The voltage-gated sodium channel, Nav1.7, is involved in the propagation of pain signals from the peripheral nervous system. Genomic data from individuals with non-functional Nav1.7 expression strongly suggest it has potential to be the target of novel analgesics; loss of Nav1.7 function completely abolishes pain sensations in otherwise healthy phenotypes.
The focus of this thesis is the development of chemical tools to elucidate mechanisms of Nav1.7 modulation in the cell. The design, synthesis, characterisation and potency data of photocrosslinking probes that target two distinct Nav1.7 domains is reported. Domain II is targeted by photoprobes derived from the spider venom inhibitory cystine knot peptide Huwentoxin-IV. Moreover, a photoprobe based on the novel family of Nav1.7-selective aryl sulfonamide inhibitors targets domain IV of Nav1.7. Determining the binding sites that lead to modulation of gating was firstly attempted in bacterial/hNav1.7 chimeric proteins that have been purified and used for crystallographic and biophysical studies. According to gel shift assays, certain photoprobes exhibited efficient photocrosslinking capabilities and
were taken forward to proteomic mass spectrometry analysis in pursuit of photocrosslinking sites. Additionally,
a series of approaches were explored in order to optimise the identification of Nav1.7 by proteomic mass spectrometry in an engineered cell line. Finally, the maturation of induced pluripotent stem cells from patients that carry a Nav1.7 mutation was followed by quantitative proteomics as an initial approach to understand Nav1.7-related mechanisms in a disease model.