geum urbanum L. root extracts and its constituents on polymorphonuclear leucocytes functions. Significance in periodontal diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Geum urbanum L. (wood avens) root infusions and decoctions have been used externally for reducing the bleeding and inflammation of gums (gingivitis), and mucous membranes. AIM OF THE STUDY: Taking into account that primed and hyperactivated neutrophils are an important factor in the transition from gingivitis to periodontitis, we investigated the effects of phytochemically characterised (HPLC-DAD-MS(n)) extracts of different polarity from Geum urbanum root on oxidative burst, elastase, metalloproteinase 9 (MMP-9), interleukin 8 (IL-8) and 1ß (IL-1ß), tumour necrosis factor (TNF-α) release, expression of adhesion molecules (CD62L and CD11b) and delayed apoptosis in stimulated neutrophils. As gemin A is a dominating compound in a raw material, so we considered its activity in parallel with the positive control quercetin. MATERIALS AND METHODS: The extracts were characterised by HPLC-DAD- MS(n) method. The inhibition of ROS production by stimulated neutrophils was determined using luminol dependent chemiluminescence method. The effect on MMP-9, IL-1ß, TNF-α and IL-8 production by neutrophils was measured by enzyme-linked immunosorbent assay (ELISA). Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules and the apoptosis of neutrophils was analyzed with flow cytometry. RESULTS: The main compounds detected in the extract belong mainly to the group of ellagitannin: pedunculagin, stachyurin, casuarynin and gemin A, and ellagic acid derivatives. Procyanidins and one complex tannin were found as minor compounds. Gemin A significantly affected the functions of stimulated neutrophils by reducing the surface expression of CD11b, and inhibiting the release of reactive oxygen species, and proteases (elastase, MMP-9), chemokines and cytokines (interleukins IL-8, IL-1ß). Interestingly, gemin A stimulated the release of TNF-α, which may be one of the stimulators of apoptosis of neutrophil cells. The primary aqueous extract, the ethyl acetate and the butanolic fractions, all containing the highest level of gemin A, have exerted similar but weaker activity. CONCLUSION: The modulating effect on the neutrophils function of extracts, and its main constituent gemin A, support the traditional use of this plant material in cavity inflammation including mucositis, gingivitis and periodontosis.