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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010New Hybrid and Mutant PIA and MII Toxins with GreaterAffinty and Selectivity for the α6* SubtypeRenato Longhi 1 , Luca Pucci 2 , Luca Rizzi 3 , Giovanni Grazioso 3 ,Clelia Dallanoce 3 , Francesco Clementi 2 , and Cecilia Gotti 21 CNR, ICRM, Via Mario Bianco 9, Milano, 20131, Italy; 2 CNR, Istituto di Neuroscienze,Via Vanvitelli 32, Milano, 20129, Italy; 3 Dipartimento di Scienze Farmaceutiche “PietroPratesi”, Unimi, Via Mangiagalli 25, Milano, 20133, ItalyIntroductionNeuronal nicotinic receptors (nAChRs) [1] make up a heterogeneous family of ligand-gatedcation channels found in the central and peripheral nervous systems. The different nAChRsare involved in various physiological functions and the in vivo presence of a multitude ofsubtypes underlines the importance of their diversity in the fine tuning of the cholinergictransmission. The α6* receptors have aroused particular interest because recent studieshave shown that they are involved in nicotine-elicited locomotion, acute and chronicnicotine self-administration and other brain activities [2]. At present, the compounds thatare α6* selective belong to the α-conotoxin family [3]. The marine cone snail peptidesα−conotoxin PIA and MII both bind the α6β2* receptors: MII binds and antagonisesα6β2*and α3β2* subtypes with relatively high affinity, whereas the PIA toxin only bindsthe α6β2* subtype with lower affinity than MII. The lack of selectivity of MII is notsurprising given the very high sequence homology in the extracellular binding domain ofthe α6 and α3 subunits. In this study, we analysed the role of the N-terminal amino acids indefining the affinity and potency of the PIA toxin for the α6β2* subtype; then weconstructed a hybrid between the PIA and MII peptides that is more potent and selective forthe native α6* subtype. Subsequently, by means of molecular modelling analysis, weaimed at identifying amino acid residues in MII which are relevant to the molecularrecognition by the α3β2* subtype, thus achieving new structural analogues whose affinityfor the α3β2* nAChRs is significantly reduced.Results and DiscussionThe small disulfide-rich α−conotoxins PIA and MII are both competitive antagonists of theα6β2* neuronal nicotinic receptor (nAChR) subtype, albeit with different affinity andselectivity. Both toxins have a common “ω−shaped” topology but, PIA is characterized inthe N-terminal region by a tail containing three amino acids (RDP), which forms a type Iβ-turn. In order to obtain PIA and MII synthetic conotoxins of high quality, care andattention were devoted to the synthesis and peptides folding: The Cysteine residues wereorthogonally protected: 1-3 Trt, 2-4 Acm. SS connectivity 1-3 was obtained using twoequivalents of hydrogen peroxide in 60 minutes (Figure 1) and connectivity 2-4 in 30minutes using 5 equivalents of iodine in 50% aqueous methanol. We than synthesised agroup of PIA-related peptides in which RDP motif was gradually removed and the firstamino acid mutated. Binding and functional studies showed that the RDP tripeptide isessential for high affinity of the PIA at the native rat α6β2*subtype (Figure 2A), with themajor role being played by the Arginineresidue R1. A molecular modeling studyshowed that the recognition process byα6β2* nAChR is mainly guided by a saltbridge involving the guanidine group ofR1 and the highly negatively chargedD166-D167 residues of the β2 subunit.Then, we inserted the RDP sequence atthe N-terminus of the α-conotoxin MII(RDP-MII). When compared with thenative α-conotoxin, the new hybrid-typepeptide showed increased affinityFig. 1. H 2 O 2 mediated folding of RDP-MII att=0, 14, 28 and 42 min. after H 2 O 2 addition.(10-fold) and potency (5-fold) for theα6β2* nAChRs (Figure 2B) whereas itspharmacological profile was marginally574