Sirtuins are a family of NAD⁺-dependent silent information regulator 2 (Sir2) enzymes that catalyze the removal of acyl groups from ε-N-amino groups of lysine residues in the proteome. The human genome encodes seven different sirtuin isoforms (SIRT1–7), which are classified according to sequence similarity and localize to different cellular compartments. The SIRT5 enzyme isoform is a major lysine desuccinylase/deglutarylse in the mitochondria; where it contributes to the maintenance of metabolic homeostatis. Among other functions, this enzyme isoform down-regulates the production of reactive oxygen species (ROS) to protect cells from oxidative stress, and it has been suggested as a target in several cancers.

In this contribution, I will present our current progress in the evaluation of enzymatic the activity and targeting of the cellular activity of SIRT5. We have developed in vitro screening systems to address substrate specificity and photo-crosslinking probes that can be applied in cell lysates. Further, we have developed mechanism-based and covalent SuFEx-based inhibitors of SIRT5 that exhibit selectivity through substrate mimicry. It is our hope that these chemotypes can help elucidate the roles of mitochondrial deacylases in metabolic homeostasis and healthy aging.