Stichodactyla Toxin - Structure

Structure

ShK is a 35-residue basic peptide first discovered in the sea anemone Stichodactyla helianthus by Professor Olga Castaneda from the University of Havana, Cuba, and her collaborators in Sweden. The formula is C₁₆₉H₂₇₄N₅₄O₄₈S₇. It is cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32 (see figure below). The amino acid sequence of the ShK toxin is Arg-Ser-Cys-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys-Thr-Ala-Phe-Gln-Cys-Lys-His-Ser-Met-Lys-Tyr-Arg-Leu-Ser-Phe-Cys-Arg-Lys-Thr-Cys-Gly-Thr-Cys. ShK is stabilized by three disulfide bridges and consists of two short α-helices comprising residues 14-19 and 21-24. The N-terminal eight residues of ShK adopt an extended conformation, followed by a pair of interlocking turns that resemble a 3₁₀ helix, while its C-terminal Cys35 residue forms a nearly head-to-tail cyclic structure through a disulfide bond with Cys3. Protein domains with structural resemblance to ShK have been described in 402 proteins, most of them from C. elegans (IPR003582). The SMART database at the EMBL has a list of the 402 proteins containing ShK-like sequences (http://smart.embl-heidelberg.de). Other proteins containing domains with similar structures include the cysteine-rich secretory protein snake toxins natrin, triflin, and stecrisp, the Toxocara canis mucins, secreted peptides from the dog hookworm Ancylostoma caninum, and the human proteins Tpx-1 and matrix metalloprotease 23 (MMP23) The peptide binds to all four subunits in the K_v1.3 tetramer through its interaction with the shallow vestibule at the outer entrance of the ion conduction pathway. The peptide's Lysine22 residue occludes the channel pore like a "cork in a bottle". This blocks the entrance to the pore.

ShK blocks the K_v1.3 channel in T cells with a K_d of about 11 pM. It blocks the neuronal K_v1.1 and K_v1.6 channels with K_ds of 16 pM and 200 pM respectively. The K_v3.2 and K_Ca3.1 channels are more than 1000 times less sensitive to the peptide.

Several ShK analogs have been generated to enhance specificity for the K_v1.3 channel over the K_v1.1, K_v1.6 and K_v3.2 channels. The first analog that showed some degree of specificity was ShK-Dap22. Attaching a fluorescein to the N-terminus of the peptide via a hydrophilic AEEA linker (2-aminoethoxy-2-ethoxy acetic acid; mini-PEG) resulted in a peptide, ShK-F6CA, with 100-fold specificity for K_v1.3 over K_v1.1 and related channels. Based on this surprising finding additional analogs were made. ShK-170 ,contains a L-phosphotyrosine in place of the fluorescein in ShK-F6CA. It blocks K_v1.3 with a K_d of 69 pM and shows exquisite specificity for K_v1.3. However, it is chemically unstable. To improve stability a new analog, ShK-186, was made with the C-terminal carboxyl of ShK-170 replaced by an amide; ShK-186 is otherwise identical to ShK-170. In rats and squirrel monkeys, an indium-labeled ShK-186 analog called ShK-221, was slowly released from the injection site and maintained blood levels above the channel blocking dose for 3-5 days Tarcha EJ, Chi V, Muñoz-Elias EJ, Bailey D, Londono LM, Upadhyay SK, Norton KN, Olson A, Tjong I, Nguyen HM, Hu X, Rupert GW, Boley SE, Slauter R, Sams J, Knapp B, Kentala D, Hansen Z, Pennington MW, Beeton C, Chandy KG, Iadonato SP (2012). "Durable pharmacological responses from a single dose of the peptide drug ShK-186, a specific Kv1.3 channel inhibitor". J. Pharm. Exp. Therap 342: 642–653. doi:10.1124/jpet.112.191890. PMID 22637724. ShK-192 is a new analog with increased stability . It contains norleucine21 in place of methionine21 to avoid methionine oxidation, and the terminal phosphotyrosine is replaced by a non-hydrolyzable para-phosphonophenylalanine (Ppa) group. ShK-192 is effective in ameliorating disease in rat models of multiple sclerosis. The D-diasteromer of ShK is also stable but blocks K_v1.3 with 2800-fold potency than the L-form (K_d = 36 nM) and it only exhibits 2-fold specificity for K_v1.3 over K_v1.1.

K_v1.3 and K_Ca3.1 regulate membrane potential and calcium signaling of T cells. Calcium entry through the CRAC channel is promoted by potassium efflux through the K_v1.3 and K_Ca3.1 potassium channels. Blockade of K_v1.3 channels in effector-memory T cells by ShK-186 suppresses calcium signaling, cytokine production (interferon-gamma, interleukin 2) and cell proliferation. In vivo, ShK-186 paralyzes effector-memory T cells at the sites of inflammation and prevent their reactivation in inflamed tissues. In contrast, ShK-186 does not affect the homing to and motility within lymph nodes of naive and central memory T cells, most likely because these cells express the K_Ca3.1 channel and are therefore protected from the effect of K_v1.3 blockade. In proof-of-concept studies, ShK and its analogs have prevented and treated disease in rat models of multiple sclerosis, rheumatoid arthritis, and delayed type hypersensitivity. ShK-186, due to its durable pharmacological action, is effective in ameliorating disease in rat models of delayed type hypersensitivity, multiple sclerosis (experimental autoimmune encephalomyelitis) and rheumatoid arthritis (pristane induced arthritis) when administered once every 2-5 days . ShK-186 has completed non-clinical safety studies as is being evaluated in phase 1 human trials.

As ShK toxin binds to the synaptosomal membranes, it facilitates an acetylcholine release at avian neuromuscular junctions while the K_v3.2 channels are expressed in neurons that fire at a high frequency (such as cortical GABAergic interneurons), due to their fast activation and deactivation rates. By blocking K_v3.2, ShK toxin depolarises the cortical GABAergic interneurons. K_v3.2 is also expressed in pancreatic beta cells. These cells are thought to play a role in their delayed-rectifier current, which regulates glucose-dependent firing. Therefore, ShK, as a K_v3.2 blocker, might be useful in the treatment of type-2 diabetes, although inhibition of the delayed-rectifier current has not yet been observed in human cells even when very high ShK concentrations were used.