New Synthetic Caffeine Analogs as Modulators of the Cholinergic System.

Alzheimer's disease is a multifactorial neurodegenerative disorder. Since cholinergic deficit is a major factor in this disease, two molecular targets for its treatment are the acetylcholinesterase (AChE) and the nicotinic acetylcholine receptors (nAChRs). Given that caffeine is a natural compound that behaves as an AChE inhibitor and as a partial agonist of nAChRs, the aim of this work was to synthetize more potent bifunctional caffeine analogs that modulate these two molecular targets. To this end, a theophylline structure was connected to a pyrrolidine structure through a methylene chain of different lengths (3 to 7 carbon atoms) to give compounds 7-11 All caffeine derivatives inhibited the AChE, of which compound 11 showed the strongest effect. Electrophysiological studies showed that all compounds behave as agonists of the muscle and the neuronal α7 nAChR with greater potency than caffeine. To explore whether the different analogs could affect the nAChR conformational state, the nAChR conformational-sensitive probe crystal violet (CrV) was used. Compounds 9 and 10 conduced the nAChR to a different conformational state comparable with a control nAChR desensitized state. Finally, molecular docking experiments showed that all derivatives interacted with both the catalytic and anionic sites of AChE and with the orthosteric binding site of the nAChR. Thus, the new synthetized compounds can inhibit the AChE and activate muscle and α7 nAChRs with greater potency than caffeine, which suggests that they could be useful leaders for the development of new therapies for the treatment of different neurologic diseases. SIGNIFICANCE STATEMENT: In this work we synthetized caffeine derivatives which can inhibit acetylcholinesterase and activate both muscle and α7 nicotinic acetylcholine receptors (nAChRs) with higher potency than caffeine. These analogs can be divided into two groups: a non-desensitizing and a desensitizing nAChR group. From the nAChR non-desensitizing group, we propose compound 11 as the most interesting analog for further studies since it inhibits acetylcholinesterase with the highest potency and activates the nAChRs in the picomolar range without inducing receptor desensitization.

Orthosteric and Allosteric Activation of Human 5-HT3A Receptors.

The serotonin type 3 receptor (5-HT3) is a ligand-gated ion channel that converts the binding of the neurotransmitter serotonin (5-HT) into a transient cation current that mediates fast excitatory responses in peripheral and central nervous systems. Information regarding the activation and modulation of the human 5-HT3 type A receptor has been based only on macroscopic current measurements because of its low ion conductance. By constructing a high-conductance human 5-HT3A receptor, we here revealed mechanistic information regarding the orthosteric activation by 5-HT and by the partial agonist tryptamine, and the allosteric activation by the terpenoids, carvacrol, and thymol. Terpenoids potentiated macroscopic currents elicited by the orthosteric agonist and directly elicited currents with slow-rising phases and submaximal amplitudes. At the single-channel level, activation by orthosteric and allosteric agonists appeared as openings in quick succession (bursts) that showed no ligand concentration dependence. Bursts were grouped into long-duration clusters in the presence of 5-HT and even longer in the presence of terpenoids, whereas they remained isolated in the presence of tryptamine. Kinetic analysis revealed that allosteric and orthosteric activation mechanisms can be described by the same scheme that includes transitions of the agonist-bound receptor to closed intermediate states before opening (priming). Reduced priming explained the partial agonism of tryptamine; however, equilibrium constants for gating and priming were similar for 5-HT and terpenoid activation. Thus, our kinetic analysis revealed that terpenoids are efficacious agonists for 5-HT3A receptors. These findings not only extend our knowledge about the human 5-HT3A molecular function but also provide novel insights into the mechanisms of action of allosteric ligands, which are of increasing interest as therapeutic drugs in all the superfamily.

Alzheimer's Disease as a Membrane Disorder: Spatial Cross-Talk Among Beta-Amyloid Peptides, Nicotinic Acetylcholine Receptors and Lipid Rafts.

Biological membranes show lateral and transverse asymmetric lipid distribution. Cholesterol (Chol) localizes in both hemilayers, but in the external one it is mostly condensed in lipid-ordered microdomains (raft domains), together with saturated phosphatidyl lipids and sphingolipids (including sphingomyelin and glycosphingolipids). Membrane asymmetries induce special membrane biophysical properties and behave as signals for several physiological and/or pathological processes. Alzheimer's disease (AD) is associated with a perturbation in different membrane properties. Amyloid-ß (Aß) plaques and neurofibrillary tangles of tau protein together with neuroinflammation and neurodegeneration are the most characteristic cellular changes observed in this disease. The extracellular presence of Aß peptides forming senile plaques, together with soluble oligomeric species of Aß, are considered the major cause of the synaptic dysfunction of AD. The association between Aß peptide and membrane lipids has been extensively studied. It has been postulated that Chol content and Chol distribution condition Aß production and posterior accumulation in membranes and, hence, cell dysfunction. Several lines of evidence suggest that Aß partitions in the cell membrane accumulate mostly in raft domains, the site where the cleavage of the precursor AßPP by ß- and γ- secretase is also thought to occur. The main consequence of the pathogenesis of AD is the disruption of the cholinergic pathways in the cerebral cortex and in the basal forebrain. In parallel, the nicotinic acetylcholine receptor has been extensively linked to membrane properties. Since its transmembrane domain exhibits extensive contacts with the surrounding lipids, the acetylcholine receptor function is conditioned by its lipid microenvironment. The nicotinic acetylcholine receptor is present in high-density clusters in the cell membrane where it localizes mainly in lipid-ordered domains. Perturbations of sphingomyelin or cholesterol composition alter acetylcholine receptor location. Therefore, Aß processing, Aß partitioning, and acetylcholine receptor location and function can be manipulated by changes in membrane lipid biophysics. Understanding these mechanisms should provide insights into new therapeutic strategies for prevention and/or treatment of AD. Here, we discuss the implications of lipid-protein interactions at the cell membrane level in AD.

Molecular Modulation of Human α7 Nicotinic Receptor by Amyloid-ß Peptides.

Amyloid ß peptide (Aß) is a key player in the development of Alzheimer's disease (AD). It is the primary component of senile plaques in AD patients and is also found in soluble forms. Cholinergic activity mediated by α7 nicotinic receptors has been shown to be affected by Aß soluble forms. To shed light into the molecular mechanism of this effect, we explored the direct actions of oligomeric Aß1-40 and Aß1-42 on human α7 by fluorescence spectroscopy and single-channel recordings. Fluorescence measurements using the conformational sensitive probe crystal violet (CrV) revealed that in the presence of Aß α7 undergoes concentration-dependent conformational changes. Exposure of α7 to 100 pM Aß changes CrV KD towards that of the desensitized state. However, α7 is still reactive to high carbamylcholine (Carb) concentrations. These observations are compatible with the induction of active/desensitized states as well as of a novel conformational state in the presence of both Aß and Carb. At 100 nM Aß, α7 adopts a resting-state-like structure which does not respond to Carb, suggesting stabilization of α7 in a blocked state. In real time, we found that Aß is capable of eliciting α7 channel activity either in the absence or presence of the positive allosteric modulator (PAM) PNU-120596. Activation by Aß is favored at picomolar or low nanomolar concentrations and is not detected at micromolar concentrations. At high Aß concentrations, the mean duration of activation episodes elicited by ACh in the presence of PNU-120596 is significantly reduced, an effect compatible with slow open-channel block. We conclude that Aß directly affects α7 function by acting as an agonist and a negative modulator. Whereas the capability of low concentrations of Aß to activate α7 could be beneficial, the reduced α7 activity in the presence of higher Aß concentrations or its long exposure may contribute to the cholinergic signaling deficit and may be involved in the initiation and development of AD.

A novel pharmacological activity of caffeine in the cholinergic system.

Cholinergic deficit is regarded as an important factor responsible for Alzheimer's disease (AD) symptoms. Acetylcholinesterase (AChE) and nicotinic receptor (AChR) are two molecular targets for the treatment of this disease. We found here that methanolic extracts of Camellia sinensis exhibited anticholinesterase activity and induced AChR conformational changes. From bioguided fractionation we confirmed that caffeine was the active compound exerting such effects. It is well-known that caffeine acts as an inhibitor of AChE and here we explored the effect of caffeine on the AChR by combining single channel recordings and fluorescent measurements. From single channel recordings we observed that caffeine activated both muscle and α7 AChRs at low concentrations, and behaved as an open channel blocker which was evident at high concentrations. Fluorescent measurements were performed with the conformational sensitive probe crystal violet (CrV) and AChR rich membranes from Torpedo californica. Caffeine induced changes in the KD value of CrV in a concentration-dependent manner taking the AChR closer to a desentisized state. In the presence of α-bungarotoxin, an AChR competitive antagonist, high concentrations of caffeine increased the KD value of CrV, compatible with a competition with CrV molecules for the luminal channel. Our electrophysiological and fluorescent experiments show that caffeine has a dual effect on nicotinic receptors, behaving as an agonist and an ion channel blocker, probably through distinct AChR sites with quite different affinities. Thus, caffeine or its derivatives can be considered for the design of promising multitarget-directed drugs for AD treatment by modulation of different targets in the cholinergic pathway.