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A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases.

ABSTRACT: HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2?kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.

SUBMITTER: Rashid MH

PROVIDER: S-EPMC3968461 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases.

Rashid M Harunur MH Huq Redwan R Tanner Mark R MR Chhabra Sandeep S Khoo Keith K KK Estrada Rosendo R Dhawan Vikas V Chauhan Satendra S Pennington Michael W MW Beeton Christine C Kuyucak Serdar S Norton Raymond S RS

Scientific reports 20140328

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculat ...[more]

PMID: 24676092

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Project description:BACKGROUND:In T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective Kv1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases. METHODS:We combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin. RESULTS:The peptide completely blocked Kv1.3 channels currents without impairing TEM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-? and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of Kv1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery. CONCLUSIONS:The Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of Kv1.3 channels in regulating TEM lymphocyte function.

Dataset Information

A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases.

Publications

A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases.

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