Abstract
ABSTRACTDrug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2’s SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.
Highlights
Studies over the last fifteen years have identified the presence of several post-translational modifications (PTMs) within the axonal collapsin response mediator protein 2 (CRMP2) that act as a “molecular switchboard” to facilitate both canonical as well as atypical functions of this protein [1,2,3,4,5,6]
We recently identified a heptamer peptide containing the CRMP2 SUMOylation consensus motif (CSM), which when conjugated to the cellpenetrant domain of the HIV-1 tat protein, disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and inhibited NaV1.7 sodium influx in sensory neurons [11]
We used the structure of Ubc9 co-crystallized with both SUMO2 and the mammalian guanosine triphosphate (GTP)ase-activating protein (RanGAP1) – one the first proteins reported to be covalently linked to SUMO2 (PDB: 5d2m) [19] – as a structural template
Summary
Studies over the last fifteen years have identified the presence of several post-translational modifications (PTMs) within the axonal collapsin response mediator protein 2 (CRMP2) that act as a “molecular switchboard” to facilitate both canonical (neurite outgrowth, axon specification, tubulin binding) as well as atypical (regulation of ion channels) functions of this protein [1,2,3,4,5,6] Among these modifications, we demonstrated that SUMOylation (addition of a small ubiquitin-like modifier (SUMO) protein) on lysine 374 of CRMP2 controlled trafficking and activity of the voltage-gated sodium channel NaV1.7 [4,7,8].
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