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ShK domain-like
Rainbow colored cartoon diagram (N-terminus = blue, C-terminus = red) of an NMR solution structure of the ShK toxin. Sidechains of cysteine residues involved in disulfide linkages are displayed as sticks and the sulfur atoms in these links are colored yellow.
Identifiers
Symbol	ShK
Pfam	PF01549
InterPro	IPR003582
SMART	SM00254
SCOP2	1roo / SCOPe / SUPFAM
TCDB	8.B.14
OPM superfamily	475
OPM protein	2lg4
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary PDB }

Stichodactyla toxin (ShK) is a 35-residue basic peptide from the sea anemone Stichodactyla helianthus that blocks a number of potassium channels. An analogue of ShK called ShK-186 or Dalazatide is in human trials as a therapeutic for autoimmune diseases.

History

Stichodactyla helianthus is a species of sea anemone (Phylum: Cnidaria) belonging to the family Stichodactylidae. Helianthus comes from the Greek words Helios meaning sun, and anthos meaning flower, which corresponds to S. helianthus common name "sun anemone". It is sessile and uses potent neurotoxins for defense against its primary predator, the spiny lobster. The venom contains, among other components, numerous ion channel-blocking peptides. In 1995, a group led by Olga Castaneda and Evert Karlsson isolated ShK, a potassium channel-blocking 35-residue peptide from S. helianthus. The same year, William Kem and his collaborator Michael Pennington synthesized and folded ShK, and showed it blocked neuronal and lymphocyte voltage-dependent potassium channels. In 1996, Ray Norton determined the three-dimensional structure of ShK. In 2005-2006, George Chandy, Christine Beeton and Michael Pennington developed ShK-170 and ShK-186, selective blockers of K_v1.3. ShK-186, now called Dalazatide, was advanced to human trials in 2015-2017 by Shawn Iadonato and Eric Tarcha, as the first-in-man K_v1.3 blocker for autoimmune disease.References used only in figure 1

Structure

ShK is cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. The solution structure of ShK reveals two short α-helices comprising residues 14-19 and 21-24; the N-terminal eight residues adopt an extended conformation, followed by a pair of interlocking turns that resemble a 3₁₀ helix; the C-terminal Cys35 residue forms a nearly head-to-tail cyclic structure through a disulfide bond with Cys3.

Phylogenetic relationships of ShK and ShK domains

ShK is cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32 (Figure 2). The solution structure of ShK reveals two short α-helices comprising residues 14-19 and 21-24; the N-terminal eight residues adopt an extended conformation, followed by a pair of interlocking turns that resemble a 3₁₀ helix; the C-terminal Cys35 residue forms a nearly head-to-tail cyclic structure through a disulfide bond with Cys3. Figure 3 shows the three-dimensional structure of ShK with key residues, in addition to the structures of related peptides: MMP-23’s ShK domain, BmK1 (from the filarial worm), and ShK-192 (an analogue of ShK-186), and the homology model of ShK-EWSS.

Phylogenetic relationships of ShK and ShK domains

The SMART database at the EMBL, as of May 2018, lists 3345 protein domains with structural resemblance to ShK in 1797 proteins (1 to 8 domains/protein), many in the worm Caenorhabditis elegans and venomous snakes. The majority of these domains are in metallopeptidases, whereas others are in prolyl 4-hydroxylases, tyrosinases, peroxidases, oxidoreductases, or proteins containing epidermal growth factor-like domains, thrombospondin-type repeats, or trypsin-like serine protease domains. The only human proteins containing ShK-like domains are MMP-23 (matrix metalloprotease 23) and MFAP-2 (microfibril-associated glycoprotein 2).

Channel targets

The ShK peptide blocks potassium (K) ion channels K_v1.1, K_v1.3, K_v1.6, K_v3.2 and K_Ca3.1 with nanomolar to picomolar potency, and has no effect on the HERG (K_v11.1) cardiac potassium channel. The neuronal K_v1.1 channel and the T lymphocyte K_v1.3 channel are most potently inhibited by ShK.

ShK binding configuration in K channels

ShK and its analogues are blockers of the channel pore. They bind to all four subunits in the K channel tetramer by interacting with the shallow vestibule at the outer entrance to the channel pore. These peptides are anchored in the external vestibule by two key interactions. The first is Lys22, which protrudes into and occludes the channel’s pore like a "cork in a bottle" and blocks the passage of potassium ions through the channel pore. The second is the neighboring Tyr23, which together with Lys22 forms a “functional dyad” required for channel block. Many K channel-blocking peptides contain such a dyad of a lysine and a neighboring aromatic or aliphatic residue. Some K channel-blocking peptides lack the functional dyad, but even in these peptides a lysine physically blocks the channel, regardless of the position of the lysine in the peptide sequence. Additional interactions anchor ShK and its analogues in the external vestibule and contribute to potency and selectivity. For example, Arg11 and Arg29 in ShK interact with two Asp386 residues in adjacent subunits in the mouse K_v1.3 external vestibule (corresponds to Asp433 in human K_v1.3).

Target

ShK toxin blocks the K channels K_v1.1, K_v1.3, K_v1.6, K_v3.2 and K_Ca3.1, 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 Lysine 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 ShK-192 is a new analog with increased stability. It contains norleucine in place of methionine 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-diastereomer 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. ShK-K-amide is a new analog with a C-terminal lysine. It blocks Kv1.3 with roughly 50-fold greater potency (IC50 of 26 ± 3 pM) than Kv1.1 ( IC50 of 942 ± 120 pM), and suppresses proliferation of human T cells (IC50 ≈ 3 nM).

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. ShK-186 is the subject of an open Investigational New Drug (IND) application in the USA, and has completed human phase 1A and 1B trials in healthy volunteers.

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.

Toxicity

Toxicity of ShK toxin in mice is quite low. The median paralytic dose is about 25 mg/kg bodyweight (which translates to 0.5 mg per 20 g mouse). In rats the therapeutic safety index was greater than 75-fold.

ShK-Dap22 is less toxic, even a dose of 1.0 mg dose did not cause hyperactivity, seizures or mortality. The median paralytic dose was 200 mg/kg body weight.

ShK-170 does not cause significant toxicity in vitro. The peptide was not toxic to human and rat lymphoid cells incubated for 48 h with 100 nM of ShK-170 (>1200 times greater than the K_v1.3 half-blocking dose). The same high concentration of ShK-170 was negative in the Ames test on tester strain TA97A, suggesting that it is not a mutagen. ShK-170 had no effect on heart rate or heart rate variability parameters in either the time or the frequency domain in rats. It does not block the hERG (K_v11.1) channel that is associated with drug-associated cardiac arrhythmias. Repeated daily administration of the peptide by subcutaneous injection (10 µg/kg/day) for 2 weeks to rats does not cause any changes in blood counts, blood chemistry or in the proportion of thymocyte or lymphocyte subsets. Furthermore, the rats administered the peptide gain weight normally.

ShK-186 is also safe. Repeated daily administration by subcutaneous injection of ShK-186 (100 µg/kg/day) for 4 weeks to rats does not cause any changes in blood counts, blood chemistry or histopathology. Furthermore, ShK-186 did not compromise the protective immune response to acute influenza viral infection or acute bacterial (Chlamydia) infection in rats at concentrations that were effective in ameliorating autoimmune diseases in rat models. Interestingly, rats repeatedly administered ShK-186 for a month by subcutaneous injection (500 µg/kg/day) developed low titer anti-ShK antibodies. The reason for the low immunogenicity of the peptide is not well understood. ShK-186 has completed GLP (Good Laboratory Practice) non-clinical safety studies in rodents and non-human primates. ShK-186 (aka Dalazatide) which was licensed to Kineta Bio is the subject of an open Investigational New Drug (IND) application in the United States of America, and has recently completed human phase 1A and 1b trials in healthy volunteers. A second human phase 1b was recently completed in 2015 in psoriasis patients. Dalazatide was shown to significantly ameliorate symptoms in 90% patients with active plaque psoriasis with a 60 mcg weekly dose.

Many groups are developing K_v1.3 blockers for the treatment of autoimmune diseases.

Use

Because ShK toxin is a specific inhibitor of K_v1.1, K_v1.3, K_v1.6, K_v3.2 and K_Ca3.1, it may serve as a useful pharmacological tool for studying these channels. The K_v1.3 specific ShK analogs, ShK-170, ShK-186 and ShK-192, have been demonstrated to be effective in rat models of autoimmune diseases, and these or related analogs might have use as therapeutics for human autoimmune diseases.

K_v1.3 is also considered a therapeutic target for the treatment of obesity, for enhancing peripheral insulin sensitivity in patients with type-2 diabetes mellitus, and for preventing bone resorption in periodontal disease. Furthermore, because pancreatic beta cells, which have K_v3.2 channels, are thought to play a role in glucose-dependent firing, 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.

References

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6. Beeton, C; Pennington, MW; Wulff, H; Singh, S; Nugent, D; Crossley, G; Khaytin, I; Calabresi, PA; Chen, CY; Gutman, GA; Chandy, KG (2005). "Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases". Molecular Pharmacology. 67 (4): 1369-1381. doi:10.1124/mol.104.008193. {{cite journal}}: Unknown parameter |month= ignored (help)
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• Ion channel toxins
• Neurotoxins