AA sequence: Arg-Ser-Cys3-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys12-Thr-Ala-Phe-Gln-Cys17-Lys-His-Ser-Met-Lys-Tyr-Arg-Leu-Ser-Phe-Cys28-Arg-Lys-Thr-Cys32-Gly-Thr-Cys35-OH
Disulfide bonds: Cys3-Cys35, Cys12-Cys28 and Cys17-Cys32
Length (aa): 35
Molecular Weight: 4054.85 Da
Appearance: White lyophilized solid
Solubility: water and saline buffer
CAS number: 165168-50-3
Purity rate: > 97%
ShK – Stichodactyla toxin
Selective blocker of Kv1.3
ShK (Stichodactyla helianthus Neurotoxin) has been isolated from the venom of the Carribean sea anemone Stoichactis helianthus. ShK inhibits voltage-dependent potassium channels. It blocks Kv1.3 (KCNA3) potently and also Kv1.1 (KCNA1), Kv1.4 (KCNA4) and Kv1.6 (KCNA6) respectively with a Kd of 11 pM, 16 pM, 312 pM and 165 pM. Interestingly, it was also demonstrated that ShK potently inhibits the hKv3.2b channel with an IC50 value of approximately 0.6 nM.
AA sequence: Arg-Ser-Cys3-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys12-Thr-Ala-Phe-Gln-Cys17-Lys-His-Ser-Met-Lys-Tyr-Arg-Leu-Ser-Phe-Cys28-Arg-Lys-Thr-Cys32-Gly-Thr-Cys35-OH
Durable pharmacological responses from the peptide ShK-186, a specific Kv1.3 channel inhibitor that suppresses T cell mediators of autoimmune disease
The Kv1.3 channel is a recognized target for pharmaceutical development to treat autoimmune diseases and organ rejection. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the pharmacokinetic-pharmacodynamic relationship for ShK-186 in rats and monkeys. The pharmacokinetic profile of ShK-186 was evaluated with a validated high-performance liquid chromatography-tandem mass spectrometry method to measure the peptide’s concentration in plasma. These results were compared with single-photon emission computed tomography/computed tomography data collected with an ¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugate of ShK-186 to assess whole-blood pharmacokinetic parameters as well as the peptide’s absorption, distribution, and excretion. Analysis of these data support a model wherein ShK-186 is absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC₅₀ value for up to 7 days in monkeys. Pharmacodynamic studies on human peripheral blood mononuclear cells showed that brief exposure to ShK-186 resulted in sustained suppression of cytokine responses and may contribute to prolonged drug effects. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration. ShK-186’s slow distribution from the injection site and its long residence time on the Kv1.3 channel contribute to the prolonged therapeutic effect of ShK-186 in animal models of autoimmune disease.
Tarcha EJ., et al. (2012) Durable pharmacological responses from the peptide ShK-186, a specific Kv1.3 channel inhibitor that suppresses T cell mediators of autoimmune disease. J Pharmacol Exp Ther. PMID: 22637724
The Kv1.3 channel is important in the activation and function of effector memory T cells. Recently, specific blockers of the Kv1.3 channel have been developed as a potential therapeutic option for diverse autoimmune diseases. In psoriatic lesions, most lymphocytes are memory effector T cells. The aim of the present study was to detect the expression of Kv1.3 channels in these cells in psoriatic lesions as well as in human psoriasiform skin grafts using the severe combined immunodeficient (SCID) mouse model. Histological and immunohistochemical staining for Kv1.3 expression and various inflammatory markers was performed in sections obtained from six psoriatic patients and 18 beige-SCID mice with psoriasiform human skin grafts. Six grafted mice were treated with Stichodactyla helianthus neurotoxin (ShK), a known Kv1.3 blocker. The results showed an increased number of Kv1.3+ cells in the psoriatic skin as well as in the psoriasiform skin grafts as compared with normal skin and normal skin grafts. Injections of ShK showed a marked therapeutic effect in three of six psoriasiform skin grafts. A significantly decreased number of Kv1.3+ cells was observed in the responders compared with the control grafts. This pilot study, although performed in a small number of mice, reveals the possible beneficial effect of Kv1.3 blockers in psoriasis patients.
The conduction properties of the voltage-gated potassium channel Kv1.3 and its modes of interaction with several polypeptide venoms are examined using Brownian dynamics simulations and molecular dynamics calculations. Employing an open-state homology model of Kv1.3, we first determine current-voltage and current-concentration curves and ascertain that simulated results accord with experimental measurements. We then investigate, using a molecular docking method and molecular dynamics simulations, the complexes formed between the Kv1.3 channel and several Kv-specific polypeptide toxins that are known to interfere with the conducting mechanisms of several classes of voltage-gated K(+) channels. The depths of potential of mean force encountered by charybdotoxin, α-KTx3.7 (also known as OSK1) and ShK are, respectively, -19, -27, and -25 kT. The dissociation constants calculated from the profiles of potential of mean force correspond closely to the experimentally determined values. We pinpoint the residues in the toxins and the channel that are critical for the formation of the stable venom-channel complexes.
Blockade of T-lymphocyte KCa3.1 and Kv1.3 channels as novel immunosuppression strategy to prevent kidney allograft rejection
Currently, there is an unmet clinical need for novel immunosuppressive agents for long-term prevention of kidney transplant rejection as alternatives to the nephrotoxic calcineurin inhibitor cyclosporine (CsA). Recent studies have shown that K(+) channels have a crucial role in T-lymphocyte activity. We investigated whether combined blockade of the T-cell K(+) channels K(Ca)3.1 and K(v)1.3, both of which regulate calcium signaling during lymphocyte activation, is effective in prevention of rejection of kidney allografts from Fisher rats to Lewis rats. All recipients were initially treated with CsA (5 mg/kg d) for 7 days. In rats with intact allograft function, treatment was continued for 10 days with either CsA (5 mg/kg d), or a combination of TRAM-34 (K(Ca)3.1 inhibitor; 120 mg/kg d) plus Stichodactyla helianthus toxin (ShK, K(v)1.3 inhibitor; 80 microg/kg 3 times daily), or vehicle alone. Kidney sections were stained with periodic acid-Schiff or hematoxylin-eosin and histochemically for markers of macrophages (CD68), T-lymphocytes (CD43), or cytotoxic T-cells (CD8). Our results showed that treatment with TRAM-34 and ShK reduced total interstitial mononuclear cell infiltration (-42%) and the number of CD43+ T-cells (-32%), cytotoxic CD8+ T-cells (-32%), and CD68+ macrophages (-26%) in allografts when compared to vehicle treatment alone. Efficacy of TRAM-34/ShK treatment was comparable with that of CsA. In addition, no visible organ damage or other discernible adverse effects were observed with this treatment. Thus, selective blockade of T-lymphocyte K(Ca)3.1 and K(v)1.3 channels may represent a novel alternative therapy for prevention of kidney allograft rejection.
Molecular mechanism of the sea anemone toxin ShK recognizing the Kv1.3 channel explored by docking and molecular dynamic simulations
Computational methods are employed to simulate the interaction of the sea anemone toxin ShK in complex with the voltage-gated potassium channel Kv1.3 from mice. All of the available 20 structures of ShK in the Protein Data Bank were considered for improving the performance of the rigid protein docking of ZDOCK. The traditional and novel binding modes were obtained among a large number of predicted complexes by using clustering analysis, screening with expert knowledge, energy minimization, and molecular dynamic simulations. The quality and validity of the resulting complexes were further evaluated to identify a favorable complex structure by 500 ps molecular dynamic simulations and the change of binding free energies with a computational alanine scanning technique. The novel and reasonable ShK-Kv1.3 complex structure was found to be different from the traditional model by using the Lys22 residue to block the channel pore. From the resulting structure of the ShK-Kv1.3 complex, ShK mainly associates the channel outer vestibule with its second helical segment. Structural analysis first revealed that the Lys22 residue side chain of the ShK peptide just hangs between C and D chains of the Kv1.3 channel instead of physically blocking the channel pore. The obvious loss of the ShK Ser20Ala and Tyr23Ala mutant binding ability to the Kv1.3 channel is caused by the conformational change. The five hydrogen bonds between Arg24 in ShK and H404(A) and D402(D) in Kv1.3 make Arg24 the most crucial for its binding to the Kv1.3 channel. Besides the detailed interaction between ShK and Kv1.3 at the atom level, the significant conformational change induced by the interaction between the ShK peptide and the Kv1.3 channel, accompanied by the gradual decrease of binding free energies, strongly implies that the binding of the ShK peptide toward the Kv1.3 channel is a dynamic process of conformational rearrangement and energy stabilization. All of these can accelerate the development of ShK structure-based immunosuppressants.
During the last two decades since the identification and characterization of T cell potassium channels great advances have been made in the understanding of the role of these channels in T cell functions, especially in antigen-induced activation. Their limited tissue distribution and the recent discovery that different T cell subtypes carrying out distinct immune functions show specific expression levels of these channels have made T cell potassium channels attractive targets for immunomodulatory drugs. Many toxins of various animal species and a structurally diverse array of small molecules inhibiting these channels with varying affinity and selectivity were found and their successful use in immunosuppression in vivo was also demonstrated. Better understanding of the topological differences between potassium channel pores, detailed knowledge of toxin and small-molecule structures and the identification of the binding sites of blocking compounds make it possible to improve the selectivity and affinity of the lead compounds by introducing modifications based on structural information. In this review the basic properties and physiological roles of the voltage-gated Kv1.3 and the Ca2+-activated IKCa1 potassium channels are discussed along with an overview of compounds inhibiting these channels and approaches aiming at producing more efficient modulators of immune functions for the treatment of diseases like sclerosis multiplex and type I diabetes.
Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well-characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. Chinese hamster ovary (CHO)-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. Stichodactyla helianthus peptide (ShK), isolated from S. helianthus venom and a known high-affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit 86Rb+ efflux from CHO-K1.hKv3.2b (IC50 approximately 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus laevis oocytes or in planar patch-clamp studies, ShK inhibited hKv3.2b channels with IC50 values of approximately 0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic beta cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with the blockade of Kv3 channels (i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential after hyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections). Taken together, these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations.
Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases
The voltage-gated Kv1.3 K(+) 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) = 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 mug/kg) attains “steady state” blood levels of approximately 300 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.
Potassium channel blockade by the sea anemone toxin ShK for the treatment of multiple sclerosis and other autoimmune diseases
Expression of the two lymphocyte potassium channels, the voltage-gated channel Kv1.3 and the calcium activated channel IKCa1, changes during differentiation of human T cells. While IKCa1 is the functionally dominant channel in naive and “early” memory T cells, Kv1.3 is crucial for the activation of terminally differentiated effector memory (TEM) T cells. Because of the involvement of TEM cells in autoimmune processes, Kv1.3 is regarded as a promising target for the treatment of T-cell mediated autoimmune diseases such as multiple sclerosis and the prevention of chronic transplant rejection. ShK, a 35-residue polypeptide toxin from the sea anemone, Stichodactyla helianthus, blocks Kv1.3 at low picomolar concentrations. ShK adopts a central helix-kink-helix fold, and alanine-scanning and other mutagenesis studies have defined its channel-binding surface. Models have been developed of how this toxin effects K+-channel blockade and how its docking configuration might differ in ShK-Dap22, which contains a single side chain substitution that confers specificity for Kv1.3 blockade. ShK, ShK-Dap22 and the Kv1.3 blocking scorpion toxin kaliotoxin have been shown to prevent and treat experimental autoimmune encephalomyelitis in rats, a model for multiple sclerosis. A fluoresceinated analog of ShK, ShK-F6CA, has been developed, which allows the detection of activated TEM cells in human and animal blood samples by flow cytometry and the visualization of Kv1.3 channel distribution in living cells. ShK and its analogs are currently undergoing further evaluation as leads in the development of new biopharmaceuticals for the treatment of multiple sclerosis and other T-cell mediated autoimmune disorders.
An “Ala scan” analysis of ShK toxin, a 35-residue basic peptide possessing three disulfide bonds, identifies seven side chains which influence binding to brain delayed rectifier potassium channels. Additional analogs were synthesized and tested to further decipher the roles of these residues, particularly Tyr23. The inhibitory effects of these analogs on 125I-labeled dendrotoxin binding to rat brain membranes showed that replacement of Tyr23 with Ala drastically lowered the affinity of the toxin for the Kv1.2 channels. Ala substitution of Phe27 reduced potency more than 15-fold. Monosubstituted Ala analogs for Ile7, Ser20, or Lys30 each displayed 5-fold reductions in potency. Thus, aromaticity at position 23 is important for effective delayed rectifier brain K channel binding. In contrast, the aromatic residue at position 27 was not critical, since cyclohexylalanine substitution increased affinity. The solution structure of ShK toxin clusters Ile7, Arg11, Ser20, Lys22, Tyr23, and Phe27 in close proximity, forming the potassium channel binding surface of the toxin. We propose an essential binding surface on the toxin in which Lys22 and Tyr23 are major contributors, through ionic and aromatic (hydrophobic) interactions, with the potassium channel.
Pennington MW., et al. (1996) An essential binding surface for ShK toxin interaction with rat brain potassium channels. Biochemistry. PMID: 8987971
Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus
A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and alpha-dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.