Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.
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.
Pennington
M W
MW
Bachem Bioscience Inc., King of Prussia, Pennsylvania, USA.
Beeton
C
C
Galea
C A
CA
Smith
B J
BJ
Chi
V
V
Monaghan
K P
KP
Garcia
A
A
Rangaraju
S
S
Giuffrida
A
A
Plank
D
D
Crossley
G
G
Nugent
D
D
Khaytin
I
I
Lefievre
Y
Y
Peshenko
I
I
Dixon
C
C
Chauhan
S
S
Orzel
A
A
Inoue
T
T
Hu
X
X
Moore
R V
RV
Norton
R S
RS
Chandy
K G
KG
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*
D051664:Kv1.3 Potassium Channel; D010455:Peptides; D026902:Potassium Channel Blockers