Synthesis and pharmacological characterization of dibenzodiazepinone-type muscarinic M2-receptor antagonists conjugated to fluorescent dyes or small peptides.

In humans, the family of muscarinic acetylcholine receptors (mAChR, MRs) comprises five subtypes (M1R-M5R), belonging to class A of the GPCR superfamily. MRs mediate the action of the neurotransmitter acetylcholine (ACh) in the central and peripheral nervous system. Whereas the M1R, M3R and M5R preferably couple to Gq-type G-proteins, the M2 and M4 receptor mainly activate Gi/o-type G-proteins. Due to the high conservation of the orthosteric (acetylcholine) binding site within the family of MRs, the development of highly subtype selective MR ligands is difficult. Selective MR ligands are needed as molecular or pharmacological tools and also as new drug candidates anticipated to cause less adverse effects. As the vestibule of MRs, also referred to as the common allosteric site, is less conserved than the orthosteric pocket, the dualsteric ligand approach, i.e. the design of ligands, which simultaneously address the orthosteric and an allosteric binding site, was suggested as a promising strategy to develop MR ligands with high affinity and improved subtype selectivity. This work was aiming at the synthesis and pharmacological characterization of dibenzodiazepinone-type MR ligands with pronounced M2R selectivity. The tricyclic MR ligands were prepared by linking the pharmacophore DIBA via various linker moieties to several short peptides, yielding a series of DIBA-peptide conjugates (70 compounds). The linker structure was varied with respect to length, rigidity/flexibility and number of basic groups. All peptide moieties contained at least one basic amino acid. In addition to proteinogenic amino acids, also unnatural amino acids were incorporated. MR affinities and selectivity profiles of the DIBA-peptide conjugates were determined by radioligand competition binding at CHO-hMxR cells (x = 1-5) using [3H]N-methyl scopolamine as labeled ligand. M1, M2 and M4 receptor affinities were determined for all target compounds. Additionally, M3 and M5 receptor affinities were determined for selected compounds, showing either high M2R selectivity over the M1 and M4 receptor, or a very low or no preference for the M2R. The study revealed that, besides the peptide structure, the type of the linker considerably determines M2R affinity and selectivity. DIBA-peptide conjugates derived from amine precursor 35 (compounds 101-106), containing an N-acylated 4-aminopiperidine and a bisalkylated piperazine ring in the linker moiety, exhibited pronounced M2R selectivity (e.g. 105: Ki M1R/M2R/M3R/M4R/M5R = >140:1:>160:59:>140), but only moderate M2R affinity (pKi 7.05-7.67). The subset of compounds derived from amine precursor 40 (107-109), representing the higher homologue of precursor 35 (extension of the linker by one methylene group), exhibited higher M2R affinities (pKi 8.93-9.08) and displayed also high M2R selectivity (e.g. 109: Ki M1R/M2R/M3R/M4R/M5R = 70:1:11,000:48:6000). Considering M2R over M3 and M5 receptor selectivity, compounds such as DIBA-peptide conjugates 107- 109 represent the most selective M2R antagonists reported to date. Regarding M2R over M1 and M4 receptor selectivity, higher M2R selectivity compared to 103-105 or 107-109 had only been reported for one other compound class. For three selected DIBA-peptide conjugates, M2R antagonism was confirmed in a M2R miniGi recruitment assay yielding Kb values, which were in excellent agreement with the respective Ki values from radioligand competition binding studies. The investigation of three selected DIBA-peptide conjugates (3, 4, 146) with respect to stability in human plasma showed highly stability against proteolytic degradation (> 99% intact compound after 24 h at 37 °C). Moreover, six fluorescently labeled dibenzodiazepinone-type MR ligands (164-169) were prepared using various fluorescent dyes (5-TAMRA, BODIPY, pyridinium dye Py-1, and pyridinium dye Py-5). All fluorescent probes exhibited high M2R affinity (pKi (radioligand competition binding): 8.75‐9.62), a low preference for the M2R over the M1 and M4 receptor and moderate to pronounced M2R selectivity compared to the M3R and M5R. The fluorescent ligands were successfully used as probes in flow cytometric M2R saturation binding assays resulting in pKd values of 8.36-9.19. Therefore, they are considered useful molecular tools for future studies using methods such as fluorescence anisotropy and BRET based MR binding assays. In conclusion, this thesis afforded new fluorescently labeled molecular tools for the M2R and new highly selective M2R antagonists, which might serve as lead structures for the development of drug-like selective M2R antagonists, representing potential therapeutics for the treatment of diseases associated with cholinergic dysfunction such as Alzheimer’s disease

Structural characterization of a novel glycosyl-phosphatidylinositol from the protozoan Tetrahymena mimbres.

A glycolipid metabolically labelled with [14C]GlcN was isolated from the free-living protozoan Tetrahymena mimbres. The glycolipid was sensitive to a bacterial phosphatidylinositol-specific phospholipase C, and the headgroup was shown to contain a phosphorylated Man alpha 1-2Man alpha 1-4Man alpha 1-4GlcN glycan. The Tetrahymena glycolipid is structurally unique among the glycosylphosphatidylinositols that have so far been characterized, including those from several protozoan parasites of humans

Effect of polymer brush architecture on antibiofouling properties

Contains fulltext : 91751.pdf (publisher's version ) (Open Access)4 p

From Particles in Steady State Shear Bands via Micro-Macro to Macroscopic Rheology Laws

Particulate systems and granular matter are discrete systems made of many particles; they display interesting dynamic or static, fluid- or solid-like states, respectively – or both together. The challenge of bridging the gap between the particulate, microscopic picture towards their continuum description (via the so-called micro-macro transition) is one of today’s challenges of modern research. This short paper gives a brief overview of recent progress and some new insights about local granular flow rules for soft particles

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic-AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic-AMP pulses and another control element by two runs of mutagenesis. A `double bypass' mutant, HG592, was obtained which aggregated in nutrient medium where wild-type did not develop. Mutants defective in expression of the csA-glycoprotein were selected from HG592 by fluorescence-activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA-negative mutants, HG693, specifically lacked the capability of forming EDTA-stable intercellular contacts. It acquired chemotactic responsiveness and developed into fruiting bodies. Expression of the transcripts for eight developmentally regulated proteins was determined in HG693. Seven of the RNA species were normally expressed; they were recognized by cDNA clones which had been produced from poly(A)(+) RNA isolated from membrane-bound polysomes. The single RNA species which was not substantially expressed in HG693 was recognized by a cDNA clone that was obtained by screening a λgt11 library with an antibody specific for the csA-glycoprotein. When probing RNA from wild-type cells, this clone hybridized with a single developmentally regulated RNA species of 1.9 kb whose expression was strongly enhanced by cyclic-AMP pulses. Appearance of this RNA coincided with the expression of the csA-glycoprotein