Engineering a Stable and Selective Peptide Blocker of the Kv1.3 Channel in T Lymphocytes

Bachem Bioscience Inc., King of Prussia, Pennsylvania, USA.
Molecular pharmacology (Impact Factor: 4.13). 02/2009; 75(4):762-73. DOI: 10.1124/mol.108.052704
Source: PubMed


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 ShK-192 in Kv1.3 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 microg/kg, approximately 100 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 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.

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Available from: Michael W Pennington,
    • "The high selectivity of this toxin in blocking N-type calcium channels involved in excitatory neurotransmitter release from primary afferent nerve terminals enabled registration as a drug for treatment of pain (McIntosh et al. 1982; Miljanich 2004). Another heralded achievement was the engineering of ShK 192, a highly active analogue of the naturally occurring toxin peptide ShK-L5 from the sea anemone Stichodactyla helianthus, to blocking of K v 1.3 channels and raising a therapeutic potential in autoimmune diseases mediated by effector memory T cells (Pennington et al. 2009). The recent reports on the structure of bacterial voltage-gated sodium channels (Payandeh et al. 2011; Zhang et al. 2012) give hope that it remains a matter of technological breakthrough before the structure of a heterotetrameric Na v is solved, which would enable determination of fine details regarding toxin-channel interactions and design of antagonists to various channelopathies. "
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    ABSTRACT: Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Navs) at two pharmacologically distinct sites. Alpha toxins bind at receptor site 3 and inhibit channel inactivation, whereas beta toxins bind at receptor site 4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and competition for receptor sites at Nav subtypes. To elucidate the surface of interaction of the two toxin classes with Navs and clarify the molecular basis of varying toxin preferences, an efficient expression system was established. Mutagenesis accompanied by toxicity, binding, and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography, uncovered the bioactive surfaces of toxin representatives of all pharmacological groups. Exchange of external loops between channels that exhibit marked differences in sensitivity to various toxins accompanied by point mutagenesis highlighted channel determinants that play a role in toxin selectivity. These data were used in further mapping of the brain channel rNav1.2a receptor sites for the beta-toxin Css4 (from Centruroides suffusus suffusus) and the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). On the basis of channel mutations that affected Css4 activity, the known structure of the toxin and its bioactive surface, and using the structure of a potassium channel as template, a structural model of Css4 interaction with the gating module of domain II was constructed. This initial model was the first step in the identification of part of receptor site 4. In parallel, a swapping and a mutagenesis approach employing the rNav1.2a mammalian and DmNav1 insect Navs and the toxin Lqh2 as a probe were used to search for receptor site 3. The channel mapping along with toxin dissociation assays and double-mutant cycle analyses using toxin and channel mutants identified the gating module of domain IV as the site of interaction with the toxin core domain, thus describing the docking orientation of an alpha toxin at the channel surface.
    Scorpion Venoms, 01/2015: pages 471-491; , ISBN: 978-94-007-6403-3
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    • "A further potent K v 1.3 blocking toxin is found in the sea anemone Stichodactyla helianthus. Recombinant peptides that are derived from its Stichodactyla toxin (ShK) are evaluated as potential novel therapeutics for autoimmune diseases (Beeton et al., 2008; Pennington et al., 2009; Tarcha et al., 2012) but being peptides they need to be administered per injection and have unfavorable pharmacokinetics with very short half-lives. Both kaliotoxin and ShK block the potassium pore of voltage gated potassium channels with higher affinity to K v 1.3 over the other subtypes (Judge & Bever, 2006), hence providing some immune specificity. "
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    ABSTRACT: The association between vitamin D and multiple sclerosis has (re)-opened new interest in nutrition and natural compounds in the prevention and treatment of this neuroinflammatory disease. The dietary amount and type of fat, probiotics and biologicals, salmon proteoglycans, phytoestrogens and protease inhibitor of soy, sodium chloride and trace elements, and fat soluble vitamins including D, A and E were all considered as disease-modifying nutraceuticals. Studies in experimental autoimmune encephalomyelitis mice suggest that poly-unsaturated fatty acids and their 'inflammation-resolving' metabolites and the gut microflora may reduce auto-aggressive immune cells and reduce progression or risk of relapse, and infection with whipworm eggs may positively change the gut-brain communication. Encouraged by the recent interest in multiple sclerosis-nutrition nature's pharmacy has been searched for novel compounds with anti-inflammatory, immune-modifying and antioxidative properties, the most interesting being the scorpion toxins that inhibit specific potassium channels of T cells and antioxidative compounds including the green tea flavonoid epigallocatechin-3-gallate, curcumin and the mustard oil glycoside from e.g. broccoli, sulforaphane. They mostly also inhibit pro-inflammatory signaling through NF-κB or toll-like receptors and stabilize the blood brain barrier. Disease modifying functions may also complement analgesic and anti-spastic effects of cannabis, its constituents, and of 'endocannabinoid enhancing' drugs or nutricals like inhibitors of fatty acid amide hydrolase. Nutricals will not solve multiple sclerosis therapeutic challenges but possibly support pharmacological interventions or unearth novel structures. Copyright © 2014. Published by Elsevier Inc.
    Pharmacology [?] Therapeutics 11/2014; 148. DOI:10.1016/j.pharmthera.2014.11.015 · 9.72 Impact Factor
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    • "This study assessed four sea anemone neurotoxins from B. granulifera and S. helianthus. The neurotoxin crystalline structures were obtained from the Protein Data Bank (PDB) under the accession codes: 1BGK derived from B. granulifera;33 1ROO the pure ShK toxin, which belongs to S. helianthus;18 1BEI that corresponds to the mutated ShK-Dap;10 and 2K9E, the potent selective ShK-192 modified from ShK–170.32 These neurotoxins were chosen because of lack of structural information reported about their interaction with the Kv1.3 channel. "
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    ABSTRACT: Sea anemone neurotoxins are peptides that interact with Na(+) and K(+) channels, resulting in specific alterations on their functions. Some of these neurotoxins (1ROO, 1BGK, 2K9E, 1BEI) are important for the treatment of about 80 autoimmune disorders because of their specificity for Kv1.3 channel. The aim of this study was to identify the common residues among these neurotoxins by computational methods, and establish whether there is a pattern useful for the future generation of a treatment for autoimmune diseases. Our results showed eight new key common residues between the studied neurotoxins interacting with a histidine ring and the selectivity filter of the receptor, thus showing a possible pattern of interaction. This knowledge may serve as an input for the design of more promising drugs for autoimmune treatments.
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