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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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Sequences of ShK analogs described in this study.
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marinedrugs-13-00529-f003: Sequences of ShK analogs described in this study.

Mentions: A series of analogs was designed to probe the Q16K substitution along with other selectivity determinants. ShK-223 and ShK-224 (Figure 3) were designed to include a substitution that a group at Amgen had published in a patent filed in 2007; this substitution replaced Gln16 with Lys [36]. The results presented in that patent suggested that this substitution conferred on ShK a high Kv1.3 versus Kv1.1 specificity. Thus, we incorporated this substitution into ShK-192, which incorporated a non-hydrolyzable para-phosphono-Phe as the N-terminal residue, extended from the primary ShK sequence with an Aeea (aminoethyloxyethyloxyacetyl, mini-Peg™) linker, as well as amidation at the C-terminus (ShK-224). We also incorporated a Met21 to Nle substitution into this sequence to generate an analog that would be less susceptible to oxidation (ShK-223). Additionally, we recombinantly expressed two additional analogs, both with C-terminal Ala additions and the Q16K substitution (ShK-234), and one with Met21 replaced with Ile (ShK-235).


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)

Sequences of ShK analogs described in this study.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-00529-f003: Sequences of ShK analogs described in this study.
Mentions: A series of analogs was designed to probe the Q16K substitution along with other selectivity determinants. ShK-223 and ShK-224 (Figure 3) were designed to include a substitution that a group at Amgen had published in a patent filed in 2007; this substitution replaced Gln16 with Lys [36]. The results presented in that patent suggested that this substitution conferred on ShK a high Kv1.3 versus Kv1.1 specificity. Thus, we incorporated this substitution into ShK-192, which incorporated a non-hydrolyzable para-phosphono-Phe as the N-terminal residue, extended from the primary ShK sequence with an Aeea (aminoethyloxyethyloxyacetyl, mini-Peg™) linker, as well as amidation at the C-terminus (ShK-224). We also incorporated a Met21 to Nle substitution into this sequence to generate an analog that would be less susceptible to oxidation (ShK-223). Additionally, we recombinantly expressed two additional analogs, both with C-terminal Ala additions and the Q16K substitution (ShK-234), and one with Met21 replaced with Ile (ShK-235).

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.

Show MeSH
Related in: MedlinePlus