The Hsp90 Isoform‐Selective Inhibitor KUNG65 Enhances Opioid Anti‐Nociception while Reducing Tolerance

Opioids are the gold standard for pain management, however, adverse side effects like respiratory depression and addiction limit their viability. One approach to improve opioid therapy focuses on a dose-reduction strategy by amplifying opioid analgesia without boosting side effects, so that less opioid can be given. To this end, we've focused on Heat shock protein 90 (Hsp90), a central protein regulator in the cell; ongoing studies from our lab have shown that inhibition of Hsp90 in the spinal cord improves morphine anti-nociceptive potency by 2-3 fold in acute and chronic pain while reducing tolerance, rescuing established tolerance, and not changing reward and constipation, enabling a dose-reduction strategy. However, our results also showed that non-selective Hsp90 inhibitors given systemically (intravenous, IV) have the opposite effect, blocking opioid pain relief. Seeking a means to surmount this roadblock, we determined which Hsp90 isoforms regulate opioid pain relief in brain and spinal cord in mice using selective small molecule inhibitors and CRISPR/Cas9 gene editing. We found that Hsp90α alone regulated opioid signaling and pain relief in the brain. However, in the spinal cord, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all regulated opioid signaling and pain relief. This led to our hypothesis that targeting spinal cord Hsp90 with isoform-selective inhibitors given IV could boost opioid pain relief without altering side effects, enabling a dose-reduction strategy. We tested this hypothesis with the novel Grp94-selective inhibitor KUNG65, given at a 1 mg/kg dose IV in male and female CD-1 mice, followed by a 24 hr treatment time, then analysis of opioid anti-nociception and side effects. We found that systemic (IV) KUNG65 treatment resulted in a 1.9 fold increase in morphine potency to relieve tail flick pain, consistent with our earlier studies injecting inhibitor directly into spinal cord. We also found that KUNG65 boosted morphine potency in paw incision pain, with a 2.2 fold increase. Additionally, we found that KUNG65 injection could rescue established morphine tolerance in the tail flick assay, again as we found for direct spinal cord injection. These results support our hypothesis that isoform-selective Hsp90 inhibitors can selectively engage Hsp90 in the spinal cord when given systemically, resulting in improved opioid anti-nociception and side effects. These results strongly suggest that Hsp90 isoform-selective inhibitors could be a powerful new tool to improve opioid therapy through a dose-reduction strategy, and further show that this effect can be achieved through a translationally relevant dosing route.