A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

Assessing the cellular toxicity of peptide inhibitors of intracellular protein-protein interactions by microinjection.

Inhibitors of protein-protein interactions can be developed through a number of technologies to provide leads that include cell-impermeable molecules. Redesign of these impermeable leads to provide cell-permeable derivatives can be challenging and costly. We hypothesised that intracellular toxicity of leads could be assessed by microinjection prior to investing in the redesign process. We demonstrate this approach for our development of inhibitors of the protein-protein interaction between inducible nitric-oxide synthase (iNOS) and SPRY domain-containing SOCS box proteins (SPSBs). We microinjected a lead molecule into AD-293 cells and were able to perform an intracellular toxicity assessment. We also investigated the intracellular distribution and localisation of injected inhibitor using a fluorescently-labelled analogue. Our findings show that a lead peptide inhibitor, CP2, had no toxicity even at intracellular concentrations four orders of magnitude higher than its Kd for binding to SPSB2. This early toxicity assessment justifies further development of this cell-impermeable lead to confer cell permeability. Our investigation highlights the utility of microinjection as a tool for assessing toxicity during development of drugs targeting protein-protein interactions.