ABSTRACT
The α-conotoxins are peptide toxins that are identified from the venom of marine cone snails and they hold outstanding potency on various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, so nAChR dysfunctions have been involved in a variety of severe pathologies. Four types of α-3/5 conotoxins MI, MIA, MIB and MIC have been found from Conus magus. Among them, the activity and selectivity of MIA and MIB have not been well studied. In this study, four α-3/5 conotoxins MI, MIA, MIB and MIC were synthesized by solid peptide synthesis method, and the bioactivities of them were screened by double electrode voltage clamp electrophysiology. The results showed that MIA and MIB selectively inhibited muscle type acetylcholine receptors with IC50 values of 14.45 and 72.78 nmol·L-1, respectively, which are slightly weaker than MI and MIC. Molecular docking results have shown MIA and MIB interact with muscle-type nAChRs with similar mechanism. The reasons for activity differences may relate to the size of the N-terminal amino acids. Together, the conotoxins MIA and MIB may have the potential to develop as a tool for detect the function of muscle type nAChRs, as well as the diagnosis or treat of related diseases.
ABSTRACT
The cyanuric chloride linkers have been used for cyclizing polypeptide, but not used for α-conotoxin, the peptides with rich disulfide bonds and more amino acid residues. In this study, cyclic peptides c[A10L]PnIA-1-4 were synthesized efficiently by lysine assisted cyanuric chloride linkers with 28.92%-52.00% yields. The activity evaluation showed that the IC50 values of c[A10L]PnIA-1 against α7 and α3β2 nAChR subtypes were 5 and 7 times higher than [A10L]PnIA respectively, and the subtype selectivity was maintained. The results of circular dichroism show that this cyclization method had no significant effect on its secondary structure. Compared with the commonly used head-to-tail cyclization in conotoxin cyclization, this method has the advantages of rapid reaction and high yield, which is expected to be further applied to the cyclization study of various α-conotoxins.
ABSTRACT
Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) in order to contribute to the development of novel analgesic drugs and neuropharmacological probes.Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins,a novel α-conotoxin Lt1.1 was cloned from Conus litteratus.The peptide-resin was synthesized using the solid-phased method and was cleaved.The resulting linear peptide was oxidized by air to give the product containing disulfide bridges.The folding product was finally purified by HPLC.The disulfide bond connectivity was determined using the two-step oxidative folding methods.The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte.Membrane currents were recorded using the two electrode voltage-clamp technique.Results A novel α-conotoxin designated as Lt1.1(GCCSHPACNVNNPDIC-NH2) was cloned and its disulfide connectivity was C1-C3,C2-C4.Lt1.1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L,respectively.Conclusion Lt1.1 is a novel 4/7 α-conotoxin that selectively targets α3β2 and α3β4 nAChRs.
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OBJECTIVE: To investigate antagonistic activities of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nicotinic acetylcholine receptors (nAChRs). METHODS: Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human α6/α3β2β3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS: The three isomers of α-conotoxin TxIB were synthesized successfully. The retention time of each isomer of α-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass. Their hydrophilicity orders were globular > ribbon > bead. Both rat and human α6/α3β2β3 nAChRs were expressed in oocytes well. Inhibition of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human α6/α3β2β3 nAChRs with corresponding IC50 of 28.2 and 32.0 nmol·L-1 respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human α6/α3β2β3 nAChRs only with an IC50 of > 10 μmol·L-1. CONCLUSION: The synthesized globular isomer of α-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human α6/α3β2β3 nAChRs, which would be helpful for its new drug development.
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OBJECTIVE: To interrogate differential sensitivity of α-conotoxin TxID on stoichiometry of α3β4 nicotinic acetylcholine receptors(nAChRs). METHODS: Oocytes of Xenopus laevis were used to express rat α3β4 nAChRs with different stoichiometries by altering α3:β4 RNA injection ratios of 1:1, 1:10 or 10:1. Sensitivity of α-conotoxin TxID on these different stoichiometry receptors were evaluated and compared. RESULTS: The three stoichiometry receptors of α3β4 nAChRs were expressed in oocytes successfully. α-Conotoxin TxID showed differential sensitivity on α3β4 nAChR stoichiometries. Inhibition of 1:10 injection ratio by TxID was similar with regular 1:1 α3β4 nAChRs within 2-fold difference. While potency of 10:1 injection ratio by TxID decreased 5-fold significantly comparing with 1:1 α3β4 nAChRs. CONCLUSION: α-Conotoxin TxID exhibits distinct sensitivity on different stoichiometry of α3β4 nAChRs, which could reflecting different stoichiometries of α and β subunits. The RESULTS would be helpful for elucidation of structure and physiology function of α3β4 nAChRs.
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OBJECTIVE: α-Conotoxin LtIA (α-CTX LtIA, LtIA) is a specific inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) from Conus litteratus, a marine snail native to Hainan. The aim of this study was to evaluate the analgesic activity of α-CTX LtIA. METHODS: The analgesic effect of α-CTX LtIA on pain models was evaluated using mice hot-plate and tail-flick models by intracerebroventricular (icv) injection. RESULTS: In tail-flick test, the maximum analgesia percentage (PMAP) was 37.74% at 15 min after LtIA administration by icv injection with dose of 0.2 nmol per mouse. While in hot-plate test, PMAP was 48. 81% at 60 min after LtIA administration by icv injection with same dose of 0.2 nmol per mouse. α-CTX LtIA showed good analgesic activity in two pain models. CONCLUSION: α-CTX LtIA exhibits good analgesic activity by specific interaction with α3β2 nAChRs subtype. These results have great significance for the research and development of LtIA painkiller in the future.
ABSTRACT
Nicotinic acetylcholine receptors (nAChRs) ,also known as neuronal nicotinic receptors ,are widely expressed throughout the central and peripheral nervous system .nAChRs play crucial roles in pain signaling .Recently ,agonists that tar-get specific nAChR subtypes have shown substantial efficacy in a wide range of acute and chronic pain models ,contributing to developing novel analgesic drugs with low drug dependence .Positive allosteric modulators offer new approaches for increasing the potency and therapeutic window of these drugs .This review summarizes the advances in nicotinic acetylcholine receptors as analgesic targets and anticipate the future directions in this field .
ABSTRACT
Three dimensional quantitative structure activity relationship between diazabicyclo[4.2.0]octanes and nicotinic acetylcholine receptor (h alpha4beta2 and h alpha3beta4) agonists was studied using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). From 11 CoMFA and CoMSIA models, CoMSIA with steric and electrostatic fields gave the best predictive models (q2=0.926 and 0.945, r2(ncv)=0.983 and 0.988). This study can be used to develop potent h alpha4beta2 receptor agonists with low activity on h alpha3beta4 subtype.