Brain Delivery of a Kv1.3-Blocking Peptide is not Enhanced by Conjugation to Blood–Brain Barrier Shuttle Peptides

The voltage-gated potassium channel KV1.3 is upregulated on pro-inflammatory microglia, and its blockade is effective in reducing neuroinflammation in vitro and in vivo. The most potent and selective KV1.3 blockers identified to date are disulfide-rich, venom-derived peptides, which are largely excluded from the brain by the blood–brain barrier (BBB). As BBB shuttle peptides have been reported to promote receptor-mediated transport of macromolecules and nanoparticles across the BBB, we have conjugated two such shuttle peptides, MTfpep (MTf) and Angiopep-2 (Ang2), to HsTX1[R14A], a KV1.3-blocking peptide with 34 residues and four disulfide bonds, using click chemistry. NMR spectroscopy showed that the conjugates adopted the same fold as HsTX1[R14A], and electrophysiology assays demonstrated the retention of nanomolar KV1.3 inhibition. Liquid chromatography tandem mass spectrometry (LC-MS/MS) assays were developed to quantify HsTX1[R14A] and MTf-HsTX1[R14A] in buffer, mouse plasma and brain homogenate; however, sufficient analytical sensitivity could not be achieved for Ang2-HsTX1[R14A]. Assessment of peptide permeability in in vitro BBB models showed no evidence of enhanced permeability of MTf-HsTX1[R14A] relative to HsTX1[R14A]. Following intravenous administration of MTf-HsTX1[R14A] to C57BL/6 mice at 4 mg/kg, plasma concentrations were detectable up to 120 min post-dose, but brain concentrations of MTf-HsTX1[R14A] remained below the limit of detection at all timepoints measured. Our results imply that the BBB shuttle peptide MTfpep failed to facilitate brain entry of this cargo. Nevertheless, the click chemistry methods developed for HsTX1[R14A] will facilitate its modular assembly with other brain-targeting ligands and are applicable to conjugation of other disulfide-rich peptides.