| Nicotinic acetylcholine receptor (nAchR) is widely distributed in the muscle, central nervous system and peripheral nervous system. Several human diseases of the nervous system are raleted to the genetic mutations of nAchR, such as congenital myasthenia gravis, autosomal dominant nocturnal frontal lobe epilepsy.Α9α10 subtype was a new member of the nAChR family discovered in recent years and was found to be involved in the formation of the auditory system. Recent studies showed that this subtype may be a new drug target for painkiller because its antagonists-α-conotoxins (Vc1.1, RgIA have good analgesic activity in several chronic pain models in rats (CCI model, PSL model, Diabetic Neuropathy model).Based on the structure and action mechanism ofα-conotoxins Vc1.1 and RgIA, a series of newlyα-conotoxin mutants were designed and synthesized in the present study. The design principles were as follows: (1) On the basis of a large number of literature collections and sequence analyses of the different conotoxins which targeted at the nAchR, we guessed the loop I ofα-conotoxin Vc1.1 and RgIA might play key role in recognizing the nAchR, while the loopII, especially the 9-11 amino acid residues, may should different binding affinity according to the different properties of the side-chain groups. In order to increase the binding affinity and analgesic activity of Vc1.1,9-11 amino acids were mutated. (2) In order to enhance analgesic activity and pharmacokinetic properties, the peptide with high analgesic activity was modified in N-terminal with chemical groups; (3) Based on the sequence of the newly reported inhibitor of theα9α10 subtypes, ArIB[V11L;V16D](sequence:DECCSNPACRL -NNPHDCRRR) and HS-2(senquence:GCCSDPRCRYDHPEIC), two mutants were designed and synthesized.According to the above design ideas, 17α-conotoxin mutants were designed and synthesized. Two classical animal models of chronic pain, the chronic injury model(CCI model) and the partial sciatic nerve ligation model(PSL model), were choosen to evaluate the analgesic activity of the mutants. Meanwhile, the homology modeling technique was used to build the pentamer complex model of the acetylcholine receptorα9α10 subtype, and used to probe howα-conotoxin Vc1.1 and RgIA bind to the receptor by computational methods. The results were summarized as follows:1. Two mutants (HS-2 and HS-10) are higher analgesic activity compared with Vc1.1 in PSL model. They both displayed a strong analgesic effect in doses of about 25μg/kg and dose-dependent in the range of 0.5-25μg/kg. At the dosage of 3nmol per rat, HS-2 increased the pain threshold to 87.2±30.4%, about 65% higher than Vc1.1 (52.7±16.1%) while HS-10 increased about 79.3±17.3%. The same positive results in the CCI model were verified. Moreover, HS-10, midified with N-benzoyl group protection, would have better anti-enzyme ability and show a great potential for drug development.2. The relationship between structure and analgesic activity was studied. First of all, Asn9 of Vc1.1 is found to be a key amino acid. When Asn9 was replaced with Arg, the analgesic activity was significantly increased, the replacement by Ala, a neutral amino acid, half of the analgesic activity decreased. Furthermore, the replacement by the negatively charged amino acids Asp, a sharp decline in analgesic activity was found. Secondly, Tyr10 is an other key amino acid, when it was replaced by Val (HS-4) or Ser (HS-5), the analgesic activity declined sharply. But using two strong hydrophilic amino acids to replace the Tyr10 and Asp11, the activity dropped only half percentage, suggesting that its binding site may be different according to Vc1.1. Finaly, DPR motif may be the important action mode of loopI since the replacement of D with H (HS-8) results in almost lost in the analgesic activity.3. The pentamer complex model of the acetylcholine receptorα9α10 subtype was built by the homology modeling technique and the molecular interaction mechanism of the receptor andα-conotoxin (Vc1.1, RgIA) was studied. The result shows that the Arg7, Asp11, Glu4 and Tyr10of Vc1.1 are the key amino acid binding to the receptor through electrostatic and hydrogen bond and binding mode of RgIA is significantly different with Vc1.1. First of all, Tyr10 of RgIA forms several hydrogen bonds with the receptor while the Tyr10 Vc1.1 shows less hydrogen bond with the same receptor. Secondly, the electrostatic interaction between RgIa and receptor are weak because the electrostatic attraction and repulsion coexist, hydrogen bond may be more important. Finally, Vc1 .1 and RgIA display significant different binding mode in the (+)α10 (-)α10 interface, Arg7 and Glu4 of Vc1.1 form strong electrostatic attraction with this surface but RgIA does not, suggesting that RgIA may not bind to this interface. |
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