ShK toxin: history, structure and therapeutic applications for autoimmune diseases WikiJournal of Science

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(27)(28)(29) Table 1 compares the sequence of ShK with related peptides from other sea anemones, parasitic worms and human MMP-23's ShK domain; conserved residues are highlighted.Figure 4 shows the phylogenetic relationships of these peptides; the tree was generated by NCBI' s Constraint-based Multiple Alignment T ool (COBALT).The ShK peptide blocks potassium (K + ) ion channels K v 1.1, K v 1.3, K v 1.6, K v 3.2 and K Ca 3.1 with nanomolar to picomolar potency, and has no effect on the HERG (K v 11.1) cardiac potassium channel (Table 2). (46,47)The neuronal K v 1.1 channel and the T lymphocyte K v 1.3 channel are most potently inhibited by ShK. (9)K 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 (Figure 5). (7,9,15,19,23,41,46)These peptides are anchored in the external vestibule by two key interactions.The first is lysine 22 , 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 ( Figures 3 and 5). (9,11,15,46)The second is the neighboring tyrosine 23 , which together with lysine 22 forms a "functional dyad" required for channel block. (9,11,15,23,46)Many K + channel-blocking peptides contain such a dyad of a lysine and a neighboring aromatic or aliphatic residue. (46,47)Some K + channel-blocking peptides lack the functional dyad, but even in these peptides a lysine physically blocks the channel, regardless Lys22 is shown protruding into the pore and interacting with the channel' s selectivity filter.

ShK binding configuration in K + channels
of the position of the lysine in the peptide sequence. (48)Additional interactions anchor ShK and its analogues in the external vestibule and contribute to potency and selectivity. (9,11,15,23,46)For example, arginine 11 and arginine 29 in ShK interact with two aspartate 386 residues in adjacent subunits in the mouse K v 1.3 external vestibule (corresponds to aspartate 433 in human K v 1.3). (9,11,15,23,46)2).

ShK-Dap 22 :
This was the first analogue that showed some degree of specificity for K v 1.3.The pore-occluding lysine 22 of ShK is replaced by diaminopropionic acid (Dap) in ShK-Dap 22 . (9,15,18)Dap is a non-natural lysine analogue with a shorter side chain length (2.5 Å from C α ) than lysine (6.3 Å). (49) Dap 22 interacts with residues further out in the external vestibule in contrast to lysine 22 , which interacts with the channel' s selectivity filter. (15)As a consequence, the orientations of ShK and ShK-Dap 22 in the external vestibule are significantly dif ferent. (15)ShK-Dap 22 exhibits >20-fold selectivity for K v 1.3 over closely related channels in whole-cell patch clamp experiments, (9) but in equilibrium binding assays it binds K v 1.1-K v 1.2 heterotetramers with almost the same potency as ShK, which is not predicted from the study of homotetrameric K v 1.1 or K v 1.2 channels. (18)K-F6CA: 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 (fluorescein-6-carboxyl), with 100-fold specificity for K v 1.3 over K v 1.1 and related channels.(17) Attachment of a tetramethyl-rhodamine or a biotin via the AEEA linker to ShK' s Nterminus did not increase specificity for K v 1.3 over K v 1.1.(17) The enhanced specificity of ShK-F6CA might be explained by dif ferences in charge: F6CA is negatively charged; tetramethylrhodamine is positively charged; and biotin is neutral.(17) Subsequent studies with other analogues suggest that the negatively charged F6CA likely interacts with residues on the turret of the K v 1.3 channel as shown for ShK-192 and ShK-EWSS (Figure 5).(23,41) ShK-170, ShK-186, ShK-192 and ShK-EWSS: Based on ShK-F6CA additional analogues were made.Attaching a L-phosphotyrosine to the N-terminus of ShK via an AEEA linker resulted in a peptide, ShK-170, with 100-1000-fold specificity for K v 1.3 over related channels.ShK-186 [a.k.a.SL5; a.k.a.Dalazatide] is identical to ShK-170 except the C-terminal carboxyl is replaced by an amide.ShK-186 blocks K v 1.3 with an IC 50 of 69 pM and exhibits the same specificity for K v 1.3 over closely related channels as ShK-170 ( Table 2).31,32) To overcome this problem, ShK-192 and ShK-EWSS were developed.In ShK-192, the N-terminal L-phosphotyrosine is replaced by a non-hydrolyzable para-phosphonophenylalanine (Ppa), and methionine 21 is replaced by the non-natural amino acid norleucine to avoid methionine oxidation ( Figure 3, Table 2).(23,32) In ShK-EWSS, the AEEA linker and Lphosphotyrosine are replaced by the residues glutamic acid (E), tryptophan (W) and two serines (S) ( Figures 3 and 5, Table 2).(41) Both ShK-192 and ShK-EWSS are highly specific for K v 1.3 over related channels.
ShK-K18A: Docking and molecular dynamics simulations on K v 1.3 and K v 1.1 followed by umbrella sampling simulations, paved the way to the selective Kv1.3 inhibitor ShK-K18A .Due to their low molecular mass, ShK and its analogues are prone to rapid renal elimination.In rats, the half-life is ~6 min for ShK-186 and ~11 min for ShK-198, with a clearance rate of ~950 ml/kg•min. (31)In monkeys, the half-life is ~12 min for ShK-186 and ~46 min for ShK-198, with a clearance rate of ~80 ml/kg•min. (31)Gylation of ShK: Conjugation of polyethylene glycol (PEG) to ShK[Q16K], an ShK analogue, increased its molecular mass and thereby reduced renal clearance and extended plasma half-life to 15 h in mice and 64 h in cynomolgus monkeys. (40)PEGylation can also decrease immunogenicity and protect a peptide from proteolysis and non-specific adsorption to inert surfaces.PEGylated ShK[Q16K] prevented adoptive-transfer experimental autoimmune encephalomyelitis in rats, a model for multiple sclerosis. (40)njugation of ShK to larger proteins: The circulating half-life of peptides can be prolonged by coupling them to larger proteins or protein domains. (46,54,55)By screening a combinatorial ShK peptide library , novel anal ogues were identified, which when fused to the C-termini of IgG1-Fc retained picomolar potency , effectively suppressed in vivo delayed type hypersensitivity and exhibited a prolonged circulating half-life. (43)olonged effects despite rapid plasma clearance: SPECT/CT imaging studies with a 111 In-DOTA-conjugate of ShK-186 in rats and sq uirrel monkeys revealed a slow release from the injection site and blood levels above the channel blocking dose for 2 and 7 days, respectively . (31)Studies on human peripheral blood T cells showed that a brief exposure to ShK-186 was suf ficient to suppress cytokine responses. (31)These findings suggest that ShK-186, despite its short circulating half-life, may have a prolonged therapeutic ef fect.In rats, the peptide is ef fective in treating disease in animal models of autoimmune diseases when administered once a day to once in 3 days. (31)In humans, subcutaneous injections twice a week are suf ficient to ameli orate disease in patients with plaque psoriasis. (45)e low molecular mass of ShK and its analogues, combined with their high isoelectric points, makes it unlikely that these peptides will be absorbed from the stomach or intestine following oral administration.Sub-lingual delivery is a possibility.A fluorescent ShK analogue was absorbed into the blood stream at pharmacological concentrations following sublingual administration with a mucoadhesive chitosan-based gel, with or without the penetration enhancer cetrimide. (56)Delivery of the peptide as an aerosol through the lung, or across the skin, or as eye drops are also possibilities.During T cell-activation, calcium enters lymphocytes through store-operated CRAC channels (calcium release activated channel) formed as a complex of Orai and Stim proteins. (50,60)The rise in intracellular calcium initiates a signaling cascade culminating in cytokine production and proliferation. (50,60)The K v 1.3 K + channel and the calcium-activated K Ca 3.1 K + channel in T cells promote calcium entry into the cytoplasm through CRAC by providing a counterbalancing cation ef flux. (46,50,60)2)60) Blockade of K v 1.3 channels in chronically-activated CD28 null effector memory T cells that are implicated in autoimmune diseases (e.g.)(52)(53) Effector memory T cells that are CD28 + are refractory to suppression by K v 1.3 blockers when they are co-stimulated by anti-CD28 antibodies. (52)In vivo, ShK-186 paralyzes effector-memory T cells at the site of an inflammatory delayed type hypersensitivity response and prevents these T cells from activating in the inflamed tissue. (22)In contrast, ShK-186 does not affect the homing and motility of naive and T CM cells to and within lymph nodes, most likely because these cells express the K Ca 3.1 channel and are therefore protected from the effect of K v 1.3 blockade. (22)(67)(68) ShK-223, an analogue of ShK-186, decreased lipopolysaccharide (LPS) induced focal adhesion formation by microglia, reversed LPS-induced inhibition of microglial migration, and inhibited LPS-induced upregulation of EH domain containing protein 1 (EHD1), a protein involved in microglia trafficking. (44)Increased K v 1.3 expression was reported in microglia in Alzheimer plaques. (69)K v 1.3 inhibitors may have use in the management of Alzheimer's disease, as reported in a recent proof-of-concept study in which a small molecule K v 1.3 blocker (PAP-1) alleviated Alzheimer's disease-like characteristics in a mouse model of AD. (70) Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis: ShK, ShK-Dap 22 , ShK-170 and PEGylated ShK-Q16K prevent adoptive-transfer EAE in Lewis rats, a model of multiple sclerosis. (14,19,40)Since multiple sclerosis is a relapsing-remitting disease, ShK-186 and ShK-192 were evaluated in a relapsing-remitting EAE model in DA (Dark Agouti) rats.Both prevented and treated disease when administered once a day to once in three days. (22,31,32)istane-induced arthritis (PIA), a model for rheumatoid arthritis: ShK-186 was ef fective in treating PIA when administered every day or on alternate days. (19,20,22,31)(73)(74)(75) ShK, ShK-170, ShK-186, ShK-192 and ShK-IgG-Fc were all ef fective in the ovalbumin-induced delayed type hypersensitivity model (19,20,22,23,31,32,43,71), while a topical formulation of ShK-198 was ef fective in treating oxazolone-induced dermatitis. (71)oriasis: Psoriasis is a severe autoimmune disease of the skin that af flicts many people worldwide.Despite the success of recent biologics in ameliorating disease, there is still a search for safe and ef fective drugs for psoriasis.ShK treated disease in a psoriasiform (psoriasis-like) SCID ( severe combined immunodeficiency) mouse model.(30) In a recently published Phase 1b placebo-controlled clinical study in patients with plaque psoriasis, ShK-186 administered twice a week (30 or 60 mg/dose/patient) by subcutaneous injection caused improvements with a statistically significant reduction in their P ASI (Psoriasis Area and Severity Index) score between baseline and day 32.(45) These patients also exhibited reduced plasma levels of multiple inflammation markers and decreased expression of T cell activation markers on peripheral blood memory T cells.(45) Allograft transplant: Immunosuppressant drugs are used to prevent the rejection of transplanted organs.In a rat allograft renal transplant model, the combination of ShK and the K Ca 3.1 inhibitor TRAM-34 was as ef fective as cyclosporine A, a drug used clinically to prevent rejection of transplanted organs.(24) Diet-induced obesity and fatty liver disease: Obesity and diabetes are major healthcare problems globally .There is need for safe drugs for these metabolic diseases.In a mouse model of diet-induced obesity , ShK-186 counteracted the negative ef fects of increased caloric intake.It reduced weight gain, adiposity , and fatty liver; decreased blood levels of cholesterol, sugar , HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity .(36) Genetic deletion of the K v 1.3 gene has the same ef fect, indicating that ShK-186's effect is due to K v 1.3 blockade.(36,(76)(77)(78) At least two mechanisms contribute to ShK-186' s therapeutic benefits.The high calorie diet induced K v 1.3 expression in brown fat tissues. (36)By blocking K v 1.3, ShK-186 doubled glucose uptake and increased β-oxidation of fatty acids, glycolysis, fatty acid synthesis and uncoupling protein 1 expression by brown fat.(36) As a consequence of brown fat activation, oxygen consumption and energy expenditure were augmented.(36) The obesity diet also induced K v 1.3 expression in the liver, and ShK-186 caused profound alteratio ns in energy and lipid metabolism in the liver .ShK, its analogues or other K v 1.3 blockers may have use in controlling the negative consequences of high calorie diets.

Arousal and anesthesia:
The mechanisms of general anesthesia involve multiple molecular targets and pathways that are not completely understood. (37)Sevoflurane is a common anesthetic used to induce general anesthesia during surgery . (37)Rats continually exposed to sevoflurane lose their righting reflex as an index of loss of consciousness.In these rats, microinfusion of ShK into the central medial thalamic nucleus (CMT) reversed sevoflurane-induced anesthesia in rodents. (37)ShK-treated rats righted themselves fully (restored consciousness) despite being continually exposed to sevoflurane. (37)ShK-microinfusion into neighboring regions of the brain did not have this ef fect. (37)Sevoflurane enhanced potassium currents in the CMT , while ShK and ShK-186 countered this ef fect. (37)These studies suggest that ShK-sensitive K + channels in the CMT are important for suppressing arousal during anesthesia.
Preventing brain damage following therapeutic brain radiation: Brain radiation is used to treat tumors of the head, neck, and brain, but this treatment carries a significant risk of neurologic injury.Injury is, in part, due to the activation of microglia and microglia-mediated damage of neurons.Neuroprotective therapies for radiation-induced brain injury are still limited.In a mouse model of brain radiation, ShK-170 reversed neurological deficits, and protected neurons from radiation-induced brain injury by suppressing microglia. (38)K and ShK-Dap 22 : ShK peptide has a low toxicity profile in mice.ShK is ef fective in treating autoimmune diseases at 10 to 100 mg/kg bodyweight.It has a median paralytic dose of approximately 25 mg/kg bodyweight (250-2500 higher than the pharmacological dose). Inrats the therapeutic safety index is greater than 75-fold.ShK-Dap 22 displayed a lower toxicity profile.(9) A 1.0 mg dose did not induce any hyperactivity , seizures or mortality in rats.The median paralytic dose for ShK-Dap 22 is about 200 mg/kg bodyweight (2000-20000 higher than pharmacological dose).(9) PEGylated ShK[Q16K] showed no adverse toxicity in monkeys over a period of several months.(40) ShK-186/Dalazatide: ShK-186 also displays a low toxicity profile in rats.Daily administration of ShK-170 or ShK-186 (100 µg/kg/day) by subcutaneous injection over 4 weeks in rats does not induce any changes in blood counts, blood chemistry or histopathology .(19,20,31) By virtue of suppressing only T EM and T EMRA cells, ShK-186 did not compromise protective immune responses to influenza virus and chlamydial infection in rats, most likely because naïve and T CM cells unaffected by K v 1.3 blockade mounted ef fective immune responses.(22) ShK-186 is poorly immunogenic and did not elicit anti-ShK antibodies in rats repeatedly administered the peptide.(20) This is possibly because the peptide' s disulfide-bonded structure hinders processing and antigen presentation by antigen-presenting cells.ShK-186 also shares sequence and structural similarity to a ShK-like domain in matrix metalloprotease 23, (25)(26)(27)(28)(29) which may cause the immune system to assume it is a normal protein in the body .ShK-186 was safe in non-human primates.) The most common adverse events were temporary mild (Grade 1) hypoesthesia and paresthesia involving the hands, feet, or perioral area. Mil muscle

Efficacy of ShK analogues in animal models of human diseases
Toxicity of ShK and its analogues were achieved. (45)The most common adverse events were temporary mild (Grade 1) hypoesthesia and paresthesia involving the hands, feet, or perioral area.Mild muscle spasms, sensitivity of teeth, and injection site pain were also observed. (45)b7, a worm protein containing a ShK domain: In male Caenorhabditis elegans worms, the absence of a protein called Mab7 results in malformed sensory rays that are required for mating.(79) Introduction of Mab7 into these male worms restores normal development of normal sensory rays.(79) Introduction of Mab7 proteins lacking the ShK domain does not correct the defect of sensory rays, suggesting a role for the ShK-domain of Mab7 in sensory ray development.(79) HMP2 and PMP1, proteins containing ShK-like domains: HMP2 and PMP-1 are astacin metalloproteinases from the Cnidarian Hydra vulgaris and the jellyfish Podocoryne carnea that contain ShK-like domains at their C-termini.(29,80) Both these ShK-domains contain the critical pore-occluding lysine required for K + channel block.(25) HMP2 plays a critical role in foot regeneration of Hydra, (80) while PMP1 is found in the feeding organ of the jelly fish and the ShK-domain may paralyze prey after they are ingested.(29) The ShK domain in MMP-23: MMP-23' s ShK domain blocks voltage-gated potassium channels (K v 1.6 > K v 1.3 > K v 1.1 = K v 3.2 > K v 1.4, in decreasing potency) in the nanomolar to low micromolar range. (25)MMP-23 traps potassium channels sensitive to its ShK domain in the endoplasmic reticulum and reduces surface expression of these channels.(25)(26)(27) One of these channels, K v 1.3, is required for sustaining calcium signaling during activation of human T cells.(46,50,51) MMP-23 may suppress T cells and interfere with immune protection. In spport, increased expression of MMP-23 in melanoma cancer cells decreases tumor-infiltrating lymphocytes, and is associated with cancer recurrence and shorter periods of progression-free survival.(28) However, in melanomas, expression of MMP -23 does not correlate with K v 1.3 expression, suggesting that MMP-23' s deleterious effect in melanomas may not be connected with its K v 1.3 channel-modulating function.(28) Kineta Inc. has licensed a patent on ShK-186 from the University of California and is developing this peptide as a therapeutic for autoimmune diseases.

Figure 4 |
Figure 4 | Phylogenetic relationships between ShK and related peptides generated with NCBI's Constraint-based Multiple Alignment Tool (COBALT).

Figure 5 |
Figure 5 | Docking configuration of EWSS-ShK in Kv1.3.For clarity, only two of the four Kv1.3 subunits in the tetramer are shown.

Figure 6 |
Figure 6 | Expression of Kv1.3 and KCa3.1 channels during T cell activation and memory T cell generation.The numbers represent functional channel numbers per T cell determined by whole cell patch clamp.The effect of specific blockade of Kv1.3 or KCa3.1 on T cell proliferation is shown.TCM = Central memory T cell.TEM = Effector memory T cell.TEMRA = Effector memory T cell that has reacquired CD45RA.