Metabolism and ageing are intimately linked. Compared to ad libitum feeding, dietary restriction (DR) or calorie restriction (CR) consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms1,2. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits3,4. Recently, several metabolites have been identified that modulate ageing5,6 with largely undefined molecular mechanisms. Here we show that the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (α-KG) extends the lifespan of adult C. elegans. ATP synthase subunit beta is identified as a novel binding protein of α-KG using a small-molecule target identification strategy called DARTS (drug affinity responsive target stability)7. The ATP synthase, also known as Complex V of the mitochondrial electron transport chain (ETC), is the main cellular energy-generating machinery and is highly conserved throughout evolution8,9. Although complete loss of mitochondrial function is detrimental, partial suppression of the ETC has been shown to extend C. elegans lifespan10–13. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit beta and is dependent on the target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased upon starvation and α-KG does not extend the lifespan of DR animals, indicating that α-KG is a key metabolite that mediates longevity by DR. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator, and DR in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Drug Affinity Responsive Target Stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small molecules. It relies on the protection against proteolysis conferred on the target protein by interaction with a small molecule. The greatest advantage of this method is being able to use the native small molecule without having to immobilize or modify it (e.g. by incorporation of biotin, fluorescent, radioisotope, or photo-affinity labels). Here we describe in detail the protocol for performing unbiased DARTS with complex protein lysate to identify potential binding targets of small molecules and for using DARTS-Western blotting to test, screen, or validate potential small molecule targets. Although the ideas have mainly been developed from studying molecules in areas of biology that are currently of interest to us and our collaborators, the general principles should be applicable to the analysis of all molecules in nature.

Recently we discovered that the central metabolite α-ketoglutarate (α-KG) extends lifespan in C. elegans through inhibition of ATP synthase and TOR signaling. Unexpectedly, here we find that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG mediated processes, extends worm lifespan similarly. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase, and inhibit mTOR signaling; these effects are mirrored in IDH1 mutant cells, suggesting a growth suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, such as when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.

drug discovery ͉ drug target identification ͉ proteomics ͉ diabetes