Development of highly selective Kv1.3-blocking peptides based on the sea anemone peptide ShK

Abstract

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC50s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.

Figure 1

Sequence alignment of K⁺ channel blocking peptides from sea anemones. The GenBank accession codes are also indicated. Alignment of the sequences is via the Cysteine residues which are shaded in gray with the pairings indicated by the lines linking them. The yellow highlighted areas are indicating sequence identity. The cyan colored residues indicate the key pore blocking residues. The green region with the (conserved Gly residue in pink) represents a sequence insertion present in the BgK-type anemone toxins.

Figure 2

The solution structure of ShK in blue (PDB 1ROO) (A) compared with the scorpion toxin kaliotoxin in dark red (PDB 2UVS) (B). The side chains of critical residues are shown in stick format and colored navy blue (Arg), orange-red (Gln), magenta (Tyr), light-blue (Lys) and orange (Met).

Figure 3

Sequences of ShK analogs described in this study.

Figure 4

(A) RP-HPLC analysis of oxidative folding of ShK-223 after overnight folding (gradient conditions 10%–70% B in 30 min, 1 mL/min, A²²⁰ (absorbance at 220 nm), 0.1 AUFS (absorbance full scale) (B) RP-HPLC analysis of the purified ShK-223; (C) ESI-MS analysis of the multi-charged ions from ShK-223; (D) RP-HPLC analysis of oxidative folding of ShK-224 after overnight folding; (E) RP-HPLC analysis of the purified ShK-224; (F) ESI-MS analysis of the multi-charged ions from ShK-224. (Asterisk indicates correctly folded ShK peptides).

Figure 5

(A) Whole-cell Kv1.3 (left) and Kv1.1 (right) currents before (control) and after addition of ShK-223; (B) Whole-cell Kv1.3 (left) and Kv1.1 (right) currents before (control) and after addition of ShK-224; (C) Dose-response effects of ShK (▲), ShK-223 (●), and ShK-224 (○) on Kv1.3 (left) and Kv1.1 (right) currents (n = 3 to 6 cells per concentration).

Figure 6

(A) Whole-cell Kv1.3 (left) and Kv1.1 (right) currents before (control) and after addition of ShK-234; (B) Whole-cell Kv1.3 (left) and Kv1.1 (right) currents before (control) and after addition of ShK-235; (C) Dose-response effects of ShK-234 (▲) and ShK-235 (●) on Kv1.3 (left) and Kv1.1 (right) currents (n = 3 to 4 cells per concentration).