The voltage-gated Kv1.3 K ϩ channel is a novel target for immunomodulation of autoreactive effector memory T (T EM ) cells that play a major role in the pathogenesis of autoimmune diseases. We describe the characterization of the novel peptide ShK(L5) that contains L-phosphotyrosine linked via a nine-atom hydrophilic linker to the N terminus of the ShK peptide from the sea anemone Stichodactyla helianthus. ShK(L5) is a highly specific Kv1.3 blocker that exhibits 100-fold selectivity for Kv1.3 (K d ϭ 69 pM) over Kv1.1 and greater than 250-fold selectivity over all other channels tested. ShK(L5) suppresses the proliferation of human and rat T EM cells and inhibits interleukin-2 production at picomolar concentrations. Naive and central memory human T cells are initially 60-fold less sensitive than T EM cells to ShK(L5) and then become resistant to the peptide during activation by up-regulating the calcium-activated K Ca 3.1 channel. ShK(L5) does not exhibit in vitro cytotoxicity on mammalian cell lines and is negative in the Ames test. It is stable in plasma and when administered once daily by subcutaneous injection (10 g/kg) attains "steady state" blood levels of ϳ300 pM. This regimen does not cause cardiac toxicity assessed by continuous EKG monitoring and does not alter clinical chemistry and hematological parameters after 2-week therapy. ShK(L5) prevents and treats experimental autoimmune encephalomyelitis and suppresses delayed type hypersensitivity in rats. ShK(L5) might prove useful for therapy of autoimmune disorders.

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 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, ϳ100 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.

Mechanisms that fine tune the activity of potassium channels are crucial to a cell's ability to integrate and respond to a plethora of internal and external signals. Peptide toxins from venomous creatures have served as vital tools to define the molecular mechanisms underlying K ϩ channel function (1, 2). It has been suggested that toxins evolved from endogenous genes that function in normal cellular pathways (3, 4). Indeed, venomous creatures possess toxins with homology to several proteins, including acetylcholinesterases (5), phospholipases (6, 7), nerve growth factor (8), endothelins (9), Lynx-1 (10, 11), Kunitz-type serine protease inhibitors (12), and the ion channel regulatory (ICR) 5 domains of cysteinerich secretory proteins (CRISPs) (3,13,14). Mammalian proteins containing toxin-like domains (TxDs) that block K ϩ channels have not been characterized previously.BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera (15,16), and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus (17,18) are potent inhibitors of K ϩ channels. The Simple Modular Architecture Research Tool (SMART) (available on the World Wide Web) predicts the existence of a large superfamily of proteins that contain domains (referred to as ShKT domains in the SMART data base) resembling these two toxins (Fig. 1A). Many of these proteins (ϳ70 proteins) are metallopeptidases, whereas others are prolyl-4-hydroxylases, tyrosinases, peroxidases, oxidoreductases, or proteins containing epidermal growth factor-like domains, thrombospondin-type repeats, or trypsin-like serine protease domains (Fig. 1B). The only human protein containing a ShKT domain in the SMART data base is MMP23 (matrix metalloprotease 23). Matrix metalloproteases belong to the metzincin superfamily and play important roles in tissue remodeling, development, and the immune response (19).MMP23 is expressed in many tissues and exists either as a type II transmembrane protein in ER/nuclear membranes or as a secreted form following cleavage of the RRRRY motif just N-terminal to the Zn 2ϩ -dependent metalloprotease domain (20 -23). The ShKT domain of MMP23 (MMP23 TxD ) lies between the metalloprotease domain and an immunoglobulinlike cell adhesion molecule (IgCAM) domain ( Fig. 2A). MMP23 has been implicated in prostate, brain, and breast cancer (24 -26). In humans, two related sequences, MMP23A (a pseudogene) and MMP23B, are co-located on chromosome 1p36 (20). We have investigated MMP23 to gain insight into the structure and physiological functions of ShKT toxin domains and describe the solution structure of the MMP23 TxD domain, its * This work was supported, in whole or in part, by National Institutes of Health

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T EM ); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T EM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-Allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K d 36 nM (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K ؉ channels tested. D-Allo-ShK inhibited human T EM cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-AlloShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K ؉ channel studies; all-D analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.

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