DESIGN AND SYNTHESIS OF NOVEL MOLECULAR PROBES TARGETING THE ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTORS: INSIGHT INTO THE MECHANISMS OF ACTIVATION AND THE RELATED THERAPEUTIC IMPLICATIONS

Abstract - The α7 nicotinic acetylcholine receptor (nAChR) is a homopentameric ion channel, characterized by both ionotropic and metabotropic activation modes. The channel-dependent activation has been deeply investigated and its modulation was proven to be a promising therapeutic approach for diseases of the central nervous system (CNS) including Alzheimer’s disease, Parkinson’s disease, schizophrenia, autism, attention deficit hyperactivity disorder (ADHD), anxiety and depression. The additional metabotropic-like activity, instead, is now considered a key functional aspect of α7 nAChR signaling, especially in non-neuronal cells. In particular, in the immune cells, the α7 subtype plays a central role in anti-inflammatory processes via G-protein-mediated activation. The hypothesis defining a relationship between the non-ionotropic activity of α7 nAChRs and their desensitized states is an emerging research topic. The ability of a new class of compounds, defined as “silent agonists”, to stabilize the desensitized states of this receptor subtype engendering anti-inflammatory responses, most likely via a metabotropic mechanism, would seem to confirm this hypothesis. In this research project, the in-depth investigation of the alternative metabotropic-like modulation was performed through the study of structure-activity relationships of two well-known silent agonists, NS6740 and KC-1. The two model compounds were taken as reference structures owing to their relevant silent agonist behavior as well as the presence of common features in their molecular skeleton. Moreover, NS6740 showed a promising anti-inflammatory profile in both in vitro and in vivo assays. From the comparison of the structures of NS6740 (1,4-diazabicyclo[3.2.2]-non-4-yl[5-[3-(trifluoromethyl)phenyl]-2-furanyl]methanone) and KC-1 (5’-phenyl-anabaseine), a preliminary pharmacophore model had been proposed. Three main features were identified: a positively charged center, a central hydrogen-bond acceptor group and an aromatic moiety. In order to provide a deeper description of the pharmacophoric requirements and to achieve a better understanding of both steric and electronic features able to promote the silent agonist profile, several modifications were performed on the key pharmacophoric portions of NS6740 and KC-1. The NS6740 derivatives were synthesized through an acylation reaction between diverse diazabicyclic nuclei and differently substituted, previously prepared, aromatic chains. The New KC-1 derivatives were, instead, prepared via a Suzuki-Miyaura reaction between the central heteroaromatic ring and a set of differently substituted aromatic groups, followed by organolithium coupling with protected lactams. All the newly synthesized compounds were assayed employing human α7 nAChRs expressed in Xenopus laevis oocytes in two-electrode voltage clamping experiments. The results obtained from the electrophysiological investigations demonstrated that the three pharmacophoric moieties are essential to preserve the peculiar silent receptor activation mode, and that the specific distances between the positively charged center and the hydrogen-bond acceptor have to be maintained. In connection with the studies on NS6740, new promising desensitizing agents were identified as well as new potent partial agonists, possessing a good α7 nAChR selectivity profile towards the main heteromeric nicotinic receptor subtypes. NS6740 and the most interesting desensitizing derivatives were also investigated in in silico studies and a potential binding mode has been proposed. From the docking results, the interactions between the basic nucleus and the aromatic cage in the binding site were highlighted; moreover, the hydrogen bond acceptor and the aromatic chain were predicted to be involved in the receptor binding.