| Cone snails are a kind of marine gastropods and are widely distributed in tropical marine areas. Up to now, there are about 750 cone snail species in the world, 70 species of cone snails have been found in the South China Sea and Taiwan Strait. Generally, cone snails are divided into three groups depending on the prey they subdue: piscivorous, molluscivorous and vermivorous. The venom of each species contains a unique array of more than 100 peptides encoded by a large gene family. Each peptide selectively targets ion channels(Na+, K+ and Ca2+ channels) or membrane receptors(n ACh R, 5-HT3 R, NMDAR and G-protein-coupled receptors). Some conopeptides have already become neuropharmacological tools, and several conopeptides have been used as analgesic drugs or have entered into clinical trials.Calcium channels are widely distributed in mammalian excitable cells, regulating the Ca2+ current. It is involved in many important physiological processes, such as muscle excitation contraction coupling, hormone secretion and regulation, neurotransmitter release, nociceptive pain conduction, etc. N-type voltage-gated calcium channel(N-VGCC, Ca V2.2) belongs to the high voltage activated calcium channels, is mainly distributed in the presynaptic terminals of the central and peripheral nervous systems, and also plays a key role in the release of neurotransmitters like glutamate, substance P or calcitonin gene-related peptide(CGRP). Ca V2.2 has already become an important drug target for the treatment of chronic neuropathic pain and inflammatory pain.A series of ω-conopeptides have been found targeting CaV2.2. ω-MVIIA(Ziconotide) was approved by the FDA in 2004, listed as the treatment of intractable cancer pain and non cancer pain, AIDS related pain. However, its clinical application was limited by severe side effects and narrow therapeutic windows. ω-CVID(Leconotide), another CaV2.2 inhibitor, has alleviated side effects than Ziconotide. Intravenous administration of ω-CVID can alleviate the allodynia in the neuropathic pain model of rat. ω-CVIE and ω-CVIF are two new inhibitors of CaV2.2 discovered recently, and can reversibly inhibit the synaptic transmission between primary afferent nerve and the neurons in superficial dorsal horn of spinal cord. ω-conotoxin SO-3, which was found in our laboratory from Conus striatus, shows similar inhibitory activity on Ca V2.2 to MVIIA, but its side effects are lower than that of MVIIA. The preclinical trial of SO-3 has been accomplished.α-conopeptides are the smallest known conotoxin class, and consisted of 12 to 19 amino acid residues. Usually, they possess 4 cysteines and two disulfide bonds "1-3, 2-4", and mainly target n ACh R. Recently, some α-conopeptides, such as Au IB, Rg IA, Vc1.1 and Pe IA, have been found to inhibit Ca V2.2 current mediated through GABAB receptor. Vc1.1 had entered a clinical trial II, but was terminated because of the poor affinity for human body. Cyclic-Vc1.1 showed an analgesic effect and could be administered orally. Because of the shorter sequence, liable synthesis and lower in toxicity, α-conopeptides have great potential value in the treatment of neuropathic pain.Our laboratory has previously carried out the discovery, synthesis, target identification and drug development of α-conopeptides besides ω-conotoxins including SO-3. Using gene cloning method, a large numbers of novel α-conopeptide sequences have been obtained. Eb1.6 and Vt1.27 were found from Conus ebraeus and Conus vitulinus in the South China Sea, which contained 16 and 17 amino acid residues, respectively. Both of them have two disulfide bonds and belongs to framework I. Recently, our cooperating laboratory in Australian has found that Eb1.6 and Vt1.27 inhibited the ICa of Ca V2.2. This is the first report that α-conotoxin can directly interact with Ca V2.2. In this paper, we are to study their structure-activity relationships of Eb1.6 and Vt1.27, design new chimera peptides of Eb1.6 and Vt1.27 using the key motif from SO-3 and MVIIA. This study will lay the foundation for the discovery of novel Ca V2.2 inhibitors with several advantages such as shorter sequence, liable synthesis and lower toxicity. The major results of this assay are as follows:(1) The whole cell patch clamp technique testing Ca V2.2 currents was successfully established. Ca V2.2 and other calcium channels plasmids were successfully expressed in HEK293 cell, the recording method of Ca V2.2 channel current in HEK293 cell was performed very well.(2) The conopeptide Eb1.6 and its mutants had lower inhibitory activity to Ca V2.2. The inhibition ratio of 10 μM Eb1.6 was 28.96 ± 8.84 %. Based on the structure of Vt1.27, Pro13 and Asn14 were mutated. Pro13 was replaced by Asn or Gln, the inhibitory activities of Eb1.6[P13N] and Eb.16[P13Q] were 10.87 ± 2.37% and 13.32 ± 3.16%, respectively. After Asn14 was replaced by Asp or Glu, the inhibitory activity was also decreased. For example, the inhibitory activities of Eb1.6[N14D] and Eb1.6[N14E] were 3.37 ± 2.61% and 5.80 ± 1.89%, respectively. Furthermore, Asn14 was replaced by hydrophobic amino acids, the inhibitory activity was decreased as well, the inhibitory activity of Eb1.6 [N14F] and Eb1.6[N14L] were 14.09 ± 2.96% and 15.93 ± 4.49%, respectively. These results show that Pro13 and Asn14 are the key amino acid residues of Eb1.6.(3) The Ca V2.2 inhibitory activity of Vt1.27 was slightly higher than Eb1.6. The inhibition ratio of 10 μM Vt1.27 was 34.52 ± 9.45%. The inhibitory activities changed slightly when His6 was substituted by Ala or Pro, or Ile10 was substituted by Asn. The inhibitory activities of Vt1.27[H6A] and Vt1.27 [H6P, P9A] and Vt1.27[I10N] were 32.01 ± 8.29%, 30.33 ± 6.03% and 29.01 ± 4.68%, respectively. Other mutants, such as Vt1.27[D11A], Vt1.27[Y12A], Vt1.27[R14A], Vt1.27[F15A], Vt1.27[I10D, D11I], etc, have lower inhibitory activities(11.20 ± 2.76% ~ 24.19 ± 8.29%). These results showed that Asp11, Tyr12 and Arg14 are the key amino acid residues of Vt1.27.(4) Based on molecular simulation and the replacement of the loop2 of Eb1.6, Vt1.27 with the loop2 of SO-3 or MVIIA and their several alkaline residues at the N-terminal and C-terminal, 11 chimeric peptides were designed and synthesized. two Eb1.6 chimeric peptides showed lower inhibitory activities(14.46 ± 5.46%, 7.45 ± 4.69%,) than that of Eb1.6(10 μM). The inhibitory activities of nine Vt1.27 chimeric peptides(10 μM) were between 18.74 ± 4.24% ~ 36.86 ± 6.72%. Among them, a chimeric peptide showed the best activity(36.86±6.72%), in which Met4 was replaced by Lys and His6 was replaced by Arg, accompanying the replacement of the loop2 of Vt1.27 with the loop2 of MVIIA and the insertion of a Gly at the end of the loop2. However, the single replacement of Vt1.27 loop2 with the loop2 of MVIIA or SO-3 resulted in the decrease in inhibitory activity, indicating that they cannot be completely interchanged, an slightly increase the length of loop2 might elleviate the inhibitory activity. |
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