A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases

Abstract

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2 kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.

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

“…The binding free energy changes due to the R14A mutation obtained using the FEP and TI methods are compared to those obtained from the PMF calculations and experiment in Table 4. The FEP and TI results are in good agreement with that of PMF, and all the computational results are consistent with the experimental 39 Thus, using the FEP/TI method described here, it is possible to calculate accurately the free energy change due to a charge mutation in a protein-ligand complex. Preservation of the binding mode appears to be the only requirement for the success of such calculations.…”

“…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. 39 In this study, FEP and TI calculations were not performed because there was some change in the binding mode. Here we investigate in detail the R29A mutation in ShK and the R14A mutation in HsTx1 to examine under which conditions the FEP/TI calculations can be expected to predict the changes in the binding free energies due to mutations.…”

“…For each contribution, we give the results of the forward and minus the backward transformations and their average. The LJ contributions in TI results are taken from the FEP calculations.observation,39 indicating more than 2.7 kcal/mol gain in Kv1.3/Kv1.1 selectivity margin (we note that the R14A mutation has a negligible e®ect on the binding free energy of HsTx1 to Kv1.3. )…”

Enabling Noninvasive Systemic Delivery of the Kv1.3-Blocking Peptide HsTX1[R14A] via the Buccal Mucosa

“…12 After completion of this resin bound HsTX1[R14A] sequence, the resin was split in half and either processed to generate the folded purified HsTX1[R14A] or attached to a mini-PEG spacer followed by a coupling of 5-FITC to the N-terminal amino function. Use of the spacer is necessary to prevent Edman-like degradation of an a-amino acid caused by adding FITC onto the N-terminus.…”

“…9,10 An analogue of HsTX1, HsTX1[R14A], has been recently developed, that not only retains the high affinity of the naturally occurring HsTX1 but also exhibits an approximate 2000-fold greater selectivity for Kv1.3 channels over other potassium channels. 11,12 Thus, HsTX1[R14A] represents another promising potential therapeutic candidate for the treatment of autoimmune diseases.…”