Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating μ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.

Most clinically used opioids are thought to induce analgesia through activation of the mu opioid receptor (MOR). However, disparities have been observed between the efficacy of opioids in activating the MOR in vitro and in inducing analgesia in vivo . In addition, some clinically used opioids do not produce cross-tolerance with each other, and desensitization produced in vitro does not match tolerance produced in vivo . These disparities suggest that some opioids could be acting through other targets in vivo , but this has not been comprehensively tested. We thus screened 9 clinically relevant opioids (buprenorphine, hydrocodone, hydromorphone, morphine, O-desmethyl-tramadol, oxycodone, oxymorphone, tapentadol, tramadol) against 9 pain-related receptor targets (MOR, delta opioid receptor [DOR], kappa opioid receptor [KOR], nociceptin receptor [NOP], cannabinoid receptor type 1 [CB1], sigma-1 receptor [σ1R], and the monoamine transporters [NET/SERT/DAT]) expressed in cells using radioligand binding and functional activity assays. We found several novel interactions, including monoamine transporter activation by buprenorphine and σ1R binding by hydrocodone and tapentadol. Tail flick anti-nociception experiments with CD-1 mice demonstrated that the monoamine transporter inhibitor duloxetine selectively promoted buprenorphine anti-nociception while producing no effects by itself or in combination with the most MOR-selective drug oxymorphone, providing evidence that these novel interactions could be relevant in vivo . Our findings provide a comprehensive picture of the receptor interaction profiles of clinically relevant opioids, which has not previously been performed. Our findings also suggest novel receptor interactions for future investigation that could explain some of the disparities observed between opioid performance in vitro and in vivo .

Opioid activation of the mu opioid receptor (MOR) promotes signaling cascades that evoke both analgesic responses to pain and side effects like addiction and dependence. Manipulation of these cascades, such as by biased agonism, has great promise to improve opioid therapy. However, the signaling cascades of the MOR are in general poorly understood, providing few targets for drug development. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel and crucial regulator of opioid anti-nociception in the brain by promoting ERK MAPK activation. In this study, we sought to identify the molecular isoforms and co-chaperones by which Hsp90 carried out this role, which could provide specific targets for future clinical intervention. We used novel selective small molecule inhibitors as well as CRISPR/Cas9 gene editing constructs delivered by the intracerebroventricular (icv) route to the brains of adult CD-1 mice to target Hsp90 isoforms (Hsp90α/β, Grp94) and co-chaperones (p23, Cdc37, Aha1). We found that inhibition of the isoform Hsp90α fully blocked morphine anti-nociception in a model of post-surgical paw incision pain, while blocking ERK and JNK MAPK activation, suggesting Hsp90α as the main regulator of opioid response in the brain. We further found that inhibition of the co-chaperones p23 and Cdc37 blocked morphine anti-nociception, suggesting that these co-chaperones assist Hsp90α in promoting opioid anti-nociception. Lastly, we used cycloheximide treatment in the brain to demonstrate that rapid protein translation within 30 min of opioid treatment is required for Hsp90 regulation of opioid response. Together these studies provide insight into the molecular mechanisms by which Hsp90 promotes opioid anti-nociception. These findings thus both improve our basic science knowledge of MOR signal transduction and could provide future targets for clinical intervention to improve opioid therapy.

The opioid epidemic has plagued the United States with high levels of abuse and poor quality of life for chronic pain patients requiring continuous use of opioids. New drug discovery efforts have been implemented to mitigate this epidemic; however, new medications are still limited by low efficacy and/or high side effect and abuse potential. Intermittent fasting (IF) has recently been shown to improve a variety of pathological states, including stroke and neuroinflammation. Numerous animal and human studies have shown the benefits of IF in these disease states, but not in pain and opioid treatment. We thus subjected male and female CD-1 mice to 18-hour fasting intervals followed by 6-hour feed periods with standard chow for 1 week. Mice that underwent this diet displayed an enhanced antinociceptive response to morphine both in efficacy and duration using thermal tail-flick and postoperative paw incision pain models. While showing enhanced antinociception, IF mice also demonstrated no morphine reward and reduced tolerance and constipation. Seeking a mechanism for these improvements, we found that the mu-opioid receptor showed enhanced efficacy and reduced tolerance in the spinal cord and periaqueductal gray, respectively, from IF mice using a 35S-GTPγS coupling assay. These improvements in receptor function were not due to changes in mu-opioid receptor protein expression. These data suggest that a daily IF diet may improve the therapeutic index of acute and chronic opioid therapies for pain patients in the clinic, providing a novel tool to improve patient therapy and reduce potential abuse.

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The various isoforms of the chaperone protein heat shock protein 90 (Hsp90) in conjunction with specific co‐chaperones are key regulators of signal transduction. We have recently identified a novel role for Hsp90 within downstream mu opioid receptor (MOR) signaling both within the brain and spinal cord. Within the brain, we found that Hsp90α and the co‐chaperones Cdc37 and p23 promote MOR‐induced ERK phosphorylation and subsequent anti‐nociception. However, in the spinal cord Hsp90 represses MOR‐induced ERK phosphorylation and subsequent anti‐nociception, so that Hsp90 inhibitor treatment in the spinal cord results in enhanced opioid pain relief. This study aimed to identify the Hsp90 isoforms and co‐chaperones necessary for this mechanism within the spinal cord, and whether they differ from the brain. We used novel isoform and co‐chaperone selective small molecule inhibitors and in vivo gene knockdown using CRISPR/Cas9 to selectively block each of the Hsp90 isoforms and co‐chaperones in the spinal cords of male and female CD‐1 mice. We then used opioid‐induced anti‐nociception in the tail flick assay as a readout, combined with immunohistochemistry to validate CRISPR/Cas9 knockdown in the spinal cord. In contrast to our earlier brain studies, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all acted to repress opioid anti‐nociception in the spinal cord, in that inhibiting these isoforms resulted in increased opioid anti‐nociception. We similarly found that the co‐chaperones Cdc37, p23, and Aha1 all similarly repressed opioid anti‐nociception in the spinal cord. These findings suggest that Hsp90β, Grp94, and Aha1 all act within the spinal cord but not the brain to regulate opioid anti‐nociception. These findings also suggest, in line with our earlier studies, that blocking these isoforms with systemic selective inhibitors could represent a novel therapeutic approach to enhance opioid therapy, increasing analgesic efficacy while reducing side effects like tolerance and reward/addiction. Support or Funding Information These studies were supported by an Arizona Biomedical Research Commission New Investigator Award (#ADHS18‐198875) and institutional funds from the University of Arizona to JMS, and NIH R01CA213566 to BB. BB is a founder with an equity stake in Grannus Therapeutics, a virtual startup for developing novel Hsp90 inhibitors. No other author has any relevant conflicts to declare.

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The opioid epidemic has continued to plague the United States with high levels of abuse and poor quality of life for chronic pain patients requiring continuous use of opioids. Many potential solutions have been proposed to mitigate this epidemic including new medications to reduce the abuse and side effect profiles of opioids; however, new medications are plagued by low efficacy, abuse potential, and toxicities. These limitations suggest that non‐pharmacological interventions could be more efficacious with low side effect profiles. Intermittent fasting (IF) is a time restriction diet which has recently grown in popularity over the past decade in the prevention and mitigation of a variety of pathological states. Numerous animal and human studies have shown the benefits of IF in these disease states, but not in opioid treatment paradigms. We thus subjected male and female CD1 mice to 18‐hour fasting intervals followed by 6‐hour feed periods with standard chow for 1 week. Mice which underwent this diet displayed an enhanced anti‐nociceptive response to systemic morphine both in efficacy and duration using thermal tail flick and post‐operative paw incision pain models. While showing enhanced anti‐nociception, IF mice also demonstrated a reduction in the development of anti‐nociceptive tolerance using the thermal tail flick assay paired with daily systemic morphine treatment. We also found a reduction in the development of opioid induced constipation using both fecal mass and fecal pellet count in IF mice. Seeking a mechanism for our behavioral findings, we performed 35S‐GTPγS assays to assess the functionality of mu opioid receptors within the spinal cord and periaqueductal grey (PAG) tissues from IF mice with and without chronic morphine treatment. The resulting DAMGO concentration‐response curves demonstrated an increased efficacy of mu opioid receptors within the spinal cord in IF mice and an absence of the development of tolerance within the PAG in IF mice, suggesting site‐specific receptor mechanisms for the behavioral effects we observed. These data suggest that a daily IF diet may improve the therapeutic index of acute and chronic opioid therapies for pain patients in the clinic, both in improved analgesia and reduced tolerance and constipation.