Isomers Synthesize And Optimal Oxidative Folding Of α-Conotoxin TxIB As A Therapeutic Candidate Peptide

Neuronal nicotinic acetylcholine receptors (nAChRs) play a crucial role in central and peripheral nervous system and development of many neuropsychiatric diseases. Depending on specific receptor composition;, nAChR receptor subtypes have distinct pharmacological properties and physiological functions. Alpha6beta2nAChRs are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson’s disease.a-conotoxins (a-CTxs) are12-20amino acid disulfide-constrained peptides that target diverse nAChR subtypes. Prior research has identified a-CTxs as important research tools, both for studying diverse pharmacological disorders and for developing new therapeutics for treating addiction, neuropathic pain and other neuropathological conditions. Disulfide bonds of native a-CTx isomers are usually connected in a (CysⅠ-Ⅲ, CysⅡ-Ⅳ)"globular" arrangement. In contrast, an alternative "ribbon" arrangement has CysI-IV, CysⅡ-Ⅲ bonding, while a "beads" arrangement has CysⅠ-Ⅱ, CysⅢ-Ⅳ bonding. a-CTx activity against specific targets is highly dependent on cysteine bond arrangement.a-CTx TxIB is a uniquely selective ligand, which blocks a6/a3β2(33nAChRs only, but does not block the other subtypes. Therefore, a-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough a-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized a-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of a-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each a-CTx TxIB isomers’ yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH4HCO3or Tris-HCl folding buffer at4℃contained40%of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH8-8.7.