A Potent and Selective Peptide Blocker of the Kv1.3 Channel: Prediction from Free-Energy Simulations and Experimental Confirmation

Abstract

The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.

CHAPTER 10. Case Study 2: Transforming a Toxin into a Therapeutic: the Sea Anemone Potassium Channel Blocker ShK Toxin for Treatment of Autoimmune Diseases

“…42 Findings over the past decade also point to a role of Kv1.3 channels in the regulation of peripheral insulin sensitivity and glucose metabolism. 43,44 In addition to their potential application as immunomodulators, ShK and its analogues may therefore prove effective for the treatment of insulin resistance and type 2 diabetes.…”

Calculation of free energy changes due to mutations from alchemical free energy simulations

“…25 For the neutral Ala side chain (the third term in Eq. (3)), using half of these times is found to be su±cient.…”

“…These problems may be resolved by performing the mutation on the ligand in the binding site and bulk simultaneously. 25 That is, while the ligand is mutated to an analogue in the binding site, the reverse mutation is performed simultaneously on the analogue in bulk water in the same system. Finally, the force¯eld used in the FEP/ TI calculations could lead to inaccuracies.…”

“…In particular, the e®ect of the K18A mutation on the binding free energy of ShK was studied using the FEP, TI and PMF methods, and the predicted gain in selectivity free energy was con¯rmed in subsequent experiments. 25 Another mutation suggested in ShK (R29A), 37 was not pursued because it changed the binding mode. Similarly, the R14A mutation in HsTx1 was predicted to improve the Kv1.3/Kv1.1 selectivity from PMF calculations, which was con¯rmed in experiments.…”

Kv1.3 Selective Peptides Based Upon N-Terminal Extension and Internal Substitutions of ShK Toxin

“…The Q16K substitution coupled with either the Ppa-Aeea or EWSS N-terminal extensions confers the greatest level of selectivity enhancement as shown in the ShK-223, ShK-224 and ShK-237. The K18A coupled with the EWSS N-terminal extension lost more than 10x of its gain of the singly substituted ShK-K18A [5].…”