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

## TL;DR

Isonicotinamide extends yeast lifespan by disrupting nucleotide salvage pathways and triggering a stress response.

## Contribution

The study identifies nucleotide salvage inhibition and specific enzymes as mechanisms for chronological lifespan extension by isonicotinamide in yeast.

## Key findings

- Isonicotinamide inhibits nucleotidase and alkaline phosphatase activities, depleting intracellular ribonucleosides.
- Phm8 and Pho8 are required for isonicotinamide-induced chronological lifespan extension in yeast.
- Isonicotinamide perturbs nucleotide metabolism and triggers a hormetic stress response during aging.

## Abstract

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 of this single cell organism by maintaining intracellular NAD+ homeostasis. INAM also extends yeast chronological lifespan, 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 collection for mutants sensitized to growth inhibition by INAM. Significant Gene Ontology 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, and Isn1) or the alkaline phosphatase Pho8. We also find that INAM directly inhibits recombinant nucleotidase activity using cytidine or nicotinamide mononucleotide as substrates and inhibits alkaline phosphatase activity quantitated from whole cell extracts. Finally, we found that Phm8 and Pho8 are specifically required for INAM-induced chronological lifespan 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.