A Bioluminescent Assay for Direct Measurement of Sirtuin Activity in Cancer Cells - A bioluminescent assay for direct measurement of sirtuin activity in cancer cells Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases that act on histones, transcription factors, epigenetic regulators and metabolic enzymes to regulate key cellular functions such as apoptosis, senescence and energy metabolism. Depending on the cellular context, sirtuins can promote or inhibit oncogenesis, defining a critical need for methods capable of interrogating sirtuin activity in intact cells and animal models. However currently available sirtuin assays are confined to cell free systems, are prone to artifacts, and have limited sensitivity and dynamic range. The lack of assays that provide a reliable, direct and real-time readout of sirtuin activity in vitro and in vivo poses a major barrier to drug development and biological studies. To overcome the limitations of current assays we propose luciferase-based assay that reports in real- time sirtuin activity in cell free and cellular assays through generation of a bioluminescence signal by conversion of furimazine to furimamide. Our design is based on a split-NanoLuc complementation system that consists of a truncated catalytically inactive N-terminal moiety (termed 11S), which is activated by complementation with a high affinity 11-aa C-terminal peptide containing two lysine residues (K8,K9). Our premise is that acylation or other modifications of the lysine residues within the high affinity C-terminal complementation peptide inhibit its binding to the N-terminal NanoLuc moiety, preventing activation; active sirtuin enzyme removes the acyl groups, allowing for luciferase complementation and generation of a luminescence signal. Our preliminary data using SIRT5 enzyme demonstrate a robust reporter signal in cell free assays, and suggest that measurement of intracellular sirtuin activity is feasible. In Aim 1 we will adapt the split-NanoLuc assay for measuring sirtuin deacylase activity in cell free systems and determine which lysine modifications within the C-terminal complementation peptide block binding to the large NanoLuc fragment, and are removed by sirtuins 1 – 7. We will optimize assay conditions, and delineate performance parameters (sensitivity, selectivity, dynamic range, coefficient of variation). In Aim 2 we will adapt the split-NanoLuc assay for measurement of SIRT5 activity in live cells and in vivo. We will generate SIRT5 reporter cell lines, targeting the truncated NanoLuc to specific cellular compartments. We will optimize conditions for quantitative bioluminescence measurement in live cancer cells and in vivo, using CRISPR/Cas9, shRNA and inhibitors to modulate SIRT5 activity. Our design has critical advantages over current approaches: (i) Potential adaptability to any type of acyl modification; (ii) High signal- to-background ratio; (iii) Avoidance of artifact-prone chemically modified probes; (iv) Adaptability to measurement of enzyme activity in live cells and animal models. Successful completion of this project will result in the establishment of a versatile and sensitive assay for detection of sirtuin deacylase enzymatic activity in cell free systems, live cancer cells and animal models of cancer, overcoming a major barrier in the field.
