Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.aging ͉ label-free ͉ proteomics ͉ small molecules

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.

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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.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.