Development of NaV1.1-selective agonists with potential for treatment of Dravet syndrome epilepsy

Abstract

This thesis describes the functional and structural characterisation of several novel peptides that modulate the activity of voltage-gated sodium channel 1.1 (NaV1.1), a putative target for the development of therapeutics to treat Dravet syndrome (DS) epilepsy.Voltage-gated sodium (NaV) channels serve a fundamental role in carrying electrical signals throughout the body. Inherited or acquired dysfunction of NaVchannels is associated with a number of human diseases, including epilepsy, cardiac arrhythmia, and chronic pain conditions. The NaV1.1 subtype, which is expressed in peripheral sensory neurons and fast-spiking GABAergic inhibitory interneurons in the brain, governs the proper excitation/inhibition balance in neuronal networks. Consequently, heterozygous loss-of-function mutations in SCN1A, the gene encoding the ion conducting α-subunit of the NaV1.1, lead to DS. DS is a rare, pharmacoresistant childhood epilepsy characterised by early-onset seizures, cognitive deficit, autistic-like behaviours, and premature death. The NaV1.1 haploinsufficiency in DS disturbs the normal balance of excitation and inhibition in the brain. We propose that this deficit could be ameliorated by a drug that selectively activatesthe smaller-than-normal population of functional NaV1.1 channels in DS interneurons, thus restoring their ability to regulate brain excitability. The overall aim of this project was to characterise novel NaV1.1-selective agonists in order to provide drug leads with potential for treatment of DS epilepsy.Designed by Nature as chemical weapons, arthropod venoms have proven to be an untapped source of ion channel modulators that can be repurposed as pharmacological tools for studying these large integral membrane proteins, and as leads for the development of new therapeutics. Recently, two disulfide-rich peptides (Hm1a and Hm1b) were isolated from the venom of the tarantula Heteroscodra maculata, and shown to specifically activate NaV1.1. A previous study demonstrated that intracerebroventricular (ICV) infusion of Hm1a restores the function of inhibitory interneurons in SCN1Aknockout mice by selectively activating NaV1.1 channels (Richards et al., 2018). However, somewhat surprisingly for a disulfide-rich venom peptide, Hm1a was poorly stable in cerebrospinal fluid (CSF) with a half-life of ~1.7 h. Given the limited amount of crude H. maculatavenom, Chapter Two describes the optimisation of recombinant production of correctly folded Hm1b (rHm1b). Despite the high degree of sequence similarity between Hm1a and Hm1b, rHm1b is much more stable than Hm1a in both human serum and human CSF (t1/2>40 h and >70 h, respectively). Furthermore, structural studies indicated that rHm1b exhibits an inhibitor cystine knot (ICK) motif. By comparing the molecular surface of Hm1a and rHm1b, we identified a conserved hydrophobic patch in these toxins that likely mediates their interaction with the NaV1.1 channel. Although in vivo studies have thus far focused on Hm1a, the easier production and higher in vivostability of rHm1b suggest that it might represent the better molecule to progress towards human clinical trials.Chapter Three explores the pharmacological profiles of native and recombinant versions of Hm1b on human NaV1.1–1.7 channel subtypes expressed in a mammalian-cell background using an automated whole-cell patch-clamp electrophysiology system. rHm1b is a highly potent and selective modulator of NaV1.1 and NaV1.3 in the low nanomolar range. However, rHm1b potentiates NaV1.3 tolesser extent than NaV1.1, and induces a smaller sustained current. Electrophysiology studies have shown that at any given potential within the physiologically relevant range, rHm1b promotes NaV1.1 activation at the resting membrane potential and inhibits fast inactivation, indicating that rHm1b stably traps the domain IV voltage sensors of NaV1.1 and NaV1.3 in the partially activated state, where the channels would maintain a persistent open state but not sufficient to trigger inactivation.In addition to the studies on rHm1b, Chapter Four describes the isolation and characterisation of two novel peptides (Hj1a and Hj1b) from venom of the scorpion Hottentotta jayakari. The primary structures of these two peptides were determined by analysis of a venom-gland transcriptome and proteomic data. Sequence alignments revealed a high degree of similarity to previously reported α-scorpion toxins. Recombinant peptides were produced, and their pharmacological profiles were characterised by electrophysiology measurements on human NaV1.1–1.7 channel subtypes. Hj1a and Hj1b were found to modulate NaV1.1 via a mechanism similar to that of classic gating-modifier toxins by enhancing channel activation and inhibiting fast inactivation. Both peptides also demonstrate diverse agonistic activity on NaV1.4–1.7 in the low nanomolar range. The original aims of this work were in part achieved, although the NaV1.1 agonists we found are not subtype-selective.Collectively, it is anticipated that future structure-activity relationship studies will facilitate rational engineering of peptide variants with improved potency and selectivity for NaV1.1.