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. 2013 Nov 7;8(11):e78712.
doi: 10.1371/journal.pone.0078712. eCollection 2013.

A potent and selective peptide blocker of the Kv1.3 channel: prediction from free-energy simulations and experimental confirmation

M Harunur Rashid  1 , Germano Heinzelmann, Redwan Huq, Rajeev B Tajhya, Shih Chieh Chang, Sandeep Chhabra, Michael W Pennington, Christine Beeton, Raymond S Norton, Serdar Kuyucak
Affiliations

A potent and selective peptide blocker of the Kv1.3 channel: prediction from free-energy simulations and experimental confirmation

M Harunur Rashid et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: MWP and CB are inventors on a patent (WO2006042151A2) claiming ShK-186 and ShK-192 for immunomodulation. This patent was licensed to Kineta Inc. for developing ShK-186 as a therapeutic for autoimmune diseases. MWP and CB are consultants to Kineta Inc. CB is an academic editor for PLOS ONE. MWP is employed by Peptides International. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. The thermodynamic cycle used in the free energy calculations.
The superscript 0 denotes amino acids with no charges on the side chain atoms.
Figure 2
Figure 2. The K18A mutation does not change the binding modes of ShK to Kv1.1 and Kv1.3 channels.
Snapshots of ShK[K18A] (yellow with colored side chains) and ShK (transparent orange) in complex with Kv1.1 and Kv1.3 are superposed to show that there is a good overlap between the toxin backbones, and all the important interactions identified in the ShK complexes are preserved in the ShK[K18A] complexes. To give the full picture, two views of the cross sections of the complex, depicting the monomers C and A (left panel) and D and B (right panel) are presented. Only the residues involved in binding are indicated explicitly.
Figure 3
Figure 3. Convergence of the FEP and TI calculations.
(A) Convergence of the FEP calculations for discharging/charging of the K18 in ShK in the binding site/bulk. Running averages of ΔGi for windows at λ = 0 (black), λ = 0.2 (red), λ = 0.4 (green), λ = 0.6 (blue), λ = 0.8 (yellow) and λ = 0.999 (magenta) are plotted as a function of the production time for Kv1.1 (left) and Kv1.3 (right). (B) Similar to A but showing the convergence of the FEP calculations for transformation of the uncharged Lys side chain to that of Ala. (C) Convergence of the TI calculations for discharging/ charging of K18 in ShK in the binding site/bulk. Running averages of the ΔG values obtained from the TI calculations are plotted as a function of the production time for Kv1.1 (left) and Kv1.3 (right). Both the forward (black) and the negative of the backward (red) results are shown to check for hysteresis effects, which remain well below 1 kcal/mol for both channels.
Figure 4
Figure 4. Comparison of the PMFs for the unbinding of ShK and ShK[K18A] from the Kv1.1 and Kv1.3 channels.
Figure 5
Figure 5. ShK[K18A] is selective for Kv1.3 over Kv1.1 channels and preferentially targets TEM lymphocytes in vitro and in vivo.
(A) Effects of ShK (▪) and ShK[K18A] (○) on Kv1.3 currents measured by whole-cell patch-clamp on L929 fibroblasts stably transfected with mKv1.3. The two left panels show whole-cell Kv1.3 currents before (control) and after perfusion of ShK[K18A] (left panel) or ShK (middle panel). The panel on the right shows the Kv1.3 currents remaining after steady-state block is reached with the different concentrations of ShK and its analog, fitted to a Hill equation (N = 5–6 cells per concentration).(B) Effects of ShK (▪) and ShK[K18A] (○) on Kv1.1 currents measured by whole-cell patch-clamp on L929 fibroblasts stably transfected with mKv1.1. The two left panels show whole-cell Kv1.1 currents before (control) and after perfusion of ShK[K18A] (left panel) or ShK (middle panel). The panel on the right shows the Kv1.1 currents remaining after steady-state block is reached with the different concentrations of ShK and its analog, fitted to a Hill equation (N = 6–7 cells per concentration).(C) Effects of ShK (▪) and ShK[K18A] (○ and dashed line) on the proliferation of rat TEM cells measured in vitro by the incorporation of [3H] thymidine into the DNA of dividing cells (N = 3). (D) Effects of ShK (▪) and ShK[K18A] (○ and dashed line) on the proliferation of rat splenocytes (mainly naïve/TCMcells; N = 3).(E) Effects of the subcutaneous administration of 100 µg/kg ShK[K18A] on an active DTH reaction elicited against ovalbumin. Data show the difference in challenged and non-challenged ear thickness in vehicle-treated rats and ShK[K18A]-treated rats (N = 6/group).
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