Structural basis of the function of peptide inhibitors targeting voltage-gated sodium channel

Abstract

Pain is defined as an unpleasant sensory and emotional experience associated with potential or actual tissue damage. Chronic pain affects nearly one-fourth of the world’s population - leading to severe social, psychological, and biological distress. Pain medications demonstrate various side-effects and can also lead to drug abuse and drug intolerability. Therefore, with the objective of designing efficient analgesics or painkillers, recent studies have focused on understanding the mechanism-of-action of the voltage-gated sodium channel, subtype 1.7 (NaV1.7) – which is a genetically validated target of numerous pain medications.The development of analgesics has been challenging owing to limited knowledge of the mechanism-of-action of NaV1.7 inhibitors and the NaV1.7 channel. Moreover, high sequence similarity between different subtypes of NaV channels (NaV1.1- NaV1.9) further prevents the design of a subtype-selective inhibitor that targets NaV1.7 only. Finally, on achieving subtype-selectivity towards NaV1.7, another challenge in the drug development process is to increase the in vivo half-life of small molecule or peptide inhibitors.Recently, in a decade long quest of a potent and selective NaV1.7 inhibitor – a disulfide-rich venom peptide (Pn3a) from the South American tarantula was reported. Pn3a targets NaV1.7 with nanomolar potency and thus, it can be utilized as a tool to characterise the pharmacological behaviour of NaV1.7. The primary aim of this thesis was to understand the mechanism-of-action of the NaV1.7 channel through the prism of Pn3a. Moreover, to counter the issue of short in vivo half-life of Pn3a, I designed a strategy to bioconjugate Pn3a with a large polypeptide (XTEN), that in turn can increase the bioavailability of Pn3a.In chapter 2 of this thesis, I discussed a bioinformatic and rational engineering strategy to predict the pharmacophore of NaV1.7 inhibitors. The information gained from this rational engineering approach can be further utilized for designing novel peptide inhibitors. I demonstrated that hydrophobic residues at positions 4 and 5 of a peptide sequence from a family of sodium spider toxins (NaSpTx1) are critical for the inhibitory activity towards NaV1.7.Previously, it has been suggested that lipid bilayer plays an important role in the interaction of a NaV1.7 inhibitor with the NaV1.7 channel. Therefore, in chapter 3, I focused on understanding the role of the lipid bilayer in the mechanism-of-action of NaV1.7. Chapter 3 summarizes the lipid-binding behaviour of various NaV1.7 modulators. Previously, Pn3a was produced using solid-phase peptide synthesis which is an inefficient approach for large-scale production of peptides. Thus, chapter 4 discussed a recombinant technology approach to produce Pn3a. In this chapter, I also elucidated the lipid-binding interface of Pn3a using Nuclear Magnetic Resonance (NMR) and Isothermal Titration Calorimetry (ITC) experiments. These biophysical techniques (NMR and ITC) characterized the importance of the C-terminal of Pn3a in the mechanism-of-action of Pn3a.Chapter 5 focused on the method development to produce voltage-sensing domains (VSDs) of NaV channels. VSDs are functionally independent units of a sodium channel. A cell-free production methodology was utilized to express VSDs of interest. Subsequently, VSDs were incorporated in a lipid bilayer model for imparting long-term stability to VSDs. The methodology developed in this chapter lays down the foundation for characterizing the molecular interactions of Pn3a and NaV1.7. Finally, chapter 6 of this thesis discussed a bioconjugation approach for increasing the in-vivo half-life of Pn3a.To summarize, I utilized Pn3a as a template to understand the subtype-selectivity of a disulfide-rich peptide towards NaV1.7 along with designing a novel and generic strategy to increase the in vivo half-life of a disulfide-rich peptide. Therefore, this thesis provides crucial information for designing a peptide-based inhibitor that targets NaV1.7 and thus further opens multiple avenues to design novel therapeutics for alleviating chronic pain.