January 17, 2017

Pan-mTOR inhibitors may be superior to rapamycin for antiaging

Gerosuppression by pan-mTOR inhibitors

Rapamycin slows organismal aging and delays age-related diseases, extending lifespan in numerous species. In cells, rapamycin and other rapalogs such as everolimus suppress geroconversion from quiescence to senescence. Rapamycin inhibits some, but not all, activities of mTOR. Recently we and others demonstrated that pan-mTOR inhibitors, known also as dual mTORC1/C2 inhibitors, suppress senescent phenotype. As a continuation of these studies, here we investigated in detail a panel of pan-mTOR inhibitors, to determine their optimal gerosuppressive concentrations. During geroconversion, cells become hypertrophic and flat, accumulate lysosomes (SA-beta-Gal staining) and lipids (Oil Red staining) and lose their re-proliferative potential (RPP). We determined optimal gerosuppressive concentrations: Torin1 (30 nM), Torin 2 (30 nM), AZD8055 (100 nM), PP242 (300 nM), both KU-006379 and GSK1059615 (1000 nM). These agents decreased senescence-associated hypertrophy with IC50s: 20, 18, 15, 200 and 400 nM, respectively. Preservation of RPP by pan-mTOR inhibitors was associated with inhibition of the pS6K/pS6 axis. Inhibition of rapamycin-insensitive functions of mTOR further contributed to anti-hypertrophic and cytostatic effects. Torin 1 and PP242 were more “rapamycin-like” than Torin 2 and AZD8055. Pan-mTOR inhibitors were superior to rapamycin in suppressing hypertrophy, senescent morphology, Oil Red O staining and in increasing so-called “chronological life span (CLS)”. We suggest that, at doses lower than anti-cancer concentrations, pan-mTOR inhibitors can be developed as anti-aging drugs.

Rapamycin slows down aging in yeast, Drosophila, worm and mice. It also delays age-related diseases in a variety of species including humans. Numerous studies have demonstrated life extension by rapamycin in rodent models of human diseases. The maximal lifespan extension is dose-dependent. One explanation is trivial: the higher the doses, the stronger inhibition of mTOR. There is another explanation: mTOR complex 1 (mTORC1) has different affinity for its substrates. For example, inhibition of phosphorylation of S6K is achieved at low concentrations of rapamycin, whereas phosphorylation of 4EBP1 at T37 / 46 sites is insensitive to pharmacological concentrations of rapamycin. Unlike rapalogs, ATP-competitive kinase inhibitors, also known as dual mTORC1/C2 or pan-mTOR inhibitors, directly inhibit the mTOR kinase in both mTORC1 and mTORC2 complexes

Gerosuppressive effect of pan-mTOR inhibitors (as measured by RPP) was equal to that of rapamycin because it is mostly associated with inhibition of the S6K/S6 axis. Yet anti-hypertrophic effect as well as prevention of SA-beta-Gal staining and large cell morphology was more pronounced with pan-mTOR inhibitors than with rapamycin. Also, at optimal concentrations, all pan-mTOR inhibitors extended loss of re-proliferative potential in stationary cell culture more potently than rapamycin. This test determines hyper-metabolism and lactic acid production and is an equivalent of “yeast CLS”. One conclusion is that pan-mTOR inhibitors may be superior to rapamycin.

At low concentrations, pan-mTOR inhibitors acted like rapamycin, inhibiting the S6K/S6 axis and causing mobility shift of 4EBP1. With increasing concentrations, these drugs inhibited phospho-4EBP1 (T37/46) followed by inhibition of phospho-AKT (S473) and thereby further contributed to anti-hypertrophic effects (and cytostatic effect), prevention of senescent morphology as well as inhibition of CLS. Importantly, effects of pan-mTOR inhibitors varied in their resemblance to rapamycin effects. In particular, Torin 1 and PP242 were rapamycin-like. The window between inhibition of pS6K/S6 versus p4EBP1 and AKT was narrower for Torin 2 and AZD8085 than for other 4 pan-mTOR inhibitors. In general, maximal gerosuppression (as measured by RPP) was achieved at concentrations that inhibited phosphorylation of S6K and S6 and only partially inhibited rapamycin-insensitive functions of mTOR. Rapamycin-like effects achieved at lower concentrations of pan-mTOR inhibitors than rapamycin–unlike effects. Preservation of RPP depends on rapamycin-sensitive functions. Inhibition of senescent morphology (SA-beta-Gal staining, hypertrophy, flat morphology) and CLS depends on both rapamycin-sensitive and -insensitive functions of mTOR.

At gerosuppressive concentrations, pan-mTOR inhibitors should be tested as anti-aging drugs. Life-long administration of pan-mTOR inhibitors to mice will take several years. Yet, administration of pan-mTOR inhibitors can be started late in life, thus shortening the experiment. In fact, rapamycin is effective when started late in life in mice [9]. Optimal doses and schedules of administration could be selected by administration of pan-mTOR inhibitors to prevent obesity in mice on high fat diet (HFD). It was shown that high doses of rapamycin prevented obesity in mice on HFD even when administrated intermittently. Testing anti-obesity effects of pan-mTOR inhibitors will allow investigators to determine their effective doses and schedules within several months. It would be important to test both rapamycin-like agents such as Torin 1 and rapamycin-unlike agent such as Torin 2 or AZD8085. Selected doses and schedules can then be used to extend life-span in both short-lived mice, normal and heterogeneous mice as well as mice on high fat diet. These experiments will address questions of theoretical and practical importance: (a) role of rapamycin-insensitive functions of mTOR in aging. We would learn more about aging and age-related diseases. (b) can pan-mTOR inhibitors extend life span beyond the limits achievable by rapamycin. If successful, such experiments may reveal new causes of death in the absence of mTOR-driven aging, a post-aging syndrome, as mentioned previously. Given that pan-mTOR inhibitors are already undergoing clinical trials for cancer therapy, one can envision their fast application for prevention of age-related diseases by slowing down aging.

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