| MVIIA was a ω-conopeptide and consists of 25 amino acids and three disulfide bonds. It was approved by FDA in 2004 and used for the treatment of neuropathic pain and severe pain of cancer and AIDS patients. SO-3, which was discovered from Conus striatus in South China Sea by our laboratory, was a novel ω-conotoxin and also consists of 25 amino acids and three disulfide bonds. SO-3 possesses a high homology (71%) with MVIIA, in which 12 amino acids in C terminal are identical.The previous study showed that MVIIA and SO-3 had similar analgesic activity, but the toxicity of MVIIA was significantly higher than SO-3 in goldfish toxicity tests. MVIIA produces a lot of severe side effects, such as illusion, ataxia, tremor and so on, and restricts its wide clinical application. Up till now, the relationship of the structure-activity of MVIIA has been investigated widely, but the origin of its toxicity has not been studied. In addition, SO-3 is now in preclinical trial, but its relationship of structure-activity is not clear.In order to investigate the origin of MVIIA’s toxicity and the relationship of structure and activity of SO-3, we synthesized the hybrids of MVIIA and SO-3 as well as other MVIIA mutants. Using patch clamp technique, goldfish toxicity test, the experiments of tremor, spontaneous locomotor activity and motor functions in mice, the key amino acids involved in toxicity were identified. In addition, we synthesized a series of mutants of SO-3 and determined the binding activity of peptide to N-type calcium channels by using patch clamp technique. Finally, molecular modeling technique was used to analyze the binding mode of MVIIA and SO-3 with N-type calcium channels.Furthermore, patch clamp technique was set up in our lab on the basis of work of Huazhong University of Science and Technology. N-type calcium channel plasmids was successfully co-expressed in HEK293 cells and hippocampal neuron cells containing natural N-type calcium channel were isolated and cultured, several A-superfamily conotoxins that might target N-type calcium channel were also synthesized.The main results were as follow:(1) MVIIA, its 5 mutants and 12 SO-3 mutants were synthesized firstly or repeatedly. After folding at 4℃, enrichment and purification, the peptides were obtained with the purity> 95%.(2) All peptides exhibit a negative peak at 210nm in CD spectroscopy, indicating they have α β-sheet structure.(3) The binding activities of MVIIA and its 5 mutants to N-type calcium channels were determined by patch clamp technique. The results show that the replacement of loop2 or Asp14 of MVIIA with the corresponding loop2 or Asn of SO-3 resulted in the three-fold increase in the IC50 of MVIIA mutants. Substitution of Leu11 or Met12 of MVIIA in loop2 with the corresponding Ile or Ala of SO-3, respectively, reduced the binding activity.(4) The goldfish toxicities of all MVIIA mutants were lower than MVIIA. Tremor toxicity of MVIIA and its effects on spontaneous locomotor activity and motor function were greater than that of SO-3 and MVIIA mutant that the Ioop2 was replaced by the corresponding loop2 SO-3.(5) The recovery experiments and the molecular calculation of MVIIA, SO-3 and MVIIA mutants binding to N-type calcium channel showed that Met12 may mainly contribute to the toxicity. The replacement of Asp14 by Asn led to higher activity and lower toxicity of MVIIA.(6) The binding activities of SO-3 and its mutations to N-type calcium channel were determined by using patch clamp technique. The results showed that the replacement of Ala3 and Ala4 of SO-3 by Ser and Lys of ω-conotoxin CVID at the same position resulted in 1-fold increase in the IC50. While the exchange of Ala4 with Pro7 of SO-3 led to an apparent increase in the activity. Furthermore, after Asn14 was replaced by Asp or Ile11 replaced by Ala, the activity of SO-3 mutants was decreased 4 times and 60 times, respectively.(7) The patch clamp technique for the determination of N-type calcium channel was also been set up, non-co-conopeptides that may target N-type calcium channel are being screened. |
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