AMPK sits at the very heart of cellular energy bookkeeping. When energy levels run low, the enzyme springs into action, switching on energy-producing processes and shutting down energy-consuming ones such as protein synthesis and cell growth. It is a heterotrimer, built from a catalytic alpha subunit and two regulatory partners, beta and gamma, and its sequence is strikingly conserved from fungi to humans. Maximal activity requires phosphorylation of a conserved threonine in the alpha subunit, a molecular switch that has been preserved across hundreds of millions of years of evolution. Decades of work in fruit flies, nematode worms and fission yeast have shown that genetic manipulations or dietary interventions that boost AMPK activity reliably produce longer-lived animals, making the enzyme one of the most compelling therapeutic targets in the biology of ageing.

The problem, until now, has been specificity. Metformin, the most famous AMPK-linked drug, activates the enzyme only indirectly, by perturbing the cellular AMP-to-ATP ratio and hitting mitochondrial and lysosomal targets along the way. Its effects in vivo are so tangled that studies in worms and flies have shown the drug’s lifespan-extending power actually flows through the gut microbiota rather than the host itself, and even in mammals there is evidence that bacteria mediate part of the response. That complexity has left the field with an uncomfortable question: is AMPK itself the pro-longevity target, or just one bystander among many? To answer it, the research team turned to 991, a synthetic molecule that binds in the so-called ADaM site, a pocket wedged between the alpha and beta subunits of AMPK, and locks the enzyme into its active state.

The first challenge was evolutionary. 991 had only ever been tested in mammalian systems, so the researchers built homology models of the fly, worm and yeast AMPK complexes against the known human crystal structure. The modelling revealed a high degree of overlap in the drug-binding pocket, including a key serine residue in the beta subunit that coordinates 991 binding, and conserved hydrophobic residues that stabilise the interaction between the beta subunit and the kinase domain. In the laboratory, the predictions held up beautifully. 991 allosterically activated AMPK purified from fruit fly cells in a dose-dependent manner, protected the enzyme from dephosphorylation by phosphatases, and boosted AMPK activity in extracts from wild-type worms but not from mutants lacking the AMPK alpha gene aak-2. Micromolar concentrations of 991 achieved what required millimolar doses of metformin or phenformin, a thousand-fold potency advantage that underscores the difference between direct and indirect activation.

Getting the drug into living organisms required some pharmaceutical finesse. In flies, 991 activated AMPK in vivo only when delivered in a chemically defined diet, which offers better drug bioavailability than standard yeast-based food, a reminder that in ageing research the medium can matter as much as the molecule. Western blotting confirmed that dietary 991 raised levels of phosphorylated, active AMPK in flies, worms and fission yeast alike. With the target engagement established across three phyla, the team moved to the question everyone wanted answered: does directly activating AMPK actually make animals live longer?

The answer was yes, and in a remarkably consistent pattern. Flies fed 991 in defined medium lived significantly longer than controls, with the effect highly statistically significant at both low and moderate doses. Fission yeast treated with 10 micromolar 991 showed extended chronological lifespan, a measure of how long stationary-phase cells survive. Nematode worms gained longevity on two different bacterial diets, the standard OP50 strain and the alternative BW25113, and the drug did not substantially alter bacterial growth, indicating that, unlike metformin, 991 acts on the worm host directly rather than through the microbes in its gut. The drug also reshaped metabolism in both animals: flies showed dose-dependent reductions in whole-body triglyceride stores without any change in feeding rate or glycogen content, while worms displayed altered lipid staining patterns. Fecundity in the flies was untouched, ruling out the classic trade-off in which reproduction is sacrificed for survival.

The most convincing evidence came from the genetic controls. Because AMPK alpha-null flies do not survive to adulthood, the researchers tested the drug in yeast and worm mutants lacking the catalytic subunit. In both cases, the lifespan benefit vanished entirely. The ssp2 yeast mutant, which is hypersensitive to any stress, was actually harmed by the drug, and the aak-2 mutant worms showed no extension at all. That loss-of-effect in the absence of the target is exactly what a rigorous pharmacological study should show, and it distinguishes this work from much of the longevity literature, where compounds often extend lifespan through murky, multi-target mechanisms that resist genetic dissection. Higher doses of 991 shortened lifespan in flies and yeast, a dose-response caution that the authors emphasise as critical for any future therapeutic development.

To bridge the gap to mammals, the team delivered 991 to mice using biodegradable polymeric nanoparticles, injected intravenously three times a week for three weeks. Unbiased proteomic analysis of liver tissue from treated wild-type mice, and from a short-lived muscular dystrophy model, revealed a clear molecular signature of AMPK activation: increased ATP biosynthesis, inhibition of the pro-ageing mTOR pathway, and enhanced mitochondrial biogenesis. Phenotypic ontology analysis flagged survival-related processes as the most enriched category among the responding proteins. While these proteomic shifts fall short of a demonstrated lifespan extension in mammals, they are precisely the profile that pro-longevity interventions produce, and they suggest the machinery responds to direct AMPK activation in mice much as it does in the invertebrates.

There is also a pharmaceutical tailwind behind these findings. Several major companies, including Merck, Pfizer, Boehringer Ingelheim and Poxel, have developed second-generation direct AMPK activators that bind the same ADaM site as 991. These compounds have better bioavailability than 991, have activated AMPK in vivo across a range of rodent studies, improving glucose homeostasis, dyslipidemia and fatty liver disease, and have even been given to non-human primates. Most strikingly, PXL770 has already been through human clinical trials for metabolic liver disease, showing good safety and improved lipid profiles after just four weeks of treatment. The path from a worm surviving longer on a drug-laced agar plate to a human pill is long, but it is considerably shorter when the pill in question already has clinical safety data.

The authors are careful about the limits of their work. The mouse data are preliminary, delivered by injection rather than diet, and longer-term metabolic and longevity studies in mammals remain to be done, likely using the improved second-generation activators. The downstream mechanisms by which AMPK activation extends lifespan in vivo are still unknown, and additional healthspan measures will be needed to complement survival curves. Even so, the study delivers something the ageing field has rarely possessed: a direct activator of a single, evolutionarily conserved target that extends lifespan in three distantly related organisms, fails to do so when the target is genetically removed, and produces a pro-longevity molecular signature in mammals. If metformin’s tangled story taught researchers anything, it is that longevity drugs need clean mechanisms. With 991, they may finally have one.

Subject of Research: Direct pharmacological activation of AMP-activated protein kinase as a conserved mechanism for extending lifespan across species

Article Title: Direct Pharmacological Activation of AMPK Extends Lifespan in Yeast, Worms and Flies

Article References: dos Santos, E., Blickling, M., Leiper, F. C., Alao, J.-P., Lennicke, C., Foley, A., Wilson, J. R., Gamblin, S. J., Chazaud, B., Lollo, G., Mounier, R., Juban, G., Rallis, C., Carling, D., Cabreiro, F., & Cochemé, H. M. (2026). Direct Pharmacological Activation of AMPK Extends Lifespan in Yeast, Worms and Flies. Aging Cell, 25(10), Article e70721. https://doi.org/10.1111/acel.70721

Image Credits: AI Generated

DOI: 10.1111/acel.70721

Keywords: AMPK, ageing, lifespan extension, 991, metformin, Drosophila, C. elegans, fission yeast, ADaM site, geroprotectors, metabolism, proteomics