PROJECT SUMMARY Our long-term goal is to develop safe and effective drugs to treat obesity. We propose to validate ALDH1A1 as a new drug target for obesity treatment by testing the anti-obesity efficacy of a novel inhibitor of this enzyme, N42, that was recently developed by our group. Retinoic acid (RA), a metabolite of vitamin A, is synthesized by three aldehyde dehydrogenases, ALDH1A1, ALDH1A2, and ALDH1A3, that are expressed in temporally and spatially distinct yet sometimes overlapping patterns in a tissue- and developmental stage-dependent manner. ALDH1A1 is the major RA synthesis enzyme in two metabolically important tissues, liver and adipose, where RA is known to regulate expression of genes involved in glucose and lipid metabolism and adipose tissue differentiation. Aldh1a1-/- mice develop normally, are healthy and fertile, and are also protected from developing diet-induced obesity. In agreement with observations in Aldh1a1-/- mice, we demonstrated that mice treated with WIN 18,446, a pan-inhibitor of ALDH1A enzymes, had lower weight gain due to a decrease in adipose tissue mass, demonstrating the feasibility of using pharmacological inhibitors of ALDH1A to treat obesity. Although WIN 18,446 treatment is promising for weight suppression, it inhibits other RA synthesis enzymes as well as ALDH2, causing unwanted side effects such as reversible spermatogenesis inhibition and alcohol intolerance. Therefore, we developed compounds that specifically inhibit ALDH1A1 and now propose to evaluate the efficacy and potential toxicity of a novel ALDH1A1-specific inhibitor, N42, for treatment of obesity and to determine mechanisms by which ALDH1A1 regulates weight gain by using comprehensive studies of gene expression and metabolite changes in response to ALDH1A1 loss. In Aim 1, we will determine if N42 treatment can (1) suppress weight gain in obese mice when they are continuously fed a high fat diet and (2) accelerate weight loss in obese mice when they are provided reduced calorie diet. We will also investigate potential organ toxicity of N42 treatment using standard, well-established protocols of clinical and toxicologic pathology. Metabolic changes associated with ALDH1A1 loss (N42 treatment or Aldh1a1-/- mice) will be comprehensively investigated using global metabolomics and gene expression studies. Phenotypic parameters including metabolites, gene expression, retinoid levels etc. will be associated with N42-mediated efficacy to identify potential biomarkers for future clinical use of this compound. In Aim 2, the role of ALDH1A1 in liver and adipose tissues will be further explored using tissue-specific Aldh1a1-/- mice. We will also determine how ALDH1A1 loss in adults alters adipogenesis and adipocyte hypertrophy using an adipocyte linage mouse model. Finally, we will identify tissue-autonomous functions of ALDH1A1 by combining a novel perifusion method, metabolomics, and systems-biology approaches. Successful completion of the proposed stud...