PROJECT SUMMARY Alcohol use disorders (AUDs) represent a leading health issue that causes an enormous number of deaths and disabilities globally, highlighting the critical medical need for the development of novel therapeutic strategies. The Gao Lab recently showed that hepatocyte-specific Aldh2-deficient mice (Aldh2hep-/-) were resistant to alcohol- seeking behavior with elevated blood acetaldehyde levels. Interestingly, Aldh2hep-/- mice also had much less reduced energy expenditure and motility than the global Aldh2 knockout mice. These data highlight that liver- specific inhibition of ALDH2 represents a promising therapeutic approach for the treatment of AUDs with fewer unwanted side effects. In addition, the hepatocyte-specific organic anion transporting polypeptide 1 (OATP1) transporters (eg, OATP1B1 and OATP1B3) represent a major class of uptake transporters for liver-targeted drug development. Our preliminary studies have shown that small-molecule YA7068 inhibits the enzymatic activity of ALDH2 with an excellent potency (IC50 = 62 nM). Simultaneously, YA7068 functions as a substrate for OATP1B1, leading to liver-specific targeting of ALDH2. Our hypothesis is that liver-specific inhibition of acetaldehyde metabolism by targeting the enzymatic activity of ALDH2 is a novel therapeutic strategy for the treatment of AUDs. Our goal is to synthesize a series of YA7068 analogs and test them using established assays in vitro and in vivo, to validate and characterize potent and liver specific ALDH2 inhibitors in treating AUDs. To achieve our goal, we will synthesize new YA7068 analogs that have been designed using a novel computer-aided drug design (CADD) methodology SILCS (Aim 1). The synthesized compounds will be subjected to a high-throughput ALDH2 enzymatic assay to determine their potencies for ALDH2. Selected inhibitors will be further assessed for their specificity for ALDH2 against other related ALDH isozymes. The liver-specific uptake of new compounds by OATP1 transporters will be quantified by comparing intracellular inhibitor levels with or without the expression of the OATP1 transporters on the cell surface. The intracellular concentration of testing compounds will be followed by LC-MS. The cellular toxicity of compounds will be tested using primary human hepatocytes. In Aim 2, we will determine the pharmacokinetics and toxicity of the top compound selected from Aim 1. For the top compound, in vivo efficacy will subsequently be tested in AUD mouse models (see letter from Dr. Bin Gao). Our collaborative research team has a strong track record of performing CADD, lead optimization, and in vitro and in vivo evaluation of compounds. Collectively, our approach puts us in a unique position to identify, validate, and characterize first-in-class liver-specific inhibitors of ALDH2, and to determine whether this novel mechanism of action is a viable option for the development of a treatment for AUDs.