Homology‐guided mutational analysis reveals the functional requirements for antinociceptive specificity of CRMP2‐derived peptides

Background and Purpose N-type voltage-gated calcium (CaV2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Despite CaV2.2 antagonism being recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and are often mired by numerous side-effects. Collapsin response mediator protein 2 (CRMP2) targets CaV2.2 to the sensory neuron membrane, and allosterically modulates Cav2.2 functionally. A fifteen amino acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and CaV2.2 to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. In this study, we set out to map the minimal domain of CBD3 necessary for its antinociceptive potential. Experimental Approach Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons, binding between CRMP2 and CaV2.2, whole-cell voltage clamp electrophysiology, and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120 (gp120). Key Results The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, alanine (at position one) and arginine (at position four), as critical for antinociception in two pain models. Conclusions and Implications These results identify an antinociceptive scaffold core in CBD3 that can be used for development of small-molecule mimetics of CBD3.