Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes

Michael W Pennington, Christine Beeton, Charles Galea, Brian J Smith, Victor Chi, Kevin P Monaghan, Adriana Garcia, Srikant Rangaraju, Alfred Giuffrida, Denise K Plank, Geroge Crossley, Daniel Nugent, Ilya Khaytin, Yann LeFievre, Irina Peshenko, Catherine Dixon, Satendra Chauhan, Alicia Orzel, Taeko Inoue, Xueyou HuRebecca V Moore, Raymond Stanley Norton, K George Chandy

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Kv1.3 potassium channels maintain the membrane potential of effector memory (T EM) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a me- thionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys 411 of the channel. ShK-192 blocks Kv1.3 with an IC 50 of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 ?g/kg, 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T EM cells and suppresses delayed type hypersensitivity when administered at 10 or 100 ?g/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T EM cells
Original languageEnglish
Pages (from-to)762 - 773
Number of pages12
JournalMolecular Pharmacology
Issue number4
Publication statusPublished - 2009
Externally publishedYes

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