Abstract
Inhibition of mTORC1 (mechanistic Target Of Rapamycin Complex 1) with the pharmaceutical rapamycin prolongs the lifespan and healthspan of model organisms including rodents, with evidence now emerging that rapamycin and its analogs may also have rejuvenative effects in dogs and humans. However, the side effects associated with long-term rapamycin treatment, many of which are due to inhibition of a second mTOR complex, mTORC2, have seemed to preclude the routine use of rapamycin as a therapy for age-related diseases. Here, we discuss recent findings suggesting that strong, chronic inhibition of both mTOR complexes may not be necessary to realize the geroprotective effects of rapamycin. Instead, modestly but specifically inhibiting mTORC1 via a variety of emerging techniques, including intermittent or transient treatment with rapamycin derivatives, or specific dietary regimens, may be sufficient to promote health and longevity with reduced side effects. We will also discuss prospects for the development of new molecules that, by harnessing the detailed molecular understanding of mTORC1 signaling developed over the last decade, will provide new routes to the selective inhibition of mTORC1. We conclude that therapies based on the selective inhibition of mTORC1 may soon permit the safer treatment of diseases of aging.
Keywords: mTORC1, Rapamycin, Rapalog, mTORC2
Chronic Treatment With the Mechanistic Target of Rapamycin Inhibitor Rapamycin Promotes Health and Longevity, but has Significant Side Effects
The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that serves as a central integrator of nutrient and hormonal cues that signal if conditions are appropriate for cell growth and proliferation. mTOR signaling can be acutely inhibited by rapamycin, a compound isolated from bacteria found in the soil of Easter Island (1). Rapamycin, along with the rapamycin analogs (rapalogs) everolimus and temsirolimus (2), are FDA-approved as immunosuppressants and for specific complications of genetic disorders of hyperactive mTOR signaling.
Over a decade ago, following the discovery that genetic inhibition of mTOR signaling could extend the lifespan of Caenorhabditis elegans (3), Drosophila melanogaster (4), and Saccharomyces cerevisiae (5,6), it was theorized that inhibition of mTOR signaling, either genetically or pharmacologically via treatment with rapamycin, might be able to extend mammalian lifespan (7,8). This proved to be the case, and since 2009 at least eight published studies have shown that rapamycin extends the lifespan of both female and male inbred mice as well as genetically heterogeneous UW-HET3 mice of both sexes (reviewed in (9)).
In addition to its potent effects on longevity, several studies have highlighted the potential of rapamycin to promote healthspan. Rapamycin can prevent or delay the onset of age-related changes in rodent tissues including the heart, liver, kidney, and tendons (10,11), and delays the onset of cancer in both inbred wild-type mice and mutant strains particularly prone to cancer (12–14). Excitingly, rapamycin can also reverse age-related dysfunction in certain tissues, rejuvenating hematopoietic stem cells and cardiac function in aged mice (15,16). Rapamycin has also shown efficacy in preventing age-related cognitive decline in wild-type mice and Alzheimer’s disease in mouse models of this disease (17–21). While the majority of studies reported to date have been in mice, rapamycin treatment has been recently reported to promote some aspects of cardiac function in dogs (22), and may also rejuvenate the immune system in humans (23).
These exciting findings have led to widespread excitement about the potential use of rapalogs as a therapy for age-related diseases. However, there is some reluctance to utilize rapalogs clinically for chronic diseases of aging due to the side effects of these compounds. Of the most direct discomfort to patients are aphthous ulcers of the mouth and lips (24). The most concern is typically reserved for the immunosuppressive effects of rapalogs; indeed, while rapalogs are FDA-approved as immunosuppressants for organ transplants, some rapalogs have also received “black-box” warnings in part due to the risks of infection or cancer due to suppression of tumor immune surveillance. These risks have led to hospitalizations and even deaths during clinical trials of these compounds for tuberous sclerosis complex (TSC) (25,26), a condition for which rapalogs need to be taken chronically and at a high dose. Chronic treatment with rapalogs can also lead to undesirable metabolic changes, including hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, glucose intolerance, insulin resistance, and an increased risk of developing new-onset diabetes (9,25–29).
For the most part, these side effects are viewed as acceptable and manageable in the context of cancer therapy (27,30); and many (but not all) of these side effects may resolve following cessation of therapy. However, the perceived risk-benefit trade-off may differ when considering the treatment of individuals suffering from diseases of aging. Importantly, virtually all of these side effects have primarily been characterized in patients suffering from serious conditions, including cancer or TSC, and taking high doses of the compounds. Less severe side effects have typically been reported in subjects taking rapamycin at lower doses following organ transplantation; even in these subjects, however, metabolic side effects are sometimes observed (27,30). The long-term consequences of many of these side effects in humans is not clear, as physicians typically switch immunosuppressed patients who develop hyperlipidemia or hyperglycemia to non-rapalog based medications. If the risk of developing side effects from rapalogs cannot be minimized, concern over these side effects will likely preclude or limit the routine or prophylactic use of rapalogs for diseases of aging.
Rapamycin: A Single Compound With Two Molecular Targets
mTOR is found in two distinct protein complexes, each of which is sensitive to distinct environmental stimuli and hormonal cues, and each of which regulate numerous cellular processes via the phosphorylation of distinct substrates. mTOR Complex 1 (mTORC1) is sensitive to the availability of nutrients including amino acids, cholesterol, and glucose. In contrast, mTOR Complex 2 (mTORC2) is primarily sensitive to signaling from hormones including insulin, IGF-1, and leptin. The composition of these two distinct protein complexes and the molecular mechanisms by which they sense nutrients and hormonal queues has been reviewed extensively elsewhere (31,32).
mTORC1 is exquisitely sensitive to rapamycin, and genetic inhibition of mTORC1 is sufficient to extend lifespan. In contrast, mTORC2 was originally characterized as rapamycin-insensitive (33), although subsequent research has shown that when treated for prolonged periods of time, mTORC2 is also sensitive in many cell lines (34,35). While researching the basis for the effects of rapamycin on glucose tolerance, we determined that chronic rapamycin treatment causes hepatic insulin resistance via disruption of mTORC2 (36). While our analysis focused on the disruption of mTORC2 in liver, muscle, and white adipose tissue, subsequent work has revealed that chronic rapamycin treatment disrupts mTORC2 in most tissues (37). Genetic experiments suggest that mTORC2 disruption in liver, adipose tissue, and pancreatic beta cells all contribute to the effects of rapamycin on glucose intolerance (38–41), while disruption of mTORC2 in skeletal muscle impairs insulin sensitivity (42–44).
From the standpoint of the immune system, it is clear that both mTORC1 and mTORC2 play important roles in numerous cell types, including T cells, B cells, and macrophages. The immunosuppressive activity of rapamycin is believed to be mediated in part by an increase in the number and activity of T regulatory cells (Tregs). Both mTORC1 and mTORC2 activity suppress the development of Tregs (reviewed in (45)), and thus it is likely that much of rapamycin’s immunosuppressive action requires inhibition of both complexes. However, due to the numerous roles of both mTOR complexes in other types of immune cells (46–48), the overall effect of rapamycin may be one of an immunomodulator rather than simply an immunosuppressant (49–51).
In light of the contribution of mTORC2 inhibition to the side effects of rapamycin, we and others have suggested that finding ways to more specifically target mTORC1 might reduce the side effects observed with long-term and high dose or chronic treatment with rapamycin. Here, we will discuss emerging evidence that even a modest reduction of mTORC1 activity, as well as intermittent or transient rapamycin dosing regimens, is sufficient to promote health and longevity while reducing undesirable side effects. We will discuss dietary regimens that may promote health through the reduction of mTORC1 signaling. Finally, we will discuss the potential for novel molecules that selectively target the mTORC1 pathway to more safely promote health and longevity.
Moderate Inhibition of mTORC1 Signaling is Sufficient to Extend Lifespan and Healthspan
How much we need to inhibit mTORC1 to promote longevity and/or healthspan remains an open question for humans, but significant data on this question has been gathered in mice (Table 1). First, while there may be an upper limit, the effects of chronic rapamycin treatment are likely dose dependent; the NIA’s Interventions Testing Program has shown that UM-HET3 mice live longer as they are dosed with increasing levels of rapamycin, with a maximum effect on longevity at doses significantly higher than the level needed to impair glucose tolerance in these mice (52,53). In female mice, positive effects of rapamycin were observed even in mice receiving the lowest dose of rapamycin (4.7 ppm in chow) tested.
Table 1.
Moderate Genetic or Pharmaceutical Inhibition of Mechanistic Target Of Rapamycin Complex 1 (mTORC1) Signaling Extends Mice Lifespan
Strain and Sex | Modification or Treatment | Δ Lifespan (%) and Sex | Reference |
---|---|---|---|
Moderate genetic inhibition of mTORC1 signaling | |||
C57BL/6 | S6K1 -/- | 19%, female only | Selman et al. (57) |
C57BL/6 | mTOR +/- mLST8 +/- | 14.4%, female only | Lamming et al. (36) |
C57BL/6J | TSC1 tg | 12.3%, female only | Zhang et al. (56) |
Low-dose rapamycin treatment | |||
UM-HET3 (MF) | 4.7 ppm rapamycin in chow from 9 months of age, continuous | 16%, female only | Miller et al. (52) |
Intermittent rapamycin treatment | |||
129/Sv (F) | 1.5 mg/kg rapamycin (I.P.) from 2 months of age, 3×/wk, 2 wks of every 4. | ~10% | Anisimov et al. (14) |
C57BL/6J.Nia (F) | 2 mg/kg rapamycin (I.P.) from 20 months of age, 1×/5d | 7% | Arriola Apelo et al. (69) |
Transient rapamycin treatment | |||
C57BL/6J.Nia (MF) | 8 mg/kg/d rapamycin (I.P) from ~20 to 23 months of age | 14%, males only | Bitto et al. (68) |
C57BL/6J.Nia (MF) | 126 ppm rapamycin (in chow) from ~20 to 23 months of age | 9% females, 14% males | Bitto et al. (68) |
Note: This table highlights recent studies in which moderate genetic inhibition of mTORC1 treatment or low-dose, intermittent, or transient rapamycin treatment was found to extend the lifespan of wild-type mice. For a more complete listing of studies examining the effect of rapamycin on mouse lifespan, please see (9).
These results mirror findings in genetic mouse models of reduced mTORC1 signaling. While complete inactivation of mTORC1 signaling in an adult mouse is lethal (54,55), two distinct mouse models with a modest decrease in mTORC1 activity resulting from either depletion of mTOR and mLST8 (mTOR+/-mLST8+/-) or transgenic expression of TSC1 also have increased female longevity (36,56). A similar female-specific extension of lifespan is seen in mice in which the gene encoding the mTORC1 substrate S6K1 is deleted (57). However, as with the dose-dependent increase in longevity following rapamycin treatment, mice homozygous for a hypomorphic allele of mTOR have an approximately 20% increase in the longevity of both male and female mice (58). From these results, we conclude that even a modest reduction in mTORC1 signaling is sufficient to improve the longevity of females.
Measurements of healthspan sometimes also show female specificity; aged S6K1-/- females have improved glucose tolerance, insulin sensitivity, body composition, hematopoietic stem cell function, rotarod performance, and neurological function relative to age-matched controls (57,59). While not all of these phenotypes have been examined in male S6K1-/- mice, aged male S6K1-/- mice have normal glucose tolerance and insulin sensitivity despite decreased adiposity (46). In contrast to these sex-specific effects, both male and female mice that transgenically express TSC1 have improved treadmill endurance and are resistant to the induction of cardiac hypertrophy by isoproterenol (56). These results suggest that mTORC1 inhibition might be sufficient to increase the healthspan of males without increasing lifespan. In further support of this concept, it was recently shown that 4E-BP1, a transcription factor that is a well-characterized target of mTORC1, has sex-specific expression in aging and high-fat diet induced obesity (60), and genetic activation of 4E-BP1 signaling improves the metabolic health of male mice (61).
Although the reasons for the female-specific benefits of moderate mTORC1 inhibition on lifespan are not fully understood, genetic or pharmaceutical interventions in the insulin-IGF1/PI3K/mTOR signaling pathway typically show greater benefits on female longevity (62). We have observed that female mice have higher mTORC1 activity than male mice (63), with greater phosphorylation of the S6K1 substrate S6 in tissues including the liver and heart (63). Males may also be less sensitive to the beneficial effects of mTORC1 inhibition because of innate low levels of 4E-BP1, reduced phosphorylation of which following rapamycin treatment may promote healthspan. As a result, females which normally have both higher phosphorylation of mTORC1 substrates and express higher levels of 4E-BP1, may be better positioned than males to benefit from mTORC1 inhibition.
Short-term and/or Late-Life Inhibition of mTORC1 Signaling Promotes Lifespan and Healthspan
When is the most effective time in life for rapamycin to be given, and for how long does treatment need to be sustained? This is a very important question, as lifelong treatment with rapamycin likely would result in a much greater risk of developing side effects than treatment for a short period of time. Importantly, and in contrast to other geroprotective interventions such as calorie restriction, rapamycin treatment appears to be equally effective at extending lifespan when initiated in aged mice as when started at a younger age (64,65). Some have attributed this to the anti-cancer effects of rapamycin treatment; rapamycin suppresses carcinogenesis in aging wild-type mice (12,14) and also extends the survival of cancer-prone mice (13,66). An alternative or complementary explanation may be that rapamycin actually functionally rejuvenates many aged tissues, including hematopoietic stem cells and the heart, where age-related hypertrophy and diastolic dysfunction is reversed by 8 weeks of rapamycin treatment (15,16,67). Fascinatingly, short-term late life rapamycin treatment is sufficient to extend longevity—two different studies have found that treatment of mice for 2–3 months at approximately 2 years of age is sufficient to extend lifespan (15,68).
These types of studies suggest that chronic, strong inhibition of mTORC1 signaling is not required to extend lifespan and healthspan. This is a critical point, as many of the effects of rapamycin may arise after only prolonged treatment. This suggests the possibility that intermittent treatment with rapamycin could promote health and longevity with reduced side effects. Mice which received rapamycin three times per week for 2 weeks of every month, followed by a 2-week period without treatment, had a significant extension of lifespan (14). We recently tested the idea that treating mice with single doses of rapamycin, given at sufficiently long intervals such that only mTORC1 was significantly inhibited, would reduce side effects while still promoting health and longevity. We determined that this dosing regimen had reduced side effects on both glucose metabolism and immunity, yet was capable of extending the lifespan of aged mice (69,70).
While there is not yet a lot of data on the effect of chronic rapamycin treatment in healthy elderly humans, 5 of 11 rapamycin treated subjects (vs. 1 of 14 control subjects) reported side effects during a small, 8-week long randomized clinical trial of 1 mg/day rapamycin (71). While this was a small study and more, longer-term data is needed, subjects treated with rapamycin had increased levels of Tregs; rapamycin treated subjects also had increased glycated hemoglobin (within-group p = .03) and a 40% rise in triglyceride levels (within-group p = .05) (71). In contrast to these results, Dr. Joan Mannick and colleagues have combined intermittent and low-dose approaches to test several doses of the rapalog everolimus (placebo, 0.5 mg/d, 5 mg/wk, or 20 mg/wk of everolimus) in healthy humans for 6 weeks (23). After a washout period, subjects who had received everolimus had an improved immune response to vaccination against the flu, as if everolimus had rejuvenated the immune system. In agreement with the idea that short-term, intermittent rapalog treatment would be well tolerated, the dosing regimen was relatively well tolerated, with an adverse result—mouth ulcers—in only a single subject, who had received the highest dosage of the drug.
A recent follow-up study tested the longer-term efficacy of a similar 6-week regimen of low-dose everolimus in combination with the PI3K/mTOR kinase inhibitor BEZ235 in elderly subjects, followed by a washout period and influenza vaccination (72). Surprisingly, the authors reported that those subjects treated with either everolimus alone or with both compounds not only had an improved response to vaccination, but also had a reduction in overall respiratory tract infections. Overall, the treatment appeared relatively safe, with similar overall reports of serious adverse events in the treated and placebo groups; however, mouth ulcerations and other side effects likely related to mTOR inhibition occurred in treated subjects, particularly those receiving both everolimus and BEZ235 (72). These results suggest that, as with mice, intermittent or low-dose treatment with rapalogs may have reduced side effects as compared to chronic treatment with rapamycin in humans, and may be capable of rejuvenating at least certain aged tissues.
Dietary Interventions as a Rapidly Translatable Way to Inhibit mTORC1
It is widely believed that inhibition of mTORC1 signaling plays an important role in response to calorie restriction (73), but it is unlikely that most people, in a society where almost 70% of the populace is overweight or obese, will be able to adhere to a calorie restricted diet (74). However, diets that alter the level of specific macronutrients without a decrease in caloric consumption are thought to be more sustainable (75,76). The sensitivity of mTORC1 to amino acids suggests that protein restriction (PR) or the restriction of specific amino acids may be viable methods to reduce mTORC1 signaling. Indeed, protein restriction extends lifespan in rodents (77), and protein intake correlates with increased mortality, as well as an increased risk of developing cancer, diabetes, and obesity in humans (78–81). We and others have demonstrated that PR specifically reduces mTORC1 signaling in many somatic tissues, including the liver, heart, skeletal muscle, and white adipose tissue (77,82). Dietary restriction of specific essential amino acids that are potent agonists of mTORC1 signaling—including methionine, leucine, isoleucine, and valine—recapitulates many of the beneficial effects of a PR diet on metabolic health in rodents, reduces mTORC1 activity, and—at least in the case of methionine restriction—extends lifespan (83–95).
Possible ways to translate these findings to the clinic would include the prescription of a low protein diet; our randomized clinical trial suggests that such a diet is sustainable at least in the short term, and is effective in reducing blood levels of the branched-chain amino acids while also improving metabolic health (87). An alternative method would be to eat a plant-based or vegan diet; vegan diets are naturally low in methionine, and vegans have reduced blood levels of methionine (96,97). Other diets that extend lifespan, such as a low carbohydrate ketogenic diet, have also been shown to reduce mTORC1 signaling in a tissue-specific manner (98). Finally, we have hypothesized that if these types of dietary strategies are not broadly acceptable, drugs could be developed that partially block intestinal uptake of specific dietary amino acids from the intestine (99).
Selective Pharmacological Inhibition of mTORC1
In the long term, pharmacological inhibitors of mTORC1 are essential to make mTORC1-based therapies accessible to everyone. As mTOR is a nutrient, growth factor, and energy-sensing kinase that is responsible for maintaining cellular homeostasis, its activity is tightly regulated. mTORC1 activation is mediated in part by its localization to the lysosome (100,101). The major players which recruit mTORC1 to the lysosome are the Rag family of small GTPases proteins; in the presence of amino acids, RagA/RagB and RagC/RagD form heterodimers which then bind to RAPTOR of mTORC1 (102,103). The Rags localize mTORC1 to the lysosome by interacting with the Ragulator, a pentameric protein complex anchored to the lysosome (104–106). Importantly, the Rag GTPases also serve to bring mTORC1 within reach of another small GTPase, Rheb (Ras homolog enriched in brain), which is also localized to the lysosome in response to amino acids (107). Interaction of mTORC1 with GTP-bound Rheb fully activates mTORC1 (108,109).
The molecular mechanisms by which amino acids and other factors regulate the Rag-dependent recruitment of mTORC1 to the lysosome and its activation there has been an active area of research for the last decade; many of these mechanisms are shown in Figure 1. As highlighted in Figure 1 and discussed in detail below, many of these mechanisms could potentially provide targets for novel small molecules that selectively inhibit the activity of mTORC1.
Figure 1.
An overview of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) signaling pathway with areas of potential pharmaceutical inhibition highlighted. Negative regulators (CASTOR1, GATOR1, SAMTOR, Sestrin2, tuberous sclerosis complex [TSC]) and positive regulators (FLCN-FNIP2, GATOR2, KICKSTOR, LRS, RAG GTPases, RAGULATOR, Rheb, SLC38A9, v-ATPase) are shown. Potential mechanisms for the development of mTORC1 specific inhibitors include: A, B, C, D. Identifying small molecules that block the ability of amino acid sensors upstream of mTORC1 to sense the availability of leucine, arginine, or SAM; (E, F) developing compounds such as BC-LI-0186 that inhibit the GAP or GEF activities of FLCN-FNIP2, LRS, or RAGULATOR; (G) Inhibiting the interaction of mTORC1 and Rheb, the mechanism of action of NR1; and (H) Identifying rapamycin derivatives that specifically inhibit mTORC1. Figure is adapted from (145), doi:10.1242/dev.152595, with permission from The Company of Biologists Ltd.
Amino acid sensing
Amino acids act to regulate the GTP-binding status of the Rag GTPases. Ragulator itself is a GEF (guanine nucleotide exchange factor) for RagA and RagB, and there appears to be “inside-out” sensing of amino acid abundance in the lysosomal lumen by the Ragulator, which is mediated in part by its interactions with the vacuolar H+-ATPase (v-ATPase), which maintains the acidic environment of the lysosome, as well as by its interaction with a lysosomal transmembrane transporter, SLC38A9, which acts to sense lysosomal arginine levels (110–113).
In the cytosol, several amino acid sensing proteins have been identified, including CASTOR1/2, Sestrin1/2/3, and SAMTOR, which detect arginine, leucine, and the methionine metabolite SAM (S-adenosylmethionine), respectively (114–121). In the absence of amino acids, both CASTOR and the Sestrin2 are bound to a positive regulator of mTORC1 activation called GATOR2, inhibiting its function. The binding of arginine by CASTOR and leucine by the Sestrins, particularly Sestrin2, disrupts these inhibitory interactions; thus in the presence of both leucine and arginine, GATOR2 is activated and inhibits GATOR1, a protein complex which has GTPase-activating protein (GAP) activity towards RagA and RagB (122). Thus, in the absence of leucine and arginine, GATOR1 is active and inhibits the lysosomal localization of mTORC1 by the Rag GTPases.
In contrast to the Sestrins and CASTOR1/2, which bind to and inhibit GATOR2, the SAM-sensitive protein SAMTOR interacts with GATOR1 in the absence of SAM, promoting its GEF activity towards RagA and RagB (121). Unlike the Sestrins and CASTOR1/2, SAMTOR binds not directly to an amino acid, but to the methionine metabolite SAM. In the presence of SAM, the SAMTOR-GATOR1 interaction is inhibited, inactivating GATOR1 and promoting mTORC1 activity. GATOR1 itself is localized to the lysosome by KICSTOR, a protein complex that recruits GATOR1 to the lysosome and is required for amino acid deprivation to inhibit mTORC1 activity (123).
The presence of cytosolic amino acid binding sensors upstream of mTORC1 activity provides an obvious target for the development of potential therapeutics, particularly in light of recently developed structural information about the binding of leucine and arginine by, respectively, Sestrin2 and CASTOR1 (117,120,124). At least one company is developing mTORC1 activators that function by binding Sestrins (125); as highlighted in Figure 1, it is possible that similar molecules could be developed that instead of activating mTORC1, block or blunt the ability of endogenous leucine, arginine, and SAM to bind to and engage these amino acid sensors. Alternatively, molecules that mimic the binding of these sensor proteins to GATOR1 and GATOR2 could be utilized as mTORC1 inhibitors.
The leucyl-tRNA synthetase (LRS) has also been reported to function as a leucine sensor for mTORC1; it has been proposed that LRS functions as a GAP for RagD (126). As shown in Figure 1, blocking the ability of LRS to bind leucine or to act as a GAP could, therefore, be another way to specifically inhibit mTORC1. The leucinol analog (S)-4-isobutyloxazolidin-2-one can inhibit mTORC1 activity by blocking LRS leucine sensing (127); leucyladenylate sulfamate derivatives can likewise inhibit mTORC1 signaling by targeting LRS (128,129). A recent report identified a compound, BC-LI-0186, inhibits mTORC1 by binding to the RagD interacting site of LRS, blocking its GAP activity (130).
While it remains to be determined if LRS inhibition by these compounds is a feasible strategy in vivo, the diverse number of LRS-interacting compounds that inhibit mTORC1 suggest that LRS is indeed a key amino acid sensor upstream of mTORC1. However, it remains to be determined if this is mediated via RagD; a recent report has found that aminoacyl-tRNA synthetases, including LRS, can catalyze the aminoacylation of specific lysine residues, and LRS-mediated leucylation of specific residues on RagA and RagB mediates the ability of mTORC1 to respond to leucine stimulation (131). The further development of LRS inhibitors as specific mTORC1 inhibitors will likely depend on establishing the exact mechanisms by which LRS regulates mTORC1, and also identifying side effects, as inhibiting the LRS-mediated leucylation of other substrates could lead to unexpected side effects.
Rheb inhibitors
While amino acids signal to mTORC1 via the Rag GTPases as described above, many other hormones, growth factors, and environmental conditions signal to mTOR via the TSC, which acts as a hub for a variety of upstream signaling cascades that either activate or inactivate mTOR depending on anabolic or catabolic needs of the cell. The TSC complex is made up of three subunits TSC1, TSC2 and TSC1D7, and TSC functions to inhibit mTORC1 signaling by acting as a GTPase for Rheb; an allosteric interaction of mTORC1 with Rheb-GTP aligns active site residues of mTORC1 and is essential for mTORC1 activity (108,109,132–134). As highlighted in Figure 1, examples of upstream intracellular events that mTORC1 senses through TSC include signaling by insulin, growth factors, Wnt, TNFa, hypoxia, DNA damage, and energy stress.
Some have hypothesized that mTORC1 could be specifically inhibited by blocking the interaction of mTORC1 with Rheb (Figure 1), a hypothesis bolstered by the finding that point mutations in Rheb can reduce mTORC1 activity without altering the ability of Rheb to bind GTP (135). A recently reported novel compound, NR1, binds Rheb and selectively blocks the activity of mTORC1 without interfering with mTORC2 function (136). One potential barrier to the development of NR1 and similar agents as pharmaceuticals include the relatively high IC50 of NR1 for mTORC1 of 2.1 µM, which is much greater than the IC50 of rapalogs and mTOR kinase inhibitors. Rheb inhibitors may also have undesirable side effects unrelated to mTORC1 inhibition, which will need to be studied; for example, male mice haploinsufficient for Rheb1 experience seizures (136,137).
Rapalogs selectively targeting mTORC1
One of the most curious aspects surrounding the inhibition of mTORC2 by rapamycin is that there is both cell and tissue specificity with regards to how readily mTORC2 can be inhibited by rapamycin treatment. Some cell lines, such as PC3 cells, have mTORC2 activity that is extremely sensitive to rapamycin, whereas mTORC2 in other cell lines are completely resistant (34). mTORC2 in different tissues likewise shows a range of sensitivity to rapamycin treatment, with mTORC2 signaling in the thymus, kidney, and stomach not disrupted by even several weeks of 8 mg/kg/every-other-day rapamycin treatment (37). While in vivo differential exposure to rapamycin may play a role in this response, the diversity of response among cell lines argues that mTORC2 sensitivity is primarily a cell-intrinsic property.
Recent work has shown that the relative expression level of different FK506-binding proteins (FKBPs) correlates with the sensitivity of mTORC2 to rapamycin (37). Canonically, rapamycin binds to FKBP12, and this rapamycin-FKBP12 complex then specifically inhibits mTORC1 by binding to a specific domain on mTORC1 (138–140). However, it was recently shown that other FKBPs can also interact with rapamycin (141). The Kennedy lab has demonstrated that while rapamycin-FKBP12 can inhibit mTORC2 activity under chronic treatment, rapamycin-FKBP51 specifically inhibits mTORC1, and does not inhibit mTORC2 activity (37). The relative expression levels of FKBP12 and FKBP51 by a cell line or tissue, therefore, determines the sensitivity of mTORC2 in that cell line or tissue to rapamycin. These results suggest that rapamycin analogs with increased binding specificity for FKBP51, or reduced ability to complex with FKBP12, could more specifically target mTORC1 (31). There could also exist rapamycin derivatives that target mTORC1 more specifically through other, as yet undescribed, mechanisms.
Conclusions
Inhibition of mTORC1 signaling with rapamycin robustly extends the lifespan of many model organisms, and may well have the potential to promote healthy aging in humans. However, concerns regarding the side effects of rapamycin, particularly when given chronically or at high doses, will likely limit the use of rapamycin-based therapies for diseases of aging. Many of these side effects are likely due, in whole or in part, to the “off-target” inhibition of mTORC2 by rapamycin and rapalogs.
In this review, we have discussed three possible pathways by which the potential geroprotective benefits of mTORC1 inhibition can be realized while minimizing the risks of adverse events and side effects. These include the use of very low doses of rapamycin and its FDA-approved analogs, which may be administered intermittently or for only limited periods of time, and perhaps only to the elderly; emerging evidence suggests this may be sufficient to promote healthspan, lifespan, and rejuvenate tissues, while minimizing side effects. A second strategy is the use of diets with altered levels of specific dietary macronutrients, such as low protein or vegan diets, with reduced levels of mTORC1-activating amino acids. Finally, small molecules which harness our emerging knowledge about the molecular regulation of mTORC1 signaling may soon lead to a new class of mTORC1-selective agents. This is a rapidly developing field; even while this manuscript was in preparation, it was found that a commonly prescribed medication, sildenafil, can act to suppress mTORC1 activity in the heart via a previously undescribed, TSC-dependent mechanism (142).
An important consideration not discussed above is that it is unlikely to be beneficial to completely inhibit mTORC1 signaling, as genetic deletion of RagA or Raptor in adult mice is lethal (54,55). A key difference between genetic ablation of mTORC1 and treatment with rapamycin is that a number of the functions of mTORC1 are rapamycin-resistant (143,144); and thus, the best strategy in the development of novel mTORC1 inhibitors for age-related diseases may be to mimic the effects of rapamycin on mTORC1 signaling as closely as possible, rather than to develop the most potent, complete inhibitor of mTORC1 activity. Collectively, the studies described above suggest that a myriad of strategies that act to modestly reduce mTORC1 activity may soon be able to treat age-related diseases and improve healthspan and lifespan.
Funding
The Lamming Laboratory and D.W.L. are supported in part by National Institutes of Health (NIH) grants AG050135, AG051974, AG056771, and AG062328, and startup funds from the University of Wisconsin-Madison School of Medicine and Public Health and the University of Wisconsin-Madison Department of Medicine. The Lamming laboratory is supported in part by the U.S. Department of Veterans Affairs (I01-BX004031), and this work was supported using facilities and resources from the William S. Middleton Memorial Veterans Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work does not represent the views of the Department of Veterans Affairs or the United States Government.
Conflict of Interest
D.W.L. has received funding from, and is a scientific advisory board member of, Aeonian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases.
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