An expedited screening platform for the discovery of anti-ageing compounds in vitro and in vivo

Abstract

Background: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan.

Methods: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture.

Results: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo.

Conclusions: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

Keywords: Ageing; CellPopAge epigenetic Clock; CpG methylation; Drug discovery; Rapamycin; Senescence.

Fig. 1

Development of the CellPopAge Clock for monitoring subtle ageing difference in cells in culture. A Predicted age of control samples using three existing epigenetic clocks. Predicted epigenetic age for control samples across all experiments as estimated by the Multi-Tissue clock (green), the Skin and Blood clock (orange), and the PhenoAge clock (yellow). Fitted lines are shown with 95% confidence intervals (semi-transparent). All three clocks show a trend to increase in predicted age with progressing passage; however, there is considerable variability in predictions, particularly for the PhenoAge clock. The Multi-Tissue clock consistently predicted cells to have the highest epigenetic age, whilst the PhenoAge clock consistently predicted cells to have the lowest epigenetic age, which even reached below zero for several samples at various passages. B Heatmap representing 23 CpG probes that undergo hypomethylation with increasing cell passage and 19 CpGs that undergo hypermethylation with increasing cell passage. These 42 CpG probes were used to develop the CellPopAge Clock. Probes that undergo hypomethylation and hypermethylation with increasing passage were separated and ordered by their methylation values per row. The mean absolute difference between passage 20 and passage 10 among clock CpGs is 0.2. C Testing the CellPopAge Clock on HMF and HDF samples that were not used to train the clock. The grey dashed line represents the diagonal (perfect prediction). The fitted line of the actual data is shown in blue, with a 95% confidence interval (semi-transparent). Cell passages are predicted accurately

Fig. 2

Using the CellPopAge Clock for the detection of anti-ageing drugs. A Schematic illustrating the experimental set-up conducted in P9 to P20 HDFs and HMFs, passaged weekly and continuously treated with either rapamycin, trametinib, torin2, or dactolisib/BEZ235, represented as a pill. Control cells were treated with vehicle, either DMSO or ethanol. B The CellPopAge Clock predictions of human dermal fibroblasts (HDF) and C human mammary fibroblasts (HMF). Represented is predicted-actual passage for passages 16, 18, and 20, showing deceleration of the CellPopAge Clock upon treatment with anti-ageing drugs rapamycin (5 nM), trametinib (0.1 nM), torin2 (5 nM), and dactolisib/BEZ235 (10 nM) and non-treated control samples (black dots)

Fig. 3

Treatment with anti-ageing drugs decreases markers of senescence. A Schematic illustrating the experimental set-up conducted in P10 to P22 HMFs, passaged weekly. B Multi-parameter analysis of senescence markers. Robust Z scores were calculated for a panel of measures relative to the vehicle control. Colour coding used to illustrate the number of Z scores of the experimental drug value from the respective vehicle control mean. Scores highlighted in blue denote a shift towards a more proliferative phenotype and scores highlighted in yellow denote a shift to a more senescent phenotype, all with a robust Z scores of ± 0.5. White indicates no change. N = 2 for all except for SA-β-gal. C P22 HMFs stained with DAPI (blue) and Cell Mask, p21, p16, IL-6, or nucleolin (red), or SA-β-Gal (blue) following 96-day treatment with 5 nM rapamycin, 10 nM dactolisib/BEZ235, 0.1 nM trametinib, or their respective controls. Size bar, 100 μm

Fig. 4

Drugs that decelerate the CellPopAge Clock extend lifespan in vivo. A Lifespan analysis on w^Dah background wild-type flies fed with holidic food containing different concentration of rapamycin or ethanol as solvent control. For each condition, 150 flies were used. B Lifespan analysis on w^Dah background wild-type flies fed with holidic food containing different concentration of dactolisib/BEZ235 or DMSO as solvent control. For each condition, 150 flies were used. C Lifespan analysis on w^Dah background wild-type flies fed with holidic food containing different concentration of torin2 or DMSO as solvent control. For each condition, 150 flies were used. D Schematic representation of our proposed screening platform which combines our novel CellPopAge Clock and other ageing biomarkers in vitro primary human cell, together with in vivo Drosophila lifespan experiments, for a detailed and robust capture of anti-ageing drug potential