title: Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.
abstract: 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 methionine 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 microg/kg, approximately 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 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.
authors.affiliation: Bachem Bioscience Inc., King of Prussia, Pennsylvania, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Pennington
authors.forename: C
authors.initials: C
authors.lastname: Beeton
authors.forename: C A
authors.initials: CA
authors.lastname: Galea
authors.forename: B J
authors.initials: BJ
authors.lastname: Smith
authors.forename: V
authors.initials: V
authors.lastname: Chi
authors.forename: K P
authors.initials: KP
authors.lastname: Monaghan
authors.forename: A
authors.initials: A
authors.lastname: Garcia
authors.forename: S
authors.initials: S
authors.lastname: Rangaraju
authors.forename: A
authors.initials: A
authors.lastname: Giuffrida
authors.forename: D
authors.initials: D
authors.lastname: Plank
authors.forename: G
authors.initials: G
authors.lastname: Crossley
authors.forename: D
authors.initials: D
authors.lastname: Nugent
authors.forename: I
authors.initials: I
authors.lastname: Khaytin
authors.forename: Y
authors.initials: Y
authors.lastname: Lefievre
authors.forename: I
authors.initials: I
authors.lastname: Peshenko
authors.forename: C
authors.initials: C
authors.lastname: Dixon
authors.forename: S
authors.initials: S
authors.lastname: Chauhan
authors.forename: A
authors.initials: A
authors.lastname: Orzel
authors.forename: T
authors.initials: T
authors.lastname: Inoue
authors.forename: X
authors.initials: X
authors.lastname: Hu
authors.forename: R V
authors.initials: RV
authors.lastname: Moore
authors.forename: R S
authors.initials: RS
authors.lastname: Norton
authors.forename: K G
authors.initials: KG
authors.lastname: Chandy
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005260:Female; D006801:Humans; D051664:Kv1.3 Potassium Channel / Q000037:antagonists & inhibitors* / Q000502:physiology; D010455:Peptides / Q000138:chemical synthesis* / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000138:chemical synthesis* / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D011917:Rats, Inbred Lew; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*
chemical_list: D051664:Kv1.3 Potassium Channel; D010455:Peptides; D026902:Potassium Channel Blockers