Novel potassium channel blockers for the treatment of multiple sclerosis and other autoimmune diseases

Multiple sclerosis (MS) is a T-cell mediated autoimmune disease characterized by demyelination of neurons in the central nervous system, resulting in progressive interference with functions that are controlled by the nervous system. The majority of the myelin-reactive T cells isolate from the blood of MS patients are effector-memory (TEM) T cells. TEM cells express high levels of the potassium channel Kv1.3 upon activation. Blockade of this channel by the sea anemone toxin ShK inhibits T-cell proliferation and ameliorates MS symptoms in animal models. ShK is therefore a promising therapeutic peptide. ShK blocks Kv1.3 potently (IC50~11 pM) but also inhibits other closely-related ion channels such as Kv1.1 with similar affinity as a result of high sequence homology in the pore regions of both channels. Analogues of ShK have been developed with increased selectivity for Kv1.3 over the neuronal and cardiac Kv1.1 channel subtypes in order to avoid neurotoxicity and cardiotoxicity. Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, type-1 diabetes and psoriasis. However, ShK analogues suffer from significant limitations owing to the presence of non-protein adducts that are potentially immunogenic and susceptible to hydrolysis in vivo. This thesis explored the designs and development of recombinant Kv1.3-selective analogues, through structural-based approaches. A recombinant peptide expression system in Escherichia coli has been established to enable isotope-labelling of ShK and its analogues in a rapid and cost efficient manner for in-depth biophysical and pharmacological studies. Meanwhile, computational strategies were implemented to predict complexes of novel ShK analogues bound to Kv1.3 and Kv1.1. Molecular dynamic (MD) simulations and electrophysiology experiments were conducted extensively to suggest sites for mutations and generated novel Kv1.3-selective analogues. In the absence of an experimental structure of ShK in complex with Kv1.3, MD simulations were exceedingly useful in visualizing the precise positioning of ShK side chains in relation to the channel turret and vestibule regions. Novel ShK analogues developed in this work maintained high potency and selectivity for Kv1.3 over Kv1.1. The backbone dynamics of ShK was also investigated and mutations of key residues were explored experimentally to probe the binding mechanism of ShK. In addition, novel ShK-like peptides, AcK1 and BmK1, in parasitic worms were identified and characterized. These ShK-like peptides not only share structural similarity with ShK but also selectively suppress the function of TEM cells in vitro and in vivo. This study assists to elucidate the functions of ShK-like peptides and proteins from parasitic helminths. Overall, the Kv1.3-selective the analogues described in this thesis represent promising therapeutic leads for autoimmune diseases.