Novel Nicotinic Acetylcholine Receptor Ligands based on Cytisine, Ferruginine, Anatoxin-a and Choline : <i>In vitro </i>Evaluation and Structure-Activity Relationships

Nicotinic acetylcholine receptors (nAChRs), excitatory ligand-gated cation channels, are transmembranic allosteric proteins composed of five polypeptide chains. The subtype at the neuromuscular junction consists of α1, β1, γ and δ subunits whereas the neuronal subtypes are formed by a combination of different α (α2 to α10) and β(β2 to β4) subunits (heteromeric type) or identical α subunits (homomeric type). NAChRs are widely distributed in the central nervous system and in the periphery. In recent years, there has been increasing interest in nAChR ligands as potential analgesics and therapeutics for the treatment of various neurological and mental disorders related to a decrease in cholinergic function such as Alzheimer’s and Parkinson’s diseases, attention deficit/hyperactivity disorder, schizophrenia, and depression. Natural toxic alkaloids like (-)-cytisine, (+)-ferruginine, and (+)-anatoxin-a possess high affinity for α4β2 nAChR, representing the major population of nACh receptors in mammalian brain, but no appropriate selectivity. Structure-activity relationship studies for these ligands are missing for other subtypes, like α7, α3β4 and (α1)2β1γδ. In order to evaluate the affinities of novel analogues of (-)-cytisine, (+)-ferruginine, and (+)-anatoxin-a, in vitro radioligand binding studies were performed for four different nAChR subtypes: α4β2*, α7*, α3β4* and (α1)2β1γδ nAChRs, using membranes of native tissues (rat brains, calf/pig adrenals and Torpedo calif. electroplax). Important information could be obtained concerning the structural requirements that enhance selectivity of these novel analogues for α4β2* nAChR over other nAChRs investigated. In general, since the molecular recognition between ligands and nAChRs might be based on cation-pi interactions and a hydrogen bond formation between the receptor site and the ligand (HBD-HBA interaction), all high affinity compounds bear the cationic and HBA motifs. Beside the search and development of novel nAChR ligands exhibiting selectivity for the α4β2* nAChR, interest is growing to develop novel compounds selective for the α7 and α3-containing subtypes. Choline has proved to be a selective ligand for the α7, showing also neuroprotective actions. In this study, choline derivatives are evaluated in in vitro radioligand binding studies to gain more information about the structural requirements to enhance the selectivity for α7* versus the α4β2* nicotinic receptor

Abstracts from the 23rd Italian congress of Cystic Fibrosis and the 13th National congress of Cystic Fibrosis Italian Society

Cystic Fibrosis (CF) occurs most frequently in caucasian populations. Although less common, this disorder have been reported in all the ethnicities. Currently, there are more than 2000 described sequence variations in CFTR gene, uniformly distributed and including variants pathogenic and benign (CFTR1:www.genet.sickkids.on.ca/). To date,only a subset have been firmily established as variants annotated as disease-causing (CFTR2: www.cftr2.org). The spectrum and the frequency of individual CFTR variants, however, vary among specific ethnic groups and geographic areas. Genetic screening for CF with standard panels of CFTR mutations is widely used for the diagnosis of CF in newborns and symptomatic patients, and to diagnose CF carrier status. These screening panels have an high diagnostic sensitivity (around 85%) for CFTR mutations in caucasians populations but very low for non caucasians. Developed in the last decade, Next-Generation Sequencing (NGS) has been the last breakthrough technology in genetic studies with a substantial reduction in cost per sequenced base and a considerable enhancement of the sequence generation capabilities. Extended CFTR gene sequencing in NGS includes all the coding regions, the splicing sites and their flankig intronic regions, deep intronic regions where are localized known mutations,the promoter and the 5'-3' UTR regions. NGS allows the analysis of many samples concurrently in a shorter period of time compared to Sanger method . Moreover, NGS platforms are able to identify CFTR copy number variation (CNVs), not detected by Sanger sequencing. This technology has provided new and reliable approaches to molecular diagnosis of CF and CFTR-Related Disorders. It also allows to improve the diagnostic sensitivity of newborn and carrier screeningmolecular tests. In fact, bioinformatics tools suitable for all the NGS platforms can filter data generated from the gene sequencing, and analyze only mutations with well-established disease liability. This approach allows the development of targeted mutations panels with a higher number of frequent CF mutations for the target populationcompared to the standard panels and a consequent enhancement of the diagnostic sensitivity. Moreover, in the emerging challenge of diagnosing CF in non caucasians patients, the possibility of customize a NGS targeted mutations panel should increase the diagnostic sensitivity when the target population has different ethnicities