Repurposing Drugs for Senotherapeutic Effect: Potential Senomorphic Effects of Female Synthetic Hormones.

Repurposing previously approved drugs may fast track the route to the clinic for potential senotherapeutics and improves the inefficiency of the clinical drug development pipeline. We performed a repurposing screen of 240 clinically approved molecules in human primary dermal fibroblasts for their effects on CDKN2A expression. Molecules demonstrating effects on CDKN2A expression underwent secondary screening for senescence-associated beta galactosidase (SAB) activity, based on effect size, direction, and/or molecule identity. Selected molecules then underwent a more detailed assessment of senescence phenotypes including proliferation, apoptosis, DNA damage, senescence-associated secretory phenotype (SASP) expression, and regulators of alternative splicing. A selection of the molecules demonstrating effects on senescence were then used in a new bioinformatic structure-function screen to identify common structural motifs. In total, 90 molecules displayed altered CDKN2A expression at one or other dose, of which 15 also displayed effects on SAB positivity in primary human dermal fibroblasts. Of these, 3 were associated with increased SAB activity, and 11 with reduced activity. The female synthetic sex hormones-diethylstilboestrol, ethynyl estradiol and levonorgestrel-were all associated with a reduction in aspects of the senescence phenotype in male cells, with no effects visible in female cells. Finally, we identified that the 30 compounds that decreased CDKN2A activity the most had a common substructure linked to this function. Our results suggest that several drugs licensed for other indications may warrant exploration as future senotherapies, but that different donors and potentially different sexes may respond differently to senotherapeutic compounds. This underlines the importance of considering donor-related characteristics when designing drug screening platforms.

Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels.

The rs2802292, rs2764264 and rs13217795 variants of FOXO3 have been associated with extreme longevity in multiple human populations, but the mechanisms underpinning this remain unclear. We aimed to characterise potential effects of longevity-associated variation on the expression and mRNA processing of the FOXO3 gene. We performed a comprehensive assessment of FOXO3 isoform usage across a wide variety of human tissues and carried out a bioinformatic analysis of the potential for longevity-associated variants to disrupt regulatory regions involved in isoform choice. We then related the expression of full length and 5' truncated FOXO3 isoforms to rs13217795 genotype in peripheral blood and skeletal muscle from individuals of different rs13217795 genotypes. FOXO3 isoforms displayed considerable tissue specificity. We determined that rs13231195 and its tightly aligned proxy variant rs9400239 may lie in regulatory regions involved in isoform choice. The longevity allele at rs13217795 was associated with increased levels of full length FOXO3 isoforms in peripheral blood and a decrease in truncated FOXO3 isoforms in skeletal muscle RNA. We suggest that the longevity effect of FOXO3 SNPs may in part derive from a shift in isoform usage in skeletal muscle away from the production of 5' truncated FOXO3 isoforms lacking a complete forkhead DNA binding domain, which may have compromised functionality.