The discovery of safe and effective analgesics is an urgent societal need and a centerpiece of NIH’s HEAL initiative. Here, we test the hypothesis that the enzyme N-Acylethanolamine Acid Amidase (NAAA) offers a novel target for analgesic medications devoid of abuse potential. NAAA hydrolyzes palmitoylethanolamide (PEA), a lipid messenger that suppresses nociception by engaging the nuclear receptor PPAR-α. The substantial antinociceptive properties of NAAA inhibitors have been recently recognized by a meta-analysis of the preclinical literature conducted by the International Association for the Study of Pain (IASP), but the cellular substrates underlying such properties are still unknown. We found that Naaa-knock out (ko) mice (i) have markedly reduced nocifensive responses to formalin, compared to wild-type littermates; and (ii) fail to develop persistent sensory abnormalities in the chronic constriction injury (CCI) model. Conversely, Naaa- overexpressing and Ppara-ko mice exhibit robust nocifensive behavior even when given a subthreshold formalin dose. The reduced sensitivity of Naaa-ko mice to pain does not result from developmental compensation, because it can be phenocopied by administration of the NAAA inhibitor ARN19702. Importantly, ARN19702 does not exert positive motivational effects in the mouse conditioned place preference (CPP) test, which is suggestive of a lack of rewarding properties. These data point to NAAA as a promising target for the discovery of non-addictive analgesics. We will test this hypothesis in two specific aims: Aim 1. Validate NAAA as a molecular target for analgesic drug discovery. Three questions will be addressed: (1) What cell types are directly targeted by NAAA inhibitors? We will generate cell-specific NAAA-ko mouse lines and evaluate, in the formalin and CCI models, pain-related behaviors and sensitivity to NAAA blockade. In parallel, we will determine the impact of pathological pain on NAAA-regulated signaling in dorsal root ganglia (DRG) and spinal cord (SC) of wild-type mice. (2) What cell types mediate NAAA-regulated antinociceptive signaling? We will identify PPAR-α-expressing cells involved in NAAA-dependent signaling by creating cell-specific PPAR-α-ko mouse lines and evaluating their pain-related responses and their sensitivity to NAAA inhibitors in the formalin and CCI tests. (3) Is the pharmacodynamic profile of NAAA inhibitors compatible with safe and effective use in pain therapy? We will determine whether ARN19702 (i) alleviates spontaneous nociception, (ii) produces tolerance after repeated administration, and (iii) exhibits rewarding and/or addicting potential. Aim 2. Develop improved NAAA inhibitors and examine their efficacy as analgesic agents. Better NAAA inhibitors are needed to assess NAAA’s possible role in pain therapy. We will combine computational structure-based drug and structure-activity relationship studies to discover novel agents with improved pharmacodynamic and pharmacokineti...