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

Pennington MW, Chang SC, Chauhan S, Huq R, Tajhya RB, Chhabra S, Norton RS, Beeton C - Mar Drugs (2015)

Bottom Line: In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus.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.

View Article: PubMed Central - PubMed

Affiliation: Peptides International Inc., 11621 Electron Drive, Louisville, KY 40065, USA. mpennington@pepnet.com.

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.

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(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).
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marinedrugs-13-00529-f006: (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).

Mentions: We used patch-clamp electrophysiology to assess the effects of ShK-223, ShK-224, ShK-234 and ShK-235 on Kv1.1 and Kv1.3 channels and compare them to the parent peptide, ShK. Mouse fibroblasts stably expressing homotetramers of Kv1.1 or Kv1.3 were patch-clamped in the whole-cell configuration and steady-state block was measured after addition of different concentrations of the peptides. As published previously, ShK inhibited Kv1.3 currents with an IC50 of 13.3 ± 1.40 pM and Kv1.1 currents with an IC50 of 21.5 ± 2.26 pM (Figure 5, Table 1) [15,36]. ShK-224 exhibited a 12-fold loss of efficacy on Kv1.3 relative to ShK, ShK-235 a 5-fold loss, and ShK-234 a 2-fold loss, whereas the potency of ShK-223 on Kv1.3 was similar to that of ShK (Figure 5 and Figure 6, Table 1). In contrast to ShK, ShK-234 exhibited a 580-fold selectivity for Kv1.3 over Kv1.1, and ShK-235 a 2250-fold selectivity, while neither ShK-223 nor ShK-224 had any effect on Kv1.1 currents at concentrations up to 100 nM, representing a selectivity for Kv1.3 of >10,000-fold.


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

Pennington MW, Chang SC, Chauhan S, Huq R, Tajhya RB, Chhabra S, Norton RS, Beeton C - Mar Drugs (2015)

(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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306950&req=5

marinedrugs-13-00529-f006: (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).
Mentions: We used patch-clamp electrophysiology to assess the effects of ShK-223, ShK-224, ShK-234 and ShK-235 on Kv1.1 and Kv1.3 channels and compare them to the parent peptide, ShK. Mouse fibroblasts stably expressing homotetramers of Kv1.1 or Kv1.3 were patch-clamped in the whole-cell configuration and steady-state block was measured after addition of different concentrations of the peptides. As published previously, ShK inhibited Kv1.3 currents with an IC50 of 13.3 ± 1.40 pM and Kv1.1 currents with an IC50 of 21.5 ± 2.26 pM (Figure 5, Table 1) [15,36]. ShK-224 exhibited a 12-fold loss of efficacy on Kv1.3 relative to ShK, ShK-235 a 5-fold loss, and ShK-234 a 2-fold loss, whereas the potency of ShK-223 on Kv1.3 was similar to that of ShK (Figure 5 and Figure 6, Table 1). In contrast to ShK, ShK-234 exhibited a 580-fold selectivity for Kv1.3 over Kv1.1, and ShK-235 a 2250-fold selectivity, while neither ShK-223 nor ShK-224 had any effect on Kv1.1 currents at concentrations up to 100 nM, representing a selectivity for Kv1.3 of >10,000-fold.

Bottom Line: In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus.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.

View Article: PubMed Central - PubMed

Affiliation: Peptides International Inc., 11621 Electron Drive, Louisville, KY 40065, USA. mpennington@pepnet.com.

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.

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Related in: MedlinePlus