Regulation of Opioid Sensitivity and Tolerance by Ubiquitin Ligase Signaling

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SUMMARY Opioid drugs are the most widely used analgesics, but also abused substances. The adverse actions of these drugs, including peripheral side effects, dependence, tolerance and withdrawal, severely limit their utility for long term pain management. The -opioid receptor (MOR) is the primary target of opioid analgesia, addiction and withdrawal. Thus, efforts aimed at developing safer opioid treatments and managing problematic side effects, such as addiction and withdrawal, require a much deeper understanding of the genetic and molecular mechanisms that regulate MOR and the behavioral effects of opioid drugs. Our long-term goal is to use unbiased, large-scale forward genetics and proteomics, as well as targeted biochemistry and pharmacology to understand how ubiquitin ligase signaling affects opioid sensitivity and tolerance at a behavioral level. Towards this goal, we deploy a transgenic MOR (tgMOR) model, in which mammalian MOR is expressed in the nervous system of C. elegans. This imbues C. elegans with opioid- sensitive behaviors that we evaluate using computationally automated assays. Importantly, tgMOR animals exhibit the behavioral hallmarks of opioid responses in higher organisms including acute depressant effects, desensitization and tolerance. We previously used forward genetics and our tgMOR platform to unveil a novel, conserved anti-opioid system that functions from C. elegans through rodent behavioral models. This approach has now identified a ubiquitin ligase that regulates opioid sensitivity and behavioral tolerance to repeated opioid exposure. Our proposal represents an unprecedented opportunity to decipher how ubiquitin ligase signaling affects opioid drug responses on a behavioral genetic level. Our first aim will use two primary approaches to determine how this ubiquitin ligase shapes opioid behavioral responses. 1) We will use unbiased, large-scale forward suppressor genetics combined with whole- genome sequencing and CRISPR/Cas9 editing to identify mutations that rescue abnormal opioid responses in tgMOR ubiquitin ligase mutants. This will identify genes and genetic networks that are inhibited by ubiquitin ligase activity to influence opioid responses. 2) We will deploy unbiased, large-scale targeted proteomics with this ubiquitin ligase that will determine its interactome, binding proteins and putative substrates. Importantly, this aim features two approaches that are unbiased and large-scale but also complementary. In the second aim, we use in depth biochemical and structural studies to evaluate candidate substrates for this ubiquitin ligase. Using orthologous human ubiquitin ligases provides an important translational component to our proposal. We further aim to develop a high throughput screen (HTS) compatible assay for these human ubiquitin ligases. Finally, we use this HTS compatible assays in a pilot screen for small molecule inhibitors of these ubiquitin ligases. Our aims represent first of their kind studies on how ubiquitin ligase signaling affects opioid responses using an innovative new whole animal behavioral model.