Molecular and Cellular Characterization of SIRT1 Allosteric Activators
SIRT1 is an NAD+-dependent lysine deacetylase that promotes healthy aging and longevity in diverse organisms. Small molecule allosteric activators of SIRT1 such as resveratrol and SRT2104 directly bind to the N-terminus of SIRT1 and lower the Km for the protein substrate. In rodents, sirtuin-activating compounds (STACs) protect from age-related diseases and extend life span. In human clinical trials, STACs have a high safety profile and anti-inflammatory activities. Here, we describe methods for identifying and characterizing STACs, including production of recombinant protein, in vitro assays with recombinant protein, and cellular assays based on mitochondrial dynamics. The methods described in this chapter will facilitate this discovery of improved STACs, natural and synthetic, in the pursuit of interventions to treat age-related diseases.
KeywordsSirtuin SIRT1 Histone deacetylase (HDAC) Deacylase Deacetylase NAD+ Nicotinamide ADP-ribose Aging Longevity Metabolism Epigenetics Histone p53 Mitochondria Membrane potential Reactive oxygen species (ROS) Sirtuin-activating compound (STAC) Resveratrol Allosteric activator Recombinant protein
We are grateful for support from the Paul F. Glenn Foundation for Medical Research. This work was supported by NIH R37 AG028730, R01 AG019719, R01 DK100263 and R21 DE027490. J.A.A. is recipient of scholarship from the Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia (PD/BD/114173/2016).
Disclosure: D.A.S. is a founder, equity owner, board member, advisor to, director of, consultant to, investor in and/or inventor on patents licensed to Vium, Jupiter Orphan Therapeutics, Cohbar, Galilei Biosciences, GlaxoSmithKline, OvaScience, EMD Millipore, Wellomics, Inside Tracker, Caudalie, Bayer Crop Science, Longwood Fund, Zymo Research, EdenRoc Sciences (and affiliates Arc-Bio, Dovetail Genomics, Claret Bioscience, Revere Biosensors, UpRNA and MetroBiotech (an NAD booster company), Liberty Biosecurity), Life Biosciences (and affiliates Selphagy, Senolytic Therapeutics, Spotlight Biosciences, Animal Biosciences, Iduna, Immetas, Prana, Continuum Biosciences, Jumpstart Fertility (an NAD booster company), and Lua Communications). D.A.S. sits on the board of directors of both companies. D.A.S. is an inventor on a patent application filed by Mayo Clinic and Harvard Medical School that has been licensed to Elysium Health; his personal royalty share is directed to the Sinclair lab. For more information see https://genetics.med.harvard.edu/sinclair-test/people/sinclair-other.php. Y.L. is an equity owner of Iduna.