ABSTRACT The management of chronic pain is clinically challenging and relies heavily on opioid drugs like morphineand oxycodone. However opioids are plagued by numerous side effects that impact quality of life like toleranceconstipation and reward/addiction contributing to an opioid abuse addiction and overdose crisis. These clinicaland social challenges highlight the vast medical need for new approaches to pain management. To this end wehave pioneered an investigation into the role of Heat shock protein 90 (Hsp90) in regulating opioid signaltransduction anti-nociception and side effects. We have found that Hsp90 regulates mu opioid receptor (MOR)signal transduction to different effect in brain vs. spinal cord. In brain Hsp90 promotes MOR signaling and anti-nociception so that Hsp90 inhibition in brain blocks opioid anti-nociception. In spinal cord Hsp90 blocks MORsignaling and anti-nociception so that Hsp90 inhibition in spinal cord enhances opioid anti-nociception. In furtherstudies we found that Hsp90 inhibition in spinal cord increases morphine anti-nociceptive potency 2-3 foldin acute and chronic pain reduces tolerance and rescues established tolerance all without altering thepotency of constipation and reward. These results suggest that spinal Hsp90 inhibition could be used as anopioid dose-reduction strategy to improve or maintain analgesic efficacy while reducing side effects. Howeverone challenge to this approach is our finding that non-selective Hsp90 inhibitors when given systemically gaveresults similar to the brain blocking opioid anti-nociception. Seeking a way around this limitation we found thatHsp90 isoforms differ between brain and spinal cord with Hsp90 alone acting in brain while Hsp90 Hsp90and Grp94 all act in spinal cord. Hypothesizing that an isoform-selective Hsp90 inhibitor could be used to targetspinal cord-specific isoforms we found that the Hsp90-selective inhibitor KUNB106 enhanced morphine anti-nociception while rescuing established morphine tolerance when given systemically. These results stronglysuggest that Hsp90-selective inhibitors could be used as a novel first-in-class opioid dose-reduction therapy.However KUNB106 is a first generation compound with poor solubility and pharmacokinetics (PK) and anuncertain therapeutic profile. In this proposal we will thus optimize KUNB106 to create a new therapeutic toenhance opioid therapy and reduce opioid side effects like reward/addiction. In Aim 1 we will utilize cutting edgemedicinal chemistry approaches using Hsp90 isoform co-crystallized structures to create optimized compoundsbased on the KUNB106 scaffold. In Aim 2 we will test these compounds for Hsp90 isoform selectivity ADMETparameters off-target interactions and in vivo PK in mice aiming to identify highly selective soluble and orallybioavailable compounds. In Aim 3 we will test the best of these compounds for their efficacy in enhancing opioidanti-nociception in acute and chronic pain models in mice while reducing tolerance constipation reward andrespiratory depression. Top candidates will be tested for off-target side effects and toxicity. Through this projectwe aim to create optimized candidates for further development as new therapeutics for patient pain management.