Chronological lifespan extension and nucleotide salvage inhibition in yeast by isonicotinamide supplementation

Isonicotinamide (INAM) is an isomer of the NAD + precursor nicotinamide (NAM) that stimulates the enzymatic activity of Sir2, an NAD + -dependent histone deacetylase from the budding yeast, Saccharomyces cerevisiae . Supplementing INAM into growth media promotes the replicative lifespan (RLS) of this single cell organism by maintaining intracellular NAD + homeostasis. INAM also extends yeast chronological lifespan (CLS), but the underlying mechanisms remain largely uncharacterized. To identify cellular pathways potentially impacted by INAM, in this study we perform a chemical genomics screen of the yeast knockout (YKO) collection for mutants sensitized to growth inhibition by INAM. Significant Gene Ontology (GO) terms for candidate genes include transcription elongation factors, metabolic pathways converging on one-carbon metabolism, and de novo purine biosynthesis, collectively suggesting that INAM perturbs nucleotide metabolism. Indeed, INAM causes dose-dependent depletion of intracellular cytidine, uridine, and guanosine, ribonucleosides derived from the breakdown of nucleotide monophosphates (NMPs) via nucleotidases (Phm8, Sdt1, Isn1) or the alkaline phosphatase Pho8. We also find that INAM directly inhibits recombinant nucleotidase activity using cytidine or nicotinamide mononucleotide (NMN) as substrates and inhibits alkaline phosphatase activity quantitated from whole cell extracts. Lastly, we find that Phm8 and Pho8 are specifically required for INAM-induced CLS extension, implicating them as likely functional targets in vivo. Taken together, the findings suggest a model whereby partial impairment of nucleotide and/or NAD + salvage pathways by INAM can trigger a hormetic stress response that supports enhanced quiescence during chronological aging.