DESIGN, SYNTHETIC APPROACHES, BIOPHARMACOLOGICAL INVESTIGATION, AND STRUCTURE ACTIVITY RELATIONSHIPS OF NOVEL LIGANDS TARGETING NEURONAL NICOTINIC RECEPTOR SUBTYPES

In the central nervous system (CNS), ligand-gated ion channels can be found presynaptically, in nerve terminals, where they control neurotransmitter release. Among them, neuronal nicotinic acetylcholine receptors (nAChRs) constitute a particular example, since nAChR-mediated stimulation of neurotransmitter release is more pronounced than the relatively low numbers of nAChRs might predict. Accordingly, this signal amplification has been hypothesized as the main function of nAChRs in the brain. Thus, nAChRs were found to exert mostly a modulatory role in the CNS, in contrast to that displayed in neuromuscular junctions and autonomic ganglia, where they mediate postsynaptic, fast, excitatory neurotransmission. Neuronal nAChRs are pentameric combinations of α and α/β subunits, with a high degree of complexity conferred by 12 different α (α2-α10) and β (β2-β4) subunits. Hence, a large repertoire of molecular architectures can be generated, and the functional relevance of these subunit compositions has yet to be fully clarified. Although neuronal nAChRs play an array of critical roles in the central nervous system (CNS), only in the last two decades a rapidly growing understanding of subtype localization has been associated with potential therapeutic applications. Indeed, the most abundant nAChR subtypes in the CNS are α4β2* heteromers, belonging to the group of α-bungarotoxin-insensitive subtypes, and α7 homomers, which are α-bungarotoxin-sensitive receptors. Functionally, α7 channels are distinguished from α4β2-containing receptors mainly for their lower affinity for acetylcholine and their relatively higher permeability to calcium. Even though the α4β2* and α7 subtypes are differently distributed in the CNS, both are expressed in brain regions (cortex, hippocampus) that are involved in cognitive processes, through the modulation of the release of neurotransmitters (besides acetylcholine, glutamate, GABA, dopamine, and serotonin) that control an array of CNS functions. Recently, much attention has been paid to the ganglionic α3β4* receptor subtype, which, owing to its CNS expression in the medial habenula and in the interpeduncolar nucleus, participates to the regulation of dopamine levels in the mesolimbic area. The improved knowledge of the role played by α4β2*, α7 and α3β4* subtypes in cognitive processes (attention, memory), mood, nociception, and neuroprotection has encouraged the development of subtype-selective compounds designed for different pathologies of the CNS, including Alzheimer’s and Parkinson’s diseases, attention deficit hyperactivity disorder (ADHD), schizophrenia, epilepsy, Tourette’s syndrome, anxiety, depression, pain, and nicotine addiction. In brief, central α7 nAChRs, rapidly opening and rapidly desensitizing ion channels, are implicated in learning, memory, and information processing, including gating deficits that are common in schizophrenia. Conversely, peripheral α7 nAChRs have been shown to play a role in the inflammation response. Central α4β2* nAChRs have been identified as key mediators of the analgesic effects of nicotine agonists, but they are also involved in learning, memory and attention-related effects. On the other hand, the habenular α3β4* subtype is considered a potential novel target for the treatment of nicotine and other drugs addiction as well as of ethanol abuse disorders. However, the involvement of α3β4* receptors in the reward circuit it is far from being completely understood. It has been proposed that α3β4* channels mediate the lateral habenular glutamatergic pathway which is directly linked with the ventrategmental area of the mesolimbic dopamine system. The habenular region, by tuning the dopamine release, could contribute to modulating the sense of reward and the subsequent withdrawal syndrome of nicotine and other drugs of abuse. During my experimental activity, I’ve deepened the study of the structure-activity relationships (SAR) of nicotinic ligands endowed with different molecular skeletons. On this grounds, I exploited the results of both literature studies and molecular modeling evaluations to design and synthesize new potential nicotinic ligands provided with high affinity and subtype selectivity. Worth noting, in a first series of novel target compounds the protonatable scaffold is a 2,5diazabicycle[2.2.2]octane in which the nature and the position of substituents on both the heteroaromatic moiety and the basic nitrogen are sharply influencing the affinity for the α7 or the α4β2 nAChR subtypes. A second series of target compounds has been designed and synthesized as novel quinuclidine derivatives. This ring system is a first choice scaffold for several α7 nAChR selective ligands. Moreover, a methyl-3-pyridyl side pendant has been inserted in position 2, as shown by peculiar α7 nAChR selective ligands studied as anti-schizophrenia agents. Preliminary binding assays at native α7 and α4β2 nAChRs expressed in rat cortical membranes showed how, indeed, an appropriate substitution pattern has led to notable nAChRs affinity and selectivity. Meta-substituted alkoxypyridine derivatives has displayed high affinity and selectivity at the α4β2 nAChRs subtype in a pico-molar range. Conversely, when a bulkier aromatic ring is introduced in para position on a pyridazine nucleus, the target compounds have shifted their affinity and selectivity towards the homomeric α7 nicotinic channel. The quinuclidine derivatives has exhibited only modest affinity at the tested receptors. In a parallel study, I’ve designed and prepared a group of derivatives of Nicotine, Epibatidine and Anabaseine, which are natural unselective nicotinic ligands, characterized by an hydroxyl or an hydroxymethyl group. Recent computational studies suggested that water molecules were present in the ligand binding domain (LBD) of nAChRs, and favored the interaction of the above mentioned natural nicotinic agonists with their complementary receptor channel. The hydroxyl substituents should mimic a water molecule in the interaction with the target receptor protein. To further evaluate the role of water molecules in the ligand-receptor interaction, I’ve designed and prepared additional compounds bearing hydrophobic groups, such as methyl and halides. In this way the mentioned compounds should exclude the water molecule from the interaction with the receptor protein. From the preliminary binding evaluations at native α4β2, α3β4 and α7 nAChRs expressed in rat cortical membranes, controversial and interesting results have been obtained. All the tested compounds have showed a loss of α4β2 protein affinity, while the interaction with the two other nAChRs subtypes was retained. Particularly, the anabaseine derivatives were characterized by a great affinity at the α3β4 nicotinic subtype in the low nano-molar range, with a massive selectivity towards the α4β2 heteromeric receptor. Further studies are ongoing to better understand both the putative role of the water molecule and the molecular requirements able to improve the α3β4 vs. α7 selectivity.