For a number of years now, mechanistic target of rapamycin (mTOR) has been the focus of a fair amount of research into aging. Goals include gaining a better understanding of the way in which metabolism determines natural variations in longevity, and also establishing means by which the pace of aging might be modestly slowed via long-term pharmaceutical alteration of metabolic processes. I don’t consider this to be the most effective way forward for longevity science, but evidently a lot of people do. mTOR appears to be a factor in a range of genetic and other interventions shown to slow aging to varying degrees in laboratory animals, but for most of these so many changes take place in cellular biochemistry that it remains a challenge to talk definitively about root causes or most important mechanisms.


So far the search for drug candidates to target mTOR has produced few if any outstanding new leads. Rapamycin is the starting point, and has been shown to extend life span in mice, but it has side-effects that make it undesirable for widespread use in humans. Researchers have been exploring the expanding suite of rapalogs, drugs with similar structures and effects, but so far nothing has jumped to the fore by virtue of a large enough improvement to demand immediate clinical development. mTOR forms two complexes in the course of interactions relevant to aging, mTORC1 and mTORC2. There is a school of thought that suggests the problems inherent in rapamycin and similar compounds arise because they affect both of these complexes. There is evidence to suggest that targeting mTORC1 while leaving mTORC2 alone would capture beneficial outcomes without many of the problem side-effects – but easier said than done with pharmaceuticals given the tools to hand. The real issues in the biochemistry are also probably more complex than this simplistic view of the situation.


The paper linked below is characteristic of continued exploration of pharmaceutical databases in search of better options, as well as the increasing complexity of the underlying theory that steers this exploration. The biochemistry of aging, the intricacy with which it progresses from moment to moment, is enormously complex. The paper is also characteristic of an increasing interest in cellular senescence in all areas of the aging research community. With the proof that removal of senescence cells extends life in mice, and increasing evidence for the role of senescent cells in specific age-related diseases, researchers now have to fit these findings into the many and varied existing views of aging, or give senescence greater prominence where already present. In the case of mTOR, researchers demonstrated last year that mTOR inhibition appears to slow the approach of cells towards replicative senescence, the state that occurs at the Hayflick limit on cell replication, which is one of the reasons why it appears here as a yardstick for measuring the effects of alternatives to rapamycin.


Gerosuppression by pan-mTOR inhibitors



Rapamycin slows down aging in yeast, Drosophila, worms, 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 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.



In cell culture, induction of senescence requires two events: cell cycle arrest and mTOR-dependent geroconversion from arrest to senescence. In proliferating cells, mTOR is highly active, driving cellular mass growth. When the cell cycle gets arrested, then still active mTOR drives geroconversion: growth without division (hypertrophy) and a compensatory lysosomal hyperfunction (beta-Gal staining). So senescence can be caused by forced arrest in the presence of an active mTOR. Senescent cells lose re-proliferative potential (RPP): the ability to regenerate cell culture after cell cycle arrest is lifted. Quiescence or reversible arrest, in contrast, is caused by deactivation of mTOR. When arrest is released, quiescent cells re-proliferate. In one cellular model of senescence (cells with IPTG-inducible p21), IPTG forces cell cycle arrest without affecting mTOR. During IPTG-induced arrest, the cells become hypertrophic, flat, SA-beta-Gal positive and lose RPP. When IPTG is washed out, such cells cannot resume proliferation. Loss of RPP is a simple quantitative test of geroconversion. Treatment with rapamycin during IPTG-induced arrest preserves RPP. When IPTG and rapamycin are washed out, cells re-proliferate.



Recently, we have shown that Torin 1 and PP242 suppresses geroconversion, preventing senescent morphology and loss of RPP. In agreement, reversal of senescent phenotype was shown by another pan-mTOR inhibitor, AZD8085. Pan-mTOR inhibitors have been developed as cytostatics to inhibit cancer cell proliferation. Cytostatic side effects in normal cells are generally acceptable for anti-cancer drugs. However, cytostatic side effects may not be acceptable for anti-aging drugs. Gerosuppressive (anti-aging) effects at drug concentrations that are only mildly cytostatic are desirable. Pan-mTOR inhibitors differ by their affinity for mTOR complexes and other kinases. Here we studied 6 pan-mTOR inhibitors (in comparison with rapamycin) and investigated effects of 6 pan-mTOR inhibitors on rapamycin-sensitive and -insensitive activities of mTOR, cell proliferation and geroconversion: Torin 1, Torin 2, AZD8055, PP242, KU-006379 and GSK1059615.



As predicted by theory of TOR-driven aging, rapamycin extends life span and prevents age-related diseases. Yet, rapamycin (and other rapalogs such as everolimus) does not inhibit all functions of mTOR. Inhibition of both rapamycin-sensitive and -insensitive functions of mTOR may be translated in superior anti-aging effects. However, potential benefits may be limited by undesirable effects such as inhibition of cell proliferation (cytostatic effect) and cell death (cytotoxic effect). In fact, pan-mTOR inhibitors have been developed to treat cancer, so they are cytostatic and cytotoxic at intended anti-cancer concentrations. Yet, the window between gerosupressive and cytotoxic effects exists. At optimal gerosuppressive concentrations, pan-mTOR inhibitors caused only mild cytostatic effect. For Torin 1 and PP242, the ratio of gerosuppressive (measured by RPP) to cytostatic concentrations was the most favorable. The ratio of anti-hypertrophic to cytostatic concentration was similar for all pan-mTOR inhibitors. 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.



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. 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.



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