PROJECT SUMMARY Opioids such as morphine, codeine, and oxycodone are the most potent analgesics widely used in clinical practice. Unfortunately, their utility is severely limited by serious side effects that include development of addiction and tolerance following prolonged use. Opioids exert their clinically significant effects through the activation of the mu-opioid receptor (MOR). We have identified that the critical role in controlling the extent of MOR signaling and dissociating beneficial (analgesia) from detrimental (tolerance and dependence) effects of opioids belongs to a complex of proteins composed of Regulator of G protein Signaling type 7 (RGS7) and its subunit: RGS7 Binding Protein (R7BP). Based on these observations we hypothesize that targeting RGS- R7BP complex pharmacologically may offer a strategy to mitigate undesirable effects of opioids. We have developed an innovative biochemical assay to track rearrangement within the RGS7-R7BP complex by Time Resolved Forster Resonance Energy Transfer (TR-FRET). The assay was adapted for the high-throughput screening (HTS) setting and validated in pilot experiments by screening libraries of small compounds. We further developed a suite of orthogonal and secondary approaches to evaluate the effects of pharmacological inhibition of RGS7-R7BP complex mimicking its physiological action in the endogenous setting. The specific focus of this proposal is on the implementation of the full HTS screen of the ~640,000 compound library to identify first-in-class inhibitors of RGS7-R7BP complex function followed by comprehensive validation of identified leads. Our approach is based on combining thorough understanding of the RGS biology and wealth of accumulated experimental tools obtained by studying their mechanisms over the last decade with the proven expertise of the drug discovery team at The Scripps Research Institute. Our team includes experts in assaying cell signaling, experts in HTS and lead identification as well as medicinal chemistry and pharmacokinetic resources. It is anticipated that successful accomplishment of these goals will provide a critical step towards making opioid medications safer. Outside of the translational aspects, chemical probes targeting RGS complexes is expected to prove useful for uncovering fundamental mechanisms that regulate neurotransmitter actions as small molecule ligands for these proteins are not currently available.