Leading Compound Screening And Functional Studies Of L-superfamily Conotoxin Of Conus Litteratus

Conotoxins are Gastropoda mollusks that exists wordwide, and there are about500species which can be divided into three categories according to their feeding habits.Conotoxins that secreted by conus are actually a small peptide which contain about10to40amino acids, especially rich in cysteine. Because of their high conservation ofthe signal peptide and the cysteine framework, conotoxins can be classified to manysuperfamilies such as the A, M, O, P, S, T and so on. They can also be classified bytheir respective pharmaceutic targets. Conotoxins are very effective probe inNeuroscience and play am important role in the research of ion channels. They arevaluable also for their wide perspective on the diagnosis or cure of disease.Our lab has discovered a rich EST sequence in the database of the cDNA of theConus litteratus. It was then confirmed that this coding gene has4cysteine aminoacids and its’ scattered cysteine framework is basically the same with conotoxin14(C-C-C-C). As its’ signal peptide is different from any other families that have beenalready discovered, we named lt14a, which belongs to a new L-superfamily. lt14a is a13-amino-acid peptide and it is an antagonist of the neuronal nicotinic acetylcholineReceptor (nAChR). This receptor is associated with the transmission of pain, andappropriate suppression of this receptor can have an analgesic action.The Intrathecal injection expreiment in rats showed that the three dose groups hada relatively good analgesic effect in the first day of the administration. And the increase of the pain threshold is greater than tramadol, one of the positive control, butweaker than morphine. Meanwhile, the rats’ brain tissues from the intrathecal injectionexperiments were used as templates in the semi-quantitative PCR experiments for thetest of expression profiles of genes that related to certain kinds of pain. We found outthat the total expression of the nos and c-fos gene were supressed, and the inhibitaleffect of lt14a is better than the control groups.In order to research the relationship between the peptide’s structure and function,based on the original sequence of lt14a, we decided to mutate the seventh amino acid,which was Lys, after a careful analysis of the peptide’s structure and theconformational changes of the ligand-receptor binding. We chose the solid-phasesynthesis method and gave the whole syntetical process a comprehensive exploration.The purity that we obtained was more than90%after the high-performance liquidchromatography and mass spectrometry. The results of the formalin inflammatorypain model and the contraction of frog gastrocnemius experiments showed that[K7A]-lt14ahas a better analgesic activity in blocking the nerve conduction than lt14a.The pain-related gene expression profile results showed that mutant is capable ofinhibiting the genes’ over-expression that caused by the induction of formalin.Meanwhile, Conotoxins have a positive stimulus impact on zebrafish embryonicdevelopment and eucell.This research was done on the animal and molecular level. It has made acomparison in terms of activity between lt14a and its mutant [K7A]-lt14a, in order toachieve the goal of screening leading compounds of drugs. Also we have established asynthetical process to acquire the mutant. Results suggested that this screening methodis basically a successul one and it has laid a foundation for the subsequent screening ofthe other conotoxins.