Obesity is a known driver of type 2 diabetes (T2D) and diabetic complications. While white adipose tissue (WAT) stores energy, brown adipose tissue (BAT), through the action of uncoupling protein 1 (UCP1) and a larger thermogenic program, releases energy as heat. BAT is now known to be present and modifiable in adult humans, including the prospect of inducing BAT-like characteristics in WAT (?browning'). Loss of retinaldehyde dehydrogenase 1 (ALDH1a1) function potently induces UCP1, causing browning of WAT and protecting against diet-induced obesity and diabetes, as first we, and subsequently others, have shown in multiple in vivo models. Unlike many BAT-activating targets, ALDH1a1 inhibition decreases both subcutaneous and visceral adipose tissue (VAT), although its higher expression in visceral fat does foster pronounced effects in this particularly pathogenic depot. Independent of adiposity, ALDH1a1 deficiency also decreases hepatic gluconeogenesis and steatosis, common T2D abnormalities. ALDH1a1 converts the substrate retinaldehyde (Rald) to retinoic acid (RA). In vitro, either ALDH1a1 inhibition or direct Rald stimulation modulates expression of key thermogenic and gluconeogenic mediators. Data supports a relationship between ALDLH1a1 levels and adiposity in humans. Thus, a strong rationale exists for pursuing ALDH1a1 as a novel therapeutic target for decreasing adiposity and improving T2D, as our interdisciplinary, multiple PI team has undertaken, resulting in the strong preliminary data underlying this application. Aim 1 seeks to optimize, further develop, and test already identified lead small molecule ALDH1a1 inhibitors and Rald mimetics. Initial focus will be on four ALDH1a1 scaffold lead candidates found in our primary chemical library screen (650,000 compounds/validated ALDH1a1 activity assay/nanomolar IC50) using iterative chemical analysis and structural modifications coupled to in vitro and in vivo testing. Since the metabolic benefits of ALDH1a1 inhibition involve increased Rald levels, a novel, orthogonal therapeutic strategy explored here involves synthetic Rald mimetics; lead analogs are already designed, made and induce UCP1 expression. Hydrogen/deuterium exchange (HDX) and ALDH1a1 co-crystallography will also be leveraged to generate additional insights into ALDH1a1 modulator structure-activity relationships. Aim 2, tightly integrated with Aim 1 compound progression, will test the functional in vitro and in vivo effects of lead ALDH1a1 modulators on diabetes through changes in thermogenesis, energy balance, gluconeogenesis and steatosis. Taken together, ALDH1a1 modulation is well matched to this NIDDK PAR seeking ?early-stage pharmacological validation of novel targets and pre- therapeutic leads?: a new pathway with compelling, validated prior data establishing clinically-relevant, unique effects on critical, unaddressed pathogenic diabetic mechanisms; an interdisciplinary, collaborative team with the requisite background and tools for the proposed work; extensive progress to date, including lead compounds that support ALDH1a1 inhibition as a potential therapeutic target for treating T2D.