Synthesis and biological activity of cyclolinopeptide A analogues modified with γ4-bis(homo-phenylalanine).

Cyclolinopeptide A (CLA), an immunosuppressive nonapeptide derived from linen seeds, was modified with S or R-γ4-bis(homo-phenylalanine) in positions 3 or 4, or both 3 and 4. These modifications changed the flexibility of new analogues and distribution of intramolecular hydrogen bonds. Analogues 11 c(Pro1-Pro2-Phe3-S-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9), 13 c(Pro1-Pro2-S-γ4-hhPhe3-R-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9) and 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) existed as a mixture of stable cis/trans isomers of Pro-Pro peptide bond. The comparison of the relative spatial orientations in crystal state of the two carbonyl groups, neighboring γ-amino acids, revealed conformational similarities to α-peptides. The addition of two -CH2- groups in γ-amino acids led to a more rigid conformation, although a more flexible one was expected. A significant difference in the relative orientation of the carbonyl groups was found for cyclic γ-peptides with a dominance of an antiparallel arrangement. As carbonyl groups may be engaged in the interactions with plausible receptors through hydrogen bonds, a similar biological activity of the modified peptides was expected. Our biological studies showed that certain cyclic, but not the corresponding linear peptides, lowered the viability of peripheral blood mononuclear cells (PBMC) at 100µg/mL concentration. The proliferation of PBMC induced by phytohemagglutinin A (PHA) was strongly inhibited by cyclic peptides only, in a dose-dependant manner. On the other hand, lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production in whole blood cell cultures was inhibited by both linear and cyclic peptides. Peptide 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) blocked the expression of caspase-3, inhibited the expression of caspases-8 and -9 in 24h culture of Jurkat cells, and caused DNA fragmentation in these cells, as an indicator of apoptosis. Thus, we revealed a new mechanism of immunosuppressive action of a nonapeptide.

Prebiotics and age, but not probiotics affect the transformation of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) by fecal microbiota - An in vitro study.

Heterocyclic aromatic amines (HAAs) are carcinogens which are formed in meat cooked using high-temperature methods. The human gastrointestinal (GI) microbiota plays a crucial role in maintaining health in humans of different ages, and especially in the elderly. However, the GI microbiota, whose metabolism and composition changes with age, may also be responsible for the activation of mutagenic substances reaching the colon with diet. Probiotics and prebiotics are promising in terms of reducing the destructive effects of HAAs. The aim of the study was to determine if fecal microbiota derived from the feces of 27 volunteers: infants (up to 18 months), adults (aged 23-39 years), the sub-elderly (aged 64-65 years), and the elderly (aged 76-87 years), and the presence of probiotics or prebiotics, affected the transformation of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) to 7-OH-IQ (2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one). The compounds were identified using LC-MS(n), NMR, and FTIR. Their genotoxicity was compared in the comet assay. Individual strains capable of IQ transformation were also identified. 7-OH-IQ was detected in six persons (two children and four elderly individuals). The degree of IQ conversion ranged from 26% (4-month-old girl) to 94% (81-year-old woman) of the initial quantity. Four Enterococcus isolates: two Enterococcus faecium and two Enterococcus faecalis strains, as well as one Clostridium difficile strain (LOCK 1030, from the culture collection) converted IQ to 7-OH-IQ. The genotoxicity of samples containing 7-OH-IQ was even three times higher (P < 0.05) than those with IQ and was correlated with the degree of IQ conversion and 7-OH-IQ concentration.

The structure of cyclolinopeptide A in chloroform refined by RDC measurements.

Three-dimensional structures of molecules traditionally assigned from nuclear Overhauser effects and vicinal coupling constants are recently complemented by measurements of residual dipolar couplings. Residual dipolar couplings measured in a stretched poly(dimethylsiloxane) gel were used to determine the structure of cyclolinopeptide A in chloroform solution at -50 °C. After structure refinement, conformational details of main cluster were discussed in relation to crystal and nuclear Overhauser effect derived structures.

Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and µ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73.

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer.  Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation.  Two p53 homologues, p63 and p73, have been described in humans.  Unlike p53, these proteins regulate developmental processes rather than genome stability.  Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx.  Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined.  Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.