Disulfide-rich peptides in the venom of spiders and scorpions are highly active and selective against various ion channels. Besides, they have high resistance to digestive enzymes, making them a promising basic scaffold for peptide drugs. The PERISS (intra periplasm secretion and selection) is a peptide selection method based on evolutionary molecular engineering using periplasmic display technology, which enables rapid screening of disulfide-rich peptides (DRPs) that bind to an ion channel.
In this study, we conducted to discover novel DRPs that inhibit a human voltage-gated sodium channel Nav1.7, which plays a key role in regulating peripheral pain, to develop novel pain therapeutic peptide drugs.
In order to conduct the PERISS screening, gene constructs of a chimeric human potassium channel Kv2.1 in which each voltage-sensing domain of Nav1.7 was transferred to Kv2.1 were prepared and expressed on the inner membrane of E. coli. The disulfide-rich peptide GTx1-15 derived from tarantula venom was used as the scaffold of the DRP library, and its genetic library was constructed using in silico calculation based on the three-dimensional structural model. Next, we performed the PERISS screening to identify Nav1.7-binding DRP sequences using these gene constructs. Then, the activity of the hit DRPs identified by the PERISS method was measured by the two-electrode voltage fixation method using an oocyte system of Xenopus laevis, we identified the DRPs with Nav1.7 inhibitory activity.
The PERISS method we have developed could provide novel active DRPs against various ion channels shorter than the conventional method that screens a chemical library with a limited number of DRP variants.