Analoghi del Neuropeptide S modificati in posizione 5

Neuropeptide S (NPS) is the last neuropeptide identified via Reverse Pharmacology techniques. NPS selectively binds and activates a previously orphan GPCR 154, now named NPSR, producing intracellular calcium mobilization and cAMP levels. Biological functions modulated by the NPS/NPSR system include anxiety, arousal, locomotion, food intake, memory, and drug addiction. The primary sequence of NPS (in humans SFRNGVGTGMKKTSFQRAKS) is highly conserved among vertebrates especially at the N-terminus. Ala- and D-scan together with truncation studies demonstrated that the N-teminal sequence of the molecule is crucial for biological activity. Focused structure–activity investigations performed on Phe2, Arg3, and Asn4 confirmed this indication and revealed the chemical requirements of these positions for NPSR binding and activation. The sequence Gly5-Val6-Gly7 seems to be important for shaping the bioactive conformation of the peptide. Structure–activity studies on Gly5 enabled identification of the first generation of peptidergic NPSR pure antagonists including [D-Cys(tBu)5]NPS and [D-Val5]NPS whose antagonist properties were confirmed in vivo. This PhD thesis is focused on the structure activity relationship study of NPS position 5. In particular, in order to identify new potent and selective antagonists of the NPSR receptor, we have designed, synthesized and inserted in position 5 of NPS the following non natural amino acids: i) the chimeric Valine/Leucine amino acid, Ipv, 2-amino-3,3,4-trimethyl-pentanoic acid as racemate; ii) the R and S form of the amino acid, Ddb, 2,4-diamino-3,3-dimethylbutyric acid characterized by a branched aliphatic side chain functionalized with a primary amino group; iii) some penicilammine S-alkylate analogs. This work has allowed to investigate and apply different chemical strategies for the synthesis of alpha-amino acids characterized by branched aliphatic side chains. In addition the original synthetic scheme adopted for the synthesis of 2-amino-3,3,4-trimethyl-pentanoic acid in which the key step is the copper (I) chloride-catalyzed conjugated addition of i-propyl magnesium bromide to 2- isopropylidene-malonic acid diethyl ester together with the commercial variety of Grignard reagents available make this route suitable for further synthetic applications, including the synthesis of novel non natural valine derivatives. The pharmacological data obtained further corroborate the proposal that chirality and steric hindrance of position 5 of NPS are crucial chemical requirements for modulating peptide efficacy and potency. It has been also found that NPSR antagonists can be generated by replacing NPS position 5 with Damino acids characterized by basic side chain. Finally, the use of S-alkyl penicilammine showed that the increase in steric hindrance of position 5 is better tolerated if generated by an aromatic substituent compared to aliphatic groups suggesting that planarity is a chemical feature useful for the identification of novel NPSR antagonists.