John M. Streicher - US grants

The mu and delta opioid receptors (MOR, DOR) modulate many of the same brain processes in vivo, including tolerance and anti-nociception in response to opioid drugs. Many groups have found that inhibiting the DOR through various means decreases the side effects of MOR agonists like morphine. While the basis for this interaction is unknown, one strong possibility is the formation of a MOR-DOR heterodimer (MDOR). Many groups have shown that the MDOR can form in heterologous expression systems in vitro, with a unique pharmacology and signal transduction profile when compared to the monomeric forms. Devi and colleagues recently developed an MDOR selective antibody, and used this antibody to demonstrate MDOR upregulation in the brains of mice chronically treated with morphine. Other experiments suggested that the MDOR promotes tolerance, dependence, and drug seeking in vivo. While MDOR selective agonists have been developed, no known drug-like antagonist has ever been created to our knowledge, limiting our ability to determine the role of MDOR in vivo. To address this lack, we created a novel series of potential selective peptide MDOR antagonists by connecting low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2) and moderate affinity DOR (Tyr-Tic- OH) pharmacophores with a variable length (15-42 atom) flexible polyamide spacer. We tested this preliminary series in vitro using radioligand binding and 35S-GTP?S coupling in antagonist mode using MOR, DOR, and MDOR expressing cell lines. We found compelling evidence that our preliminary series selectively targets the MDOR, with a selectivity ratio of ~91 fold for our best compound, the 24 atom spacer length. Building from this initial success, we aim in the current proposal to explore the MDOR structure-activity relationship (SAR) of our compound series, and improve compound potency and selectivity at the MDOR by modulating pharmacophore affinity (increased MOR, decreased DOR) as well as linker rigidity (minimally rigid [gly-gly-pro], moderately rigid [gly-pro]). These studies will be performed in an iterative development process, using the best compound from each series to inform the next series, minimizing compound number. After this SAR development, we will use our most potent and selective compound to begin to test MDOR activity in vivo. This will be accomplished by intracerebroventricular (icv) and intrathecal (it) injection of MDOR antagonist into mice prior to tail-flick anti- nociception evoked by MOR (DAMGO), DOR (DSLET), and MDOR (CYM51010) selective agonists. These studies will demonstrate the selectivity of our compound in vivo. We will also pre-treat mice with MDOR antagonist prior to the induction of tolerance and dependence with morphine to begin to explore the in vivo role of the MDOR in these opioid side effects, which has been suggested by the literature. In the short term, this initial optimized series will provide a useful tool to interrogate the role of the MDOR in vivo. Long term, through modifications to improve drugability (glycosylation, etc.), these compounds may provide the basis for selective MDOR targeted therapeutics to improve the side effect profile of opioid therapy.

ABSTRACT The management of chronic pain is clinically challenging, and relies heavily on opioid drugs like morphine and oxycodone. However, opioids are plagued by numerous side effects that impact quality of life, like tolerance, constipation, and reward/addiction, contributing to an opioid abuse, addiction, and overdose crisis. These clinical and social challenges highlight the vast medical need for new approaches to pain management. To this end, we have pioneered an investigation into the role of Heat shock protein 90 (Hsp90) in regulating opioid signal transduction, 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 MOR signaling and anti-nociception, so that Hsp90 inhibition in spinal cord enhances opioid anti-nociception. In further studies, we found that Hsp90 inhibition in spinal cord increases morphine anti-nociceptive potency 2-3 fold in acute and chronic pain, reduces tolerance and rescues established tolerance, all without altering the potency of constipation and reward. These results suggest that spinal Hsp90 inhibition could be used as an opioid dose-reduction strategy, to improve or maintain analgesic efficacy while reducing side effects. However, one challenge to this approach is our finding that non-selective Hsp90 inhibitors, when given systemically, gave results similar to the brain, blocking opioid anti-nociception. Seeking a way around this limitation, we found that Hsp90 isoforms differ between brain and spinal cord, with Hsp90? alone acting in brain while Hsp90?, Hsp90?, and Grp94 all act in spinal cord. Hypothesizing that an isoform-selective Hsp90 inhibitor could be used to target spinal 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 strongly suggest 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 an uncertain therapeutic profile. In this proposal, we will thus optimize KUNB106 to create a new therapeutic to enhance opioid therapy and reduce opioid side effects like reward/addiction. In Aim 1 we will utilize cutting edge medicinal chemistry approaches using Hsp90 isoform co-crystallized structures to create optimized compounds based on the KUNB106 scaffold. In Aim 2, we will test these compounds for Hsp90 isoform selectivity, ADMET parameters, off-target interactions, and in vivo PK in mice, aiming to identify highly selective, soluble, and orally bioavailable compounds. In Aim 3, we will test the best of these compounds for their efficacy in enhancing opioid anti-nociception in acute and chronic pain models in mice, while reducing tolerance, constipation, reward, and respiratory depression. Top candidates will be tested for off-target side effects and toxicity. Through this project, we aim to create optimized candidates for further development as new therapeutics for patient pain management.

ABSTRACT Chronic pain is a serious and worsening epidemic in the United States and worldwide, seriously degrading patient quality of life. Opioid drugs like morphine are the ?gold standard? for treating moderate to severe chronic pain, however, they are burdened by major side effects, especially addiction liability, which has contributed to a paralell epidemic of opioid addiction, abuse, and overdose. In addition, opioids are ineffective in some pain types, most notably neuropathic pain. In the search for alternatives, phytocannabinoids from Cannabis sativa have been heavily studied. However, cannabinoids have generally been shown to have modest to poor efficacy, and have their own side effects, especially psychoactive side effects with ?9-tetrahydrocannabinol treatment. This has led again to a search for methods to improve cannabinoid therapy. For this reason, research has focused on the ~150 terpene compounds found in Cannabis, which impart flavor and aroma to the plant. Limited evidence suggests that terpenes produce pain relief on their own, and they have also been proposed to modulate and potentially improve the effects of cannabinoids like THC, termed the ?entourage effect? hypothesis. However the quality of evidence on terpene efficacy is in general poor, limited by poorly-defined and complex extracts, and few mechanistic studies. We thus performed a preliminary study on the Cannabis terpenes ?- humulene, ?-pinene, geraniol, and linalool. We found that all 4 terpenes produced anti-nociception in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) comparable or better than morphine. At the same time, geraniol and linalool produced no reward or aversion, suggesting no addictive or aversive liability. Seeking mechanistic insight, we found that all 4 terpenes produced tail flick anti- nociception by a cannabinoid receptor type 1 (CB1) mechanism, and further synergized with the cannabinoid WIN55,212, providing evidence for the entourage effect hypothesis. We further identified CB2, Adenosine A2a, and anti-inflammatory activity as potential mechanisms of action. In this proposal, we will extend these studies to evaluate therapeutic potential and mechanisms of action of these terpenes in neuropathic pain, providing potential support to the use of these ligands as improved non-opioid pain therapeutics. In Aim 1, we will fully test the terpenes in a mouse model of CIPN, including dose/response, alternate neuropathy models, side effects like tolerance and reward/aversion, synergy with other analgesics such as opioids and cannabinoids, and terpene impact on side effects of these other analgesics (especially opioid reward). In Aim 2, we will identify molecular mechanisms for terpene action in CIPN, focusing on 1) CB1/2, 2) A2a, and 3) anti-inflammatory activity. We will use selective antagonists and CRISPR gene editing, identify sites of action (e.g. brain, spinal cord, periphery), measure tissue response to terpene (e.g. cytokine production), and use in vitro models to confirm these mechanisms. Together these studies will provide a rigorous evaluation of the potential use of terpenes as efficacious and low side-effect therapeutics for neuropathic pain.