Accumulation of acetaldehyde from ethanol metabolism results in a number of unpleasant symptoms that cause natural aversion to alcohol consumption. Aversion pharmacotherapy for treatment of alcoholism is based on blocking the metabolism of ethanol-derived acetaldehyde by disulfiram, an inhibitor of aldehyde dehydrogenase (ALDH). Disulfiram acts as a prodrug; through its metabolites it modifies irreversibly active site cysteine residues of ALDH. Because disulfiram is a non-specific alkylating agent, other enzymes with critical cysteine residues are also inhibited, and serious side effects are manifested. The long-term objective of this grant application is to identify and characterize new potent and specific inhibitors of the high affinity mitochondrial ALDH isozyme 2 (ALDH2) as therapeutic agents for alcoholism. The specific aims of this proposal are: i) Selection of peptides from phage-displayed combinatorial peptide libraries that bind specifically to recombinant ALDH2 (rat, human) by an iterative biopanning procedure and identification of conserved structural motifs by DNA sequencing. ii) Screening of motif-carrying phage peptides for binding to ALDH2 and enzymatic inhibition. Phage peptides with inhibitory properties will be substituted with synthetic soluble peptides that can be delivered to cells, and the mechanism of ALDH2 inhibition will be elucidated. iii) Validation of lead peptide inhibitors in a rat hepatoma cell line transfected with rat or human ALDH2. Inhibitors will be delivered to mitochondria by lipids and peptide carriers, and with viral (retroviral and adenoviral) vectors.