Opioid drugs are highly effective analgesics but suffer from serious on-target side-effects. Principle among these are addition liability and respiratory depression that have led to the current opioid crisis. These effects together with other actions, including constipation and nausea and vomiting, also reduce the effectiveness of opioids in the pain clinic. Thus, there is a vital need to develop new analgesics or to improve the clinical profile of existing medications. Morphine and related opioids exert their effects by acting at the orthosteric site on the mu-opioid receptor (MOR), i.e. the site where the endogenous opioid peptides bind. Recent advances in our knowledge of the structure of G-protein coupled receptors (GPCRs) have highlighted the possibility that GPCR function may be controlled by compounds binding at a separate, allosteric, site on the receptors. In this regard positive allosteric modulators (PAMs) that act at MOR (MOR-PAMs) have been identified. Previous experiments show these compounds act to allosterically modulate the orthosteric site and so increase the binding affinity, potency and/or maximal response of MOR agonists, including endogenous opioid peptides, in a probe-dependent manner. Preliminary experiments demonstrate that MOR- PAMs are effective antinociceptive agents in vivo in the mouse and act by enhancing endogenous enkephalin activity, thus avoiding the need for opioid drugs such as morphine and oxycodone. This in vivo activity should preserve the temporal and spatial characteristics of neuronal signaling and so avoid compensatory mechanisms induced by chronic MOR activation. This application moves the field forward by using structural biology and computational methods to identify the allosteric binding site on MOR, aided by the development of new allosteric probes to allow us to more clearly define the mechanism of allosterism, and further exploration of preliminary findings that allosteric modulators work as effective pain relieving agents in vivo. Detailed understanding of the actions of allosteric modulators of MOR will provide new information on mechanisms by which MOR function may be controlled and, together with the identification of high affinity probes, will pave the way for future drug development efforts of MOR modulators as novel analgesics that safely harness the analgesic efficacy of MOR without addiction and respiratory depression. RELEVANCE (See instructions):