New Multitarget Molecules Derived from Caffeine as Potentiators of the Cholinergic System.

Cholinergic deficit is a characteristic factor of several pathologies, such as myasthenia gravis, some types of congenital myasthenic syndromes, and Alzheimer's Disease. Two molecular targets for its treatment are acetylcholinesterase (AChE) and nicotinic acetylcholine receptor (nAChR). In previous studies, we found that caffeine behaves as a partial nAChR agonist and confirmed that it inhibits AChE. Here, we present new bifunctional caffeine derivatives consisting of a theophylline ring connected to amino groups by different linkers. All of them were more potent AChE inhibitors than caffeine. Furthermore, although some of them also activated muscle nAChR as partial agonists, not all of them stabilized nAChR in its desensitized conformation. To understand the molecular mechanism underlying these results, we performed docking studies on AChE and nAChR. The nAChR agonist behavior of the compounds depends on their accessory group, whereas their ability to stabilize the receptor in a desensitized state depends on the interactions of the linker at the binding site. Our results show that the new compounds can inhibit AChE and activate nAChR with greater potency than caffeine and provide further information on the modulation mechanisms of pharmacological targets for the design of novel therapeutic interventions in cholinergic deficit.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Production of low molecular weight chitosan by acid and oxidative pathways: Effect on physicochemical properties.

Chitosan-based biomaterials with a low molecular weight (LMW) have been drawn attention due to the promising applications in the pharmaceutical and food fields. For this reason, the aim of this work was to study the effect of two distinct depolymerization pathways on the chitosan physicochemical properties. Chitosan was submitted to depolymerization reaction to obtain chitosan with low molecular weight (LMW), using the oxidative pathway (H2O2) and the acid pathway (HCl). The molecular weight reduction was investigated by kinetic study and chain scission mechanism. Chitosan characterization was performed according to its viscosimetric average molecular weight and deacetylation degree, respectively, through the viscosimetric method and proton nuclear magnetic resonance spectroscopy (1H NMR). The structural integrity was evaluated by Fourier transform infrared (FTIR) and energy dispersive spectroscopy (EDS). The crystalline and thermal properties were investigated, respectively, by X-ray diffraction (XRD) spectroscopy and thermogravimetric (TGA)/ differential thermal (DTA)/ differential scanning calorimetry (DSC) analysis. The water-chitosan interaction study was used to estimate the chitosan solubility. The results pointed out that both pathways resulted in chitosan with low molecular weight (<50 kDa). Moreover, the structural integrity of chitosan polymeric chains was preserved after depolymerization by oxidative pathway, while the acid pathway modified the polymer chain arrangement. Therefore, the chemical pathways resulted in two distinct low molecular weight chitosans, which allows different applications in food science.