Researches On The Inhibition Of Morphine Dependence By Conantokin Peptides And The Relationship Of Structure-analgesic Acitivity Of Conopeptide SO-3

Conotoxins are a series of small, conformationally constrained peptides from Conus snails (genus Conus), most of which contain multiple disulfide bonds and could selectively target a specific voltage-gated ion channel, ligand-gated ion channel or G-protein-coupled receptor. Compared with other peptide toxins, conotoxins possess the superior chemical and pharmacological features, such as small molecular, stable structure, high activity and selectivity. Conotoxins could be used as probes for the research of ion channel and receptor as well as new drugs or lead compounds. The purpose of this paper is to explore the inhibition of morphine dependence by conantokins and variants, and to study the relationship between functional residues and analgesic acitivity of a new N-type calcium channel inhibitor SO-3.1. Synthesis, purification of conatokins and variants and the study of their inhibition morphine dependence(1) Unnatural Fmoc-protected-γ-carboxyglutamic acid for conantokin peptide synthesis was synthesized. (2) Eight conantokins and variants with different affinities on NMDA receptor and different selectivities on subunit were synthesized by solid phase synthesis method and were purified by Gel filtration chromatography, anion exchange chromatography and reverse-phase high performance liquid chromatography (RP-HPLC) with 85% or more purity. (3) The inhibitory effects of conantokin peptides on naloxone-induced withdrawal symptoms of morphine dependent mice were first reported.The results indicated that conantokins and variants could significantly reduce withdrawal jumping numbers of morphine dependent mice, and the inhibitory activtity showed dose-dependent mode. The inhibitory effects of conantokins and variants are 700 times higher than other NMDA antagonists, such as memantine and agmatine as well as NR2B inhibitor ifenprodil. Conantokins and their variants show great potential in developing new candidates for inhibiting the development of morphine dependence.Meanwhile, at dose of icv 15 nmol/kg, con-G with NR2B subunit selectivity attenuated 89.0% of the naloxone-induced withdrawal jumping, another two NR2B subunit selective inhibitors con-G[S16Y] and con-G[γ7K] completely inhibited the withdrawal jumping. Whereas con-R(1-17) and Ala-con-G, with higher inhibitory activity on NMDA receptor and without subunit selectivity, just inhibited 40.2%-58.3% of the naloxone-induced withdrawal jumping. These results demonstrated that the inhibitory activities of conantokin peptides are involved with their NR2B subunit selectivities.2. Research on structure-activity relationship ofω-conotoxin SO-3(1) Five SO-3 variants were designed b, (SO-3[R10K,I11L]); c, (SO-3[I11L,A12M]); d, (SO-3[A3S,A4K,A12M]); e, (SO-3[A3S,R10K,I11K,A12M]); f, (SO-3[A4P,P7A])) after comparisons with the functional residues of other analgesicω-conotoxins MVIIA and CVID and molecular calculations; (2) The above Linear peptides were synthesized and the folding conditions were explored, the correct folding products were purified by RP-HPLC. (3) The analgesic effects of SO-3 variants in hot-plate test, writhing test and formalin test, and the affinities of SO-3 and its variants to N-type VSCC were assessed by radioligand binding assay.The results indicated that four SO-3 variants exhibited potent analgesic activity with a dose-dependent mode in several animal models. The analgesic effects of SO-3 and its variants exhibited following order: SO-3>f>d>c>b>>e. Similarly, the affinities to N-type VSCC of SO-3 and its variants in radioligand binding assay were expressed as following order: f>SO-3>d>c>b>>e (high to low), and consistent with their analgesic effects.These results demonstrated that Ile11 is an important functional residue, substituting Ala12 with Met doesn't affect its analgesic activity, Pro7 may contributes to N-type VSCC binding and low toxicity, Pro7 replaced with Ala as well as Ala4 substituted with Pro result in the increasing toxicity of SO-3.The innovations of this paper include:(1) It is the first time to reveal the inhibition of morphine dependence by conantokins and variants.(2) We elicit the conclusion that the inhibitory activities of conantokin peptides are involved with their NR2B subunit selectivities.(3) We discovered that the analgesic activities ofω-conotoxin SO-3 and its variants related to their affinities to N-type VSCC.(4) The relationships of analgesic activities and functional residues of SO-3 are elicited, which provide basic data to further modification of SO-3.