The proposed research explores molecular approaches for the study and regulation of aberrant metalloenzyme activity in human disease, focusing on the structural and chemical biology of histone deacetylase (HDAC) isozymes 6, 8, 10, and 11. These zinc-dependent enzymes are targets for drug design efforts aimed at developing new approaches to cancer chemotherapy, treatment of neurodegenerative disease, and immune modulation. To advance our understanding of structure and function in this important family of metalloenzymes, and to enable innovative molecular approaches for new disease therapies, we aim to pursue the following lines of investigation: (1) We propose to determine crystal structures of HDAC6 complexed with fluorinated inhibitors to probe the medicinal chemistry of a fluorophilic crevice in the enzyme active site. These studies will also inform optimal approaches for the development of 18F derivatives used in PET imaging of HDAC6 and other proteins. We will also determine structures of complexes with large macrocyclic octapeptide and nonapeptide inhibitors that will map out interactions in the outer active site cleft. Analysis of these complexes will illuminate structural features in the outer active site cleft that also accommodate the binding of large protein substrates. Finally, we will explore the preparation and structure determination of a ternary complex of HDAC6, the E3 ubiquitin ligase protein cereblon, and a proteolysis-targeting chimera (PROTAC). (2) We propose to explore the structural basis of HDAC10 substrate specificity to better understand the cryptic lysine deacetylase activity observed for this polyamine deacetylase. The binding of acetyllysine- containing substrates must require conformational changes in the active site to switch between the binding of bulky peptide and protein substrates versus slender polyamine substrates. Additionally, we will determine crystal structures of complexes with HDAC10-selective inhibitors containing mercaptoacetamide zinc-binding groups. In view of the genotoxicity associated with degradation of the hydroxamate moiety, HDAC inhibitors are needed with alternative zinc-binding groups that exhibit improved stability and safety profiles. Finally, we will explore the preparation and structure determination of a ternary HDAC10-PROTAC-cereblon complex. (3) We propose to study the structural basis of the lysine-fatty acid deacylase activity of HDAC8 and HDAC11. We will determine the structure of an intact enzyme-substrate complex with HDAC8 to map out the fatty acid binding site. We will also explore the crystallization and structure determination of the sole class IV isozyme, HDAC11, which represents the last frontier of HDAC structural biology. We aim to bring the same rigor of structure-function analysis to this isozyme as we have brought to other HDAC isozymes in recent years.