The Synthesis And Functional Charaterization Of Novel A-super Family Conopeptides Derived From South China Sea

Objective:The purpose of this study is to synthesize several novel A-superfamily conopeptides, identify their targets to neuronal nicotinic cetylcholine receptor subtypes, and investigate the structure-activity relationships of some conopeptides. This work will lay the foundation for the discovery of new targets of these peptides, the development of the novel analgesic drugs and neuropharmacological probes.Methods:(1)Peptide-resins were prepared on the ABI433 A synthesizer using solid phase method, and were then cleaved. The resulting linear peptides were oxidated to form folding producs which were purified by HPLC;(2) The binding activity of conopeptides to neuronal nicotinic acetylcholine receptor subtypes expressed in xenopus oocytes was determined using the two-electrode voltage clamp technique;(3) The disulfide bond connectivities of two peptides were determined using the two-step folding methodsResults:(1)Fourteen A-superfamily conopeptides(Bt1.8, Bt1.10, Lt1.1, Lt1.2, Lt1.3, Lt1.4, Im1.2, Bt14. 10, Bt14.11, Bt14.14, Bt14.15, Bt14.19, Em14.1, Vi1.6) were synthesized.(2)The targets of eleven A-superfamily conopeptides were screened. α-conotoxins Bt1.10 and Lt1.3 selectively targets α3β2 n ACh R, with the IC50 of 52.94 n M,44.78 n M respectively. But α-conotoxins Bt1.8、Lt1.1 and Lt1.2 inhibited both α3β2 and α3β4 n ACh Rs, with the IC50 of 9.36 n M,166.76 n M,164.74 n M respectively.(3)The binding activities of four Bt1.8 mutants(Bt1.8[N5H] 、Bt1.8[I9A]、Bt1.8[N11E] and Bt1.8[Q15A]) to α3β2 n ACh R were determined. The result showed that the activity of the mutants of Bt1.8 was as follows: Bt1.8[N5H] isomer 1 > Bt1.8 > Bt1.8[Q15A] > Bt1.8[N5H] isomer 2 > Bt1.8[I9A] > Bt1.8[N11E].(4)The binding activities of four Vi1.6 mutants(Vi1.6[R1A] 、Vi1.6[D2A]、Vi1.6[H11A] and Vi1.6[+16L]) to α3β2 n ACh R were determined. The results showed that the activity of the mutants of Vi1.6 were as follows: Vi1.6[+16L] > Vi1.6[R1A] > Vi1.6 > Vi1.6[D2A] > Vi1.6[H11A].(5)The disulfide connectivity of alpha-conopeptides Bt1.8 and Vi1.6 were determined. The results showed that the two conopeptides had the disulfide connectivity of “C1-C3,C2-C4”.Conclusion:(1)Alpha-conopeptide Bt1.8 strongly inhibits α3β2 n ACh R, in which the Asn11, Ile9 and Gln15 may be important for the toxin’s unique selectivity preference and potency.(2)The structure-activity relationship of Vi1.6 demonstrated that Asp1 and His11 of Vi1.6 are the functional amino acids, and the introduction of hydrophobic residues at the position16 can significantly enhance the inhibitiory activity to α3β2 subtype.(3)Alpha-conopeptides Lt1.1 and Lt1.2 nhibit both α3β2 and α3β4 n ACh Rs, but α-conotoxins Bt1.10 and Lt1.3 selectively target α3β2 n ACh R.(4)The novel A-super family conopeptides(Bt14.11, Bt14.14, Bt14.15 and Bt14.19) with four single cysteines can not bind to the neuronal n ACh Rs subtypes.