Project Summary Neuropathic pain remains a challenging condition to treat, as all currently available drugs fail to achieve adequate pain relief in a significant proportion of patients. Although opioid-based analgesics have been proven effective in reducing the intensity of pain for many neuropathic pain conditions, their clinical utility is grossly limited due to the substantial risks involved in such therapy, including nausea, constipation, physical dependence, tolerance, and respiratory depression. Clearly, there is a critical unmet medical need for new neuropathic pain therapies with improved efficacy and side effect profiles. Cumulative evidence suggests that human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain with limited side effects due to its restricted expression in nociceptors within the peripheral nervous system. BAM8-22, a putative endogenous MRGPRX1 agonist, has shown analgesic efficacy in a variety of pain models following intrathecal injection. While these findings open up the intriguing possibility for CNS-penetrant small molecule MRGPRX1 agonists for the treatment of pain, direct activation of MRGPRX1 at peripheral terminals is expected to induce itch side effects, limiting the therapeutic utility of orthosteric MRGPRX1 agonists. Interestingly, submaximal levels of BAM22-derived peptides acting as endogenous MRGPRX1 orthosteric agonists were detected at the spinal cord dorsal horn during persistent pain but remained undetectable in the skin. This finding led to the exploration of positive allosteric modulators (PAMs) of MRGPRX1 to potentiate the effects of the endogenous agonists at the central terminals of sensory neurons without activating peripheral MRGPRX1. Indeed, intrathecal injection of a prototype MRGPRX1 PAM, ML382, effectively attenuated evoked, persistent, and spontaneous pain without causing itch side effects. Thus, the central goal of this proposal is to develop a CNS-penetrant small molecule MRGPRX1 PAM that can be given orally to treat neuropathic pain conditions. To accomplish the ultimate goal of assembling an IND application, a team of independent investigators with complementary expertise has been assembled with many years of research experience in the areas of medicinal chemistry, ADME, neuroscience, preclinical pain models, and clinical pain management. Under this milestone-driven phased cooperative agreement, the team will work closely with NIH program staff to accelerate the optimization and development of a promising MRGPRX1 PAM lead into a non-addictive therapeutic agent for the treatment of neuropathic pain conditions.