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Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain

Significance Chronic pain is one of the most pressing national healthcare problems, and as a result, chronic pain was named by the NIH as the “silent epidemic.” Consequentially, chronic pain has directly contributed to the disproportionate increase in opioid use in America. However, thus far, there has been little progress in discovery of alternative noninvasive therapeutics for chronic pain. The current report outlines the repurposing of three tetracyclines for treatment of neuropathic pain by targeting the catalytic domain of EphB1 kinase. These results can be readily applicable clinically for treatment of neuropathic pain and provide proof of concept for the development of molecules for targeting Eph/ephrin-mediated pathologies. Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.