Targeting the hKv1.3 potassium channel in the development of novel treatments for autoimmune diseases

Raymond Tang, James Milnes, Laurence Louis, Jenny Wilson, Wojciech Piotrowski, Sarah Eastwood, Simon Jones, Mushtaq Mulla, Richard Hamlyn, David Madge, John Ford
Xention Ltd., Cambridge, United Kingdom

Human T-cells express two populations of potassium (K+) ion channels, the voltage-gated Kv1.3 and the Ca2+-dependent IKCa1 (review Panyi 2005). Kv1.3 and IKCa1 play a critical role in setting and maintaining a negative membrane potential which facilitates Ca2+ entry through Ca2+ release-activated Ca2+ channels (CRAC). Sustained elevation of cytosolic [Ca2+] following cell activation results in gene transcription, cytokine secretion and proliferation. It has been shown that central memory T-cells (TCM) predominantly express the IKCa1 subtype following acute activation, whereas effector memory T-cells (TEM) substantially up-regulate and express Kv1.3 following activation. Autoreactive TEM cells are implicated in the pathogenesis of a variety of autoimmune (AI) diseases. Proof-of-concept studies have demonstrated Kv1.3 blockers to be effective in the treatment of a range of T-cell mediated AI disease animal models (Beeton et al., 2006). Current therapies for AI diseases are often associated with poor efficacy, inconvenient administration and uncertain long-term safety profiles (Goldbach-Mansky & Lipsky 2003). Consequently there is a substantial and timely need for safe, effective treatments. Kv1.3 represents a novel therapeutic target for the treatment of AI diseases. Xention’s Kv1.3 programme is aimed at developing a small molecule Kv1.3 blocker for AI diseases. Screening of a proprietary compound library identified multiple active lead series that inhibit Kv1.3 at sub-micromolar concentrations. Compound XEN-3929 represents an example compound with favourable physiochemical properties from one such active series following lead optimisation. It was the aim of this study to investigate how this compound interacts with the Kv1.3 channel. Whole-cell patch-clamp experiments were performed at room temperature using either a hKv1.3 or hERG expressing CHO cell line. XEN-3929 potently inhibits hKv1.3 current (IKv1.3) with an IC50 of 38.6 ± 12.1 nM (mean ± SEM, n = 16 cell). XEN-3929 exhibited greater than 100-fold selectivity for Kv1.3 over hERG. PAP-1, the most potent small molecule Kv1.3 inhibitor published to date (Schmitz et al., 2005) was used a reference compound and was shown to inhibit IKv1.3 with an IC50 of 10.7 nM (95% CI 8.2-13.9nM, n=10 cells). Following a train of voltage-clamp steps (-80 to +40 mV, 500 ms) to elicit IKv1.3 in control solution, either XEN-3929 or PAP-1 was applied for 60 s while the cell was held at -80 mV to maintain the channels in the closed state before repeating the voltage train in the presence of compound. Peak Kv1.3 current of the 1st and 2nd pulses in the presence of XEN-3929 were inhibited by -2 ± 3 % and 73 ± 3 % (n=3 cells) respectively and 7.9 ± 2.9 % and 81.7 % for PAP-1 respectively. The effect of XEN-3929 on the activation/inactivation of Kv1.3 was also examined. XEN-3929 produced a significant rightward shift in the Kv1.3 conductance-voltage (g-v) relationship (t-test P < 0.001, n = 5) and a significant leftward shift in the apparent V1/2 of the IKv1.3 inactivation-voltage relationship (t-test P < 0.001, n = 6). In conclusion XEN-3929 is a novel, potent, Kv1.3 inhibitor with a good cardiac safety profile

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Goldbach-Mansky & Lipsky (2003), Annu Rev Med. 54,197-216
Panyi (2005) Eur Biophys J.34,515-29
Schmitz et al. (2005) Mol Pharmacol. 68,1254-70