*3.1. ShK Toxin*

The Kv1.3 channel is an established target for the treatment of autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis [67,68]. ShK toxin from the sea anemone Stichodactyla helianthus [69,70] binds to the Kv1.3 channel with an IC50 of 11 pM [71], hence it provides an excellent lead for development of an immunosuppressant drug. However, ShK also binds to other Kv channels with high affinity, in particular Kv1.1 with an IC50 of 16 pM [71]. Lack of specificity for Kv1.3 prevents the use of ShK as a therapeutic drug. Therefore, there is an intense effort for developing analogs of ShK that have improved selectivity for Kv1.3 over Kv1.1 and other potassium channels [9,10]. Many ShK analogs have been created for this purpose, and some had the required selectivity, e.g., ShK-Dap22 [71], ShK-186 [72], and ShK-192 [73]. However, the use of non-natural amino acids or adducts in these analogs has limited their potential for development as drugs. For example, the phosphorylated Tyr residue in ShK-186 is prone to hydrolysis. Therefore, developing Kv1.3 selective analogs of ShK from natural amino acids is more desirable.

**Figure 2.** Snapshots of the Kv1.1–ShK and Kv1.3–ShK complexes showing the strongly interacting residues. Only those involved in the binding are indicated explicitly. In order to show all the residues involved in binding, two views of the complex are presented depicting the channel monomers A and C, and B and D separately.

An essential first step in searching for selective analogs using structure-based drug design methods is to construct accurate models of the protein-ligand complexes involved in the selectivity problem. This has been recently achieved for the Kv1.x–ShK complexes using docking and molecular dynamics (MD) simulations [59]. Snapshots of the equilibrated Kv1.1–ShK and Kv1.3–ShK complexes are shown in Figure 2. A more quantitative description of the strongly interacting residues is provided in Table 2, which shows the average atom-atom distances obtained from 5 ns of unrestrained MD simulations for each complex. Comparison of the results for the Kv1.3–ShK complex with the alanine scanning mutagenesis data [74] shows that the complex model accounts for all the strongly interacting residues identified in the experiment. The only exception is the mutation R24A, which is an allosteric effect. R24 is not involved in the binding but its mutation to alanine breaks the R24–K18 bond (see Figure 1), which changes the shape of the ShK mutant and its binding mode. Further evidence for the validity of the complex models are provided by the binding free energies, which are obtained from the integration of the PMFs shown in Figure 3. In all three cases, the PMF calculations of the binding free energies have reproduced the experimental values within chemical accuracy [59].

**Table 2.** List of the strongly interacting residues in the ShK–Kv1.x complexes. The average atom-atom distances obtained from MD simulations are given in units of Å (standard deviations are not listed as they are less than 0.4 Å). The N–O distances are shown for the charge interactions and the closest C–C distance for the hydrophobic ones. Bare C, N, and O refer to the backbone atoms and the subscripted ones refer to the side chain atoms. The monomer identity is given at the end of the residue number.


**Figure 3.** Comparison of the PMFs obtained from the umbrella sampling simulations for the unbinding of ShK from the Kv1.1, Kv1.2, and Kv1.3 channels.

Comparison of the binding modes of the Kv1.1–ShK and Kv1.3–ShK complexes (Figure 2 and Table 2) gives valuable hints for improving the selectivity of ShK for Kv1.3 over Kv1.1. The side chains of K18 and R29 in ShK are strongly coupled to the glutamate side chains in Kv1.1 via ionic bonds but they do not exhibit any interactions with the Kv1.3 residues. Thus mutation of the either residue in ShK to alanine could lead to a substantial selectivity gain for Kv1.3 over Kv1.1. To test this hypothesis, binding of the K18A and R29A mutants of ShK to the Kv1 channels have been studied employing the same protocols used for ShK. The R29A mutation was found to alter the binding mode in both Kv1.1 and Kv1.3, and therefore was not pursued. The K18A mutation did preserve the binding mode in both channels, and hence the effect of the K18A mutation on the selectivity free energy could be calculated using the FEP and TI methods. Because the validity of these methods for charge mutations was not well established, the binding free energies of ShK-K18A were also calculated as a test of FEP and TI. All three methods gave similar results and predicted that the K18A mutation

would improve the Kv1.3/Kv1.1 selectivity free energy by about 2 kcal/mol, which was confirmed in subsequent experiments.

An amidated analogue of ShK (ShK-K-amide) has been known to improve the Kv1.3/Kv1.1 selectivity but the mechanism of this selectivity was not clear. A similar computational study performed for ShK-K-amide showed that this mutation had negligible effect on its binding to Kv1.3 but grossly altered its binding mode to Kv1.1 [60]. The binding free energies of ShK-K-amide calculated from the respective PMFs again reproduced the experimental values within chemical accuracy, increasing the confidence in the accuracy of the computational methods.
