We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel V1.7">NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of V1.7">NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2–Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly V1.7">NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of V1.7">NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce V1.7">NaV1.7 currents and reverse neuropathic pain.
Targeting CRMP2 SUMOylation by disrupting the Ubc9 E2 SUMO-conjugating enzyme–CRMP2 interaction is a promising therapeutic strategy for chronic pain.
aDepartment of Pharmacology, Graduate Interdisciplinary Program, College of Medicine, Tucson, AZ, United States
bDepartment of Pharmacology and Toxicology, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, United States
Departments of cNeurology and
dPharmacology, the First Hospital of Jilin University, Jilin University, Changchun, Jilin Province, China
eDepartment of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ, United States,
fThe Center for Innovation in Brain Sciences, The University of Arizona Health Sciences, Tucson, AZ, United States
gDepartment of Neuroscience, Graduate Interdisciplinary Program, College of Medicine, Tucson, AZ, United States
Corresponding author. Address: Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Dr, P.O. Box 245050, Tucson, AZ 85724, United States. Tel.: (520) 626-4281; fax: (520) 626-2204. E-mail address: email@example.com (R. Khanna).
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Received January 27, 2018
Received in revised form May 16, 2018
Accepted May 19, 2018