Identification and characterization of DL001 in vitro

Although all FDA-approved rapalogs inhibit both mTOR complexes20,29, recent work investigating the molecular mechanism by which rapamycin disrupts mTORC2 (ref. 30) suggested that rapamycin derivatives with reduced activity against mTORC2 but full potency against mTORC1 might exist. We therefore screened a library of approximately 90 rapamycin analogs in order to identify compounds with reduced activity against mTORC2; for the screens, we primarily utilized PC3 cells (Supplementary Fig. 1a), a cell line in which mTORC2 is exquisitely sensitive to rapamycin31. While a majority of the compounds demonstrated strong inhibition of mTORC1 (as indicated by reduced phosphorylation of the mTORC1 downstream readout S6 S240/S244), we also identified several compounds with reduced inhibition of mTORC2. We selected DL001 (Fig. 1a) for further characterization due to its strong mTORC1 inhibitory activity.

Fig. 1 DL001 is a selective inhibitor of mTORC1. a Structure of DL001. b, c PC3 cells were treated with rapamycin or DL001 at 0.3–100 nM for 48 h and b the phosphorylation of S6K1 T389 (a mTORC1 substrate), S6 S240/S244 (a readout for mTORC1 activity), and AKT S473 (a mTORC2 substrate) was determined by western blotting; c the inhibition of AKT S473 phosphorylation was plotted (n = 3 biologically independent experiments; *p < 0.005, Sidak’s test following two-way ANOVA). Rapamycin data are plotted in black, DL001 data plotted in red. d–f The IC 50 for rapamycin (Rap) and DL001 against d mTORC1 and e mTORC2 was determined using an in vitro assay to measure the phosphorylation of S6 S240/S244 (mTORC1) and AKT S473 (mTORC2) in PC3 lysates treated for 24 h. Rapamycin data are plotted in black, DL001 data plotted in red. f The selectivity of each compound for mTORC1 was calculated from the IC 50 (n = 4 biologically independent replicates, three technical replicates each; IC 50 was calculated using Prism 7; *p < 0.05, Student’s t-test). g–i Quantitative proteomic analysis was performed on PC3 cells treated for 24 h with 100 nM Rapamycin (Rap) or DL001, or vehicle, and significantly affected (q < 0.05) proteins were analyzed with gProfiler to identify significantly altered KEGG and Reactome categories, which included g ribosome, h translation initiation, and i macroautophagy and lysosome. Error bars represent standard error. Source data are provided as a Source Data file Full size image

We compared the effect of rapamycin and DL001 on mTORC1 and mTORC2 activity in PC3 cells across a range of doses (0.3–100 nM) via western blotting, assessing the phosphorylation of the mTORC1 substrate S6K1 and its substrate S6, the phosphorylation of rapamycin-sensitive and -insensitive residues on 4E-BP1 (S65 and T37/S46, respectively), and the phosphorylation of the mTORC2 substrate AKT S473. While rapamycin and DL001 had very similar effects on mTORC1 activity (Fig. 1b, Supplementary Fig. 1b–d), the effect of these compounds on mTORC2 was dramatically different (Fig. 1b, c). The IC 50 mTORC2 for rapamycin in this assay was approximately 10 nM, while the IC 50 mTORC2 for DL001 was unable to be determined, as the experiments did not achieve greater than 50% inhibition at the levels tested, but was in excess of 100nM. We observed similar effects on other mouse and human cell lines (Supplementary Fig. 2a, b).

In order to validate the above western blot analysis and to determine accurately the IC 50 mTORC2 for DL001, we utilized a commercially available high-sensitivity chemiluminescent assay (AlphaLISA). PC3 cells treated with either vehicle or a range of doses of either rapamycin or DL001 for 24 h were analyzed to determine the phosphorylation of S6 S240/S244 and Akt S473. As shown in Fig. 1d, in our assay we found that rapamycin inhibited mTORC1 with an IC 50 of 63.3 pM, whereas DL001 inhibited mTORC1 with a very similar IC 50 of 74.9 pM. In contrast, while rapamycin inhibited mTORC2 activity with an IC 50 of 534.9 pM, DL001 inhibited with a significantly greater IC 50 of 26,245.4 pM (Fig. 1e). Thus, while both rapamycin and DL001 are similarly potent mTORC1 inhibitors, DL001 is over 430× more selective for mTORC1 than mTORC2—and is 44-fold more selective for mTORC1 than rapamycin (Fig. 1f).

Finally, to comprehensively compare the functional impact of rapamycin and DL001 treatment on cellular processes downstream of mTORC1, we performed quantitative proteomics. We observed that rapamycin and DL001 both significantly downregulated ribosomal proteins and proteins involved in translation (Fig. 1g, h), while upregulating proteins involved in macroautophagy and lysosomal function (Fig. 1i).

Rapamycin analogs act acutely to inhibit mTORC1 via a noncompetitive mechanism that involves the formation of a ternary complex between a FK506-binding protein (FKBP), rapamycin, and mTOR32. While rapamycin’s activity is most closely associated with FKBP12, it was recently shown that other FKBPs, including FKBP51, can also be induced by rapamycin to bind to and inhibit mTOR activity33. These findings, and the recent demonstration that FKBP12 is essential for the inhibition of mTORC2 by rapamycin30, have led some of us to suggest that rapamycin derivatives that do not bind to FKBP12 would be more selective for mTORC1. We tested if DL001 might act independently of FKBP12 by treating PC3 cells expressing shRNAs against either FKBP12, FKBP51, or a nonspecific control with a range of doses (0.3–100 nM) of DL001 and rapamycin, and western blotting to determine the phosphorylation of S6 and AKT S473 (Fig. 2a–d). We find that knockdown of FKBP12, but not FKBP51, inhibits the ability of both rapamycin and DL001 to inhibit the phosphorylation of S6 (Fig. 2e). Likewise, the effect of both compounds on mTORC2 was completely dependent upon FKBP12, and was unaffected by knockdown on FKBP51 (Fig. 2f).

Fig. 2 DL001 is an FKBP12-dependent mTORC1 inhibitor. a Decreased protein expression of FKBP12 and FKBP51 by shRNA was verified via western blotting. b–d PC3 cells expressing b a nonspecific control shRNA (shCtrl), c shRNA against FKBP12, or d shRNA against FKBP51 were treated with rapamycin or DL001 at 0.3–100 nM for 48 h and the phosphorylation of S6 S240/S244 (a readout for mTORC1 activity) and AKT S473 (a mTORC2 substrate) was determined by western blotting. e–f The inhibition of e S6 S240/S244 phosphorylation and f The inhibition of AKT S473 phosphorylation was plotted (n = 3 biologically independent experiments; statistics for the overall effects of knockdown of FKBP12 or FKBP51 represent the p-value from a two-way ANOVA). Rapamycin data are plotted in black, DL001 data plotted in red; squares represent cells expressing shCtrl, circles cells expressing shFKBP12, and triangles cells expressing shFKBP51. Error bars represent standard error. Source data are provided as a Source Data file Full size image

DL001 specifically inhibits mTORC1 in vivo

We and others have observed that the metabolic effects of rapamycin are apparent in mice after 2–3 weeks of chronic treatment28,34. We therefore administered DL001 to mice for 20 days, and broadly assessed the effect of DL001 on mTORC1 and mTORC2 signaling in 11 tissues. We observed robust inhibition of mTORC1 signaling by DL001 in 9 of 11 tissues, including liver, gastrocnemius, and heart (Fig. 3a–d), visceral white adipose tissue, pancreas, soleus, lung, thymus, and kidney (Supplementary Fig. 3a–f). DL001 did not inhibit mTORC1 signaling in either stomach or spleen (Supplementary Fig. 3g–h), tissues in which rapamycin also has limited or no efficacy30. As compared to mice treated in parallel with 8 mg kg−1 rapamycin30, DL001 inhibited mTORC1 with equal efficacy to rapamycin in many tissues including muscle and white adipose tissue, but less strongly in liver and heart (Fig. 3d). Strikingly, mTORC2 inhibition by DL001 in every tissue was significantly reduced relative to rapamycin. Further, in tissues where complete mTORC1 inhibition by DL001 was observed (e.g. visceral fat and thymus (Supplementary Fig. 3a, e)), AKT S473 phosphorylation was increased, consistent with expectations for a highly selective mTORC1 inhibitor due to the mTORC1-mediated feedback regulation of IRS1 (refs. 35,36).

Fig. 3 DL001 selectively inhibits mTORC1 in vivo. a–d Protein lysates were prepared from the a liver, b gastrocnemius, and c heart of female C57BL/6J mice treated with vehicle or 13 mg kg−1 DL001 every other day for 20 days, and the phosphorylation of S240/S244 S6 (a readout for mTORC1 activity) and Akt S473, an mTORC2 substrate, was determined by western blotting. d The activity of DL001 and rapamycin against mTORC1 and mTORC2 was determined by quantification of the western blots above and from mice treated in parallel with 8 mg kg−1 rapamycin 30 using NIH ImageJ (n = 4 biologically independent animals per group, *p < 0.05, Tukey–Kramer test following one-way ANOVA). Data from Vehicle-treated mice are plotted with white bars, rapamycin-treated mice with black bars, and DL001-treated mice with red bars. e, f C57BL/6J mice were treated with either vehicle, 8 mg kg−1 rapamycin or 12 mg kg−1 DL001 every other day for 5 weeks. e Quantification of phosphorylated liver proteins in vehicle, rapamycin, and DL001-treated mice (n = 17 vehicle, 16 rapamycin, and 9 DL001-treated biologically independent animals for quantification of phosphorylated S6 and S6K1, 12 vehicle, 10 rapamycin, and 6 DL0001-treated biologically independent animals for quantification of phosphorylated ULK1; Dunnett’s test following one-way ANOVA, *p < 0.05). f The integrity of mTORC2 was determined by immunoprecipitation of Rictor from liver lysate; the immunoprecipitate and lysate were probed with antibodies against the indicated proteins and quantified with NIH ImageJ. mTOR(IP) and RICTOR(IP) refer to the quantification of the mTOR and RICTOR immunoblots from the RICTOR immunoprecipitate (n = 6 vehicle, 5 rapamycin, and 3 DL001-treated biologically independent animals, *p < 0.05, Tukey–Kramer test following one-way ANOVA). e, f Data from Vehicle-treated mice are plotted with white bars, rapamycin-treated mice with black bars, and DL001-treated mice with red bars. Error bars represent standard error. Source data are provided as a Source Data file Full size image

As the effect of rapamycin on mTORC1 and mTORC2 signaling in the liver is likely to be highly important—the liver is a key tissue in the regulation of both glucose and lipid homeostasis—we examined in detail the effect of 12 mg kg−1 DL001 administered every other day for 5 weeks on hepatic mTORC1 signaling. Consistent with our findings above, we confirmed decreased mTORC1 signaling in the liver of both rapamycin and DL001-treated mice as measured via the phosphorylation of S6 S240/S244 (Fig. 3e), and also observed decreased phosphorylation of the direct rapamycin-sensitive mTORC1 substrate S6K1 T389; phosphorylation of the rapamycin-resistant mTORC1 substrate ULK1 S757 trended lower in DL001-treated mice, but was not significantly decreased by either compound (Fig. 3e). Despite dosing DL001-treated mice with higher levels of compound than rapamycin-treated mice, we observed that the phosphorylation of mTORC1 substrates in a number of tissues were lower in rapamycin-treated than in DL001-treated mice; consistent with this, and preliminary observations of a shorter half-life for DL001 than rapamycin, we observed lower levels of DL001 than rapamycin in the blood 16 h following the final administration of the compounds (Supplementary Fig. 4a).

In order to directly determine the effects of chronic rapamycin treatment on mTORC2, we immunoprecipitated the complex in order to observe the association of mTOR and Rictor, the two defining subunits of mTORC2 (refs. 28,30). Rapamycin dramatically impacted both the amount of Rictor-associated mTOR and the total amount of mTORC2 in the liver, while DL001 had no effect on mTORC2 integrity or abundance (Fig. 3f). Genetic experiments from a number of labs have also pointed to an important role for mTORC2 in several tissues, including skeletal muscle and white adipose tissue, in the regulation of glucose homeostasis37,38,39,40. In agreement with our previous observation that rapamycin disrupts the Rictor–mTOR association in skeletal muscle and white adipose tissue, we observed that rapamycin, but not DL001, disrupts mTORC2 integrity in these two tissues (Supplementary Fig. 5a, b). DL001 thus shows enhanced specificity for mTORC1 not only in cell culture, but also in vivo.

DL001 has reduced side effects relative to rapamycin

Our initial hypothesis was that a rapalog that specifically inhibits mTORC1, such as DL001, would enable us to avoid many of the side effects of rapamycin. To test this, we examined the effects of DL001 on glucose metabolism, lipid metabolism, and the immune system. Male C57BL/6J mice, which show a robust metabolic response to rapamycin and to genetic inhibition of hepatic mTORC2 signaling28,41, were treated for 5 weeks with either 8 mg kg−1 rapamycin or 12 mg kg−1 DL001. The specific doses of compounds used were chosen as a preliminary single dose pharmacokinetic study suggested that DL001, when delivered i.p. at 12 mg kg−1, has a shorter half-life in blood than rapamycin, with a t 1/2 of ~3.5 h. As we expected, while there was not a significant difference between blood levels of DL001 and rapamycin 16 h after administration, blood levels of DL001 trended ~25% lower (Supplementary Fig. 4a). Given our half-life estimate, this time point reflects a near-trough concentration (C min ) of DL001; the trough level of rapalogs is clinically monitored in patients subjected to rapalog therapy and is directly linked with rapalog exposure, efficacy, and side effects42,43. Importantly, the blood levels of both DL001 and rapamycin were substantially higher than levels observed in the blood of mice fed 14 ppm rapamycin24, which as we have previously demonstrated is sufficient to inhibit mTORC2 in vivo34,44.

After 2 weeks of treatment, a fasting glucose tolerance test (GTT) was conducted (Fig. 4a); as expected, rapamycin-treated mice had significantly impaired glucose tolerance, with increased blood glucose levels starting at 30 min, and a 33% increase in total glucose burden over the time course of the assay as measured by area under the curve (AUC). In contrast, mice treated with DL001 were indistinguishable from vehicle-treated mice at every time point and in AUC (Fig. 4a). We also performed a pyruvate tolerance test (PTT); as we have previously observed28,44, rapamycin-treated mice were pyruvate intolerant, indicating a failure to suppress hepatic gluconeogenesis (Fig. 4b). Once again, DL001-treated mice were similar to vehicle-treated mice throughout the assay, and exhibited an AUC that was indistinguishable from that of vehicle-treated control mice (Fig. 4b). Clinically, the first sign of impaired glycemic control in humans is fasting hyperglycemia, which is induced by rapamycin in both humans and mice;14 we observed a statistically significant elevation in fasting glucose levels in rapamycin-treated mice, but not in mice treated with DL001 (Fig. 4c).

Fig. 4 Unlike rapamycin, DL001 does not cause metabolic disruption. a–i C57BL/6J mice were treated with vehicle, 8 mg kg−1 rapamycin, or 12 mg kg−1 DL001 every other day. a Glucose and b pyruvate tolerance tests were performed after 2 or 3 weeks, respectively (n = 18 vehicle, 18 rapamycin, and 9 DL001-treated biologically independent animals; for GTT/PTT, Tukey–Kramer test following two-way repeated-measures ANOVA, a = p < 0.006 vs. vehicle, b = p < 0.006 vs. DL001. For AUC, means with the same letter are not significantly different from each other (Tukey–Kramer test following one-way ANOVA, p < 0.0007)). For GTT/PTT, data from Vehicle-treated mice are plotted with white squares, Rapamycin-treated mice with black squares, and DL001-treated mice with red triangles. c Fasting blood glucose was measured in mice after 4 weeks of treatment (n = 14 vehicle, 16 rapamycin, and 9 DL001-treated biologically independent animals, means with the same letter are not significantly different from each other, Tukey–Kramer test following one-way ANOVA, p < 0.05). d–f Blood was collected after 5 weeks of treatment and plasma levels of d cholesterol, e triglycerides, and f free fatty acids were determined (n = 18 vehicle, 18 rapamycin, and 9 DL001-treated biologically independent animals, means with the same letter are not significantly different from each other, Tukey–Kramer test following one-way ANOVA, p < 0.05). g–i Flow cytometry analysis (expressed as percent of total live cells) of splenocytes collected and isolated after 5 weeks of treatment (n = 18 vehicle, 17 rapamycin, and 9 DL001-treated biologically independent animals, means with the same letter are not significantly different from each other, Tukey–Kramer test following one-way ANOVA, p < 0.05). a–i Data from Vehicle-treated mice are plotted with white bars, rapamycin-treated mice with black bars, and DL001-treated mice with red bars. Error bars represent standard error. Source data are provided as a Source Data file Full size image

A prominent clinical effect of rapamycin is dyslipidemia, as defined by elevated levels of cholesterol, triglycerides, and free fatty acids; some of these effects are associated with inhibition of mTORC2 signaling, while the molecular basis of others is unclear45. We observed that while rapamycin robustly elevated plasma cholesterol, triglycerides, and free fatty acids, DL001 does not increase blood levels of these lipids (Fig. 4d–f). The physiological and molecular mechanisms which mediate rapamycin-induced hyperlipidemia and hypercholesterolemia have remained mysterious for some time46. It has been suggested that these changes may be due in part to the induction of lipolysis in white adipose tissue45; mTORC1 is an important regulator of lipolysis in adipocytes47,48, and some researchers have also proposed a role for mTORC2 in the regulation of lipolysis in adipose tissue37,40. Although DL001 does not inhibit mTORC2 in white adipose tissue (Supplementary Figs. 3a and 4a), rapamycin induces a 24% increase in ATGL protein, with a 16% increase (p = 0.1, t-test) in DL001-treated animals; rapamycin and DL001 both increase the phosphorylation of PKA substrates (Supplementary Fig. 6a, b). Our observations support the conclusion that mTORC1 regulates lipolysis in adipose tissue, but suggest that this effect is not sufficient to cause rapamycin-induced dyslipidemia.

In addition to its metabolic side effects, the immunosuppressive effects of rapamycin—mediated in part via inhibition of mTORC2 (ref. 49)—are likely a significant barrier to its application for the treatment of chronic age-related diseases. Rapamycin is associated with an increase in viral and fungal infections in humans, short courses of low-dose rapamycin impairs the defense against acute bacterial and viral infections in mice, and chronic treatment with rapamycin impairs adaptive immunity in mice50,51,52. As both mTOR complexes play important roles in adaptive immunity, in part by promoting the survival, differentiation, activation, and function of T cells49, we predicted that by more selectively targeting mTORC1, DL001 would have reduced—although not zero—effects on immune cell numbers as compared to rapamycin.

To compare the effects of rapamycin and DL001 on immune cells, we isolated splenocytes during tissue harvesting, and analyzed their population by flow cytometry20. As we and others have previously reported20,53, rapamycin-treated mice had significantly decreased T cell numbers; rapamycin reduced total CD3+ (T) cell numbers by 40%, and CD3+CD4+ (helper T) cell numbers by 47% (Fig. 4g, h). In contrast, DL001 had a significantly smaller effect on total T cell and helper T cell numbers, reducing the number of CD3+ cells and CD3+CD4+ cells by 13% and 23%, respectively. We and others have previously reported that chronic rapamycin treatment of mice results in decreased numbers of CD3+CD4+CD25+Foxp3+ T regulatory cells (Tregs)20,53,54; rapamycin reduced Tregs by 54%, while DL001 had a significantly smaller effect on Tregs, reducing the numbers by 37% (Fig. 4i). Finally, and surprisingly, while rapamycin reduced the CD3+CD8+ (suppressor/cytotoxic T) cell number by 36%, DL0001 had no effect on CD3+CD8+ cell number (Fig. 4j). While further work is required to fully assess the effect of DL001 on immune function, these observations support the idea that mTORC1-specific inhibitors such as DL001 are likely to have reduced impact on the immune system.

DL001 suppresses the hyperactive mTORC1 of cells lacking TSC

While mTORC1-specific inhibitors such as DL001 are likely to prove of use for many diseases of aging, an immediate urgent need where they may prove beneficial is for the treatment of tuberous sclerosis complex (TSC), a rare genetic disease resulting from a loss of function mutation in TSC1 or TSC2 characterized by the formation of non-malignant tumors in organs including the brain, heart, kidney, and lungs; disfiguring facial angiofibromas; and neurological symptoms including seizures and epilepsy. Functioning together, the proteins encoded by TSC1 and TSC2 normally act to inhibit the activity of mTORC1; TSC patients therefore have hyperactive mTORC1 signaling55.

Treatment with rapamycin can suppress many of the effects of TSC in animal models of the disease56, and a rapalog (everolimus) has been FDA approved for the treatment of specific symptoms of TSC57,58. However, suppression of TSC symptoms requires continuous chronic treatment with rapalogs, which can results in significant side effects. Over the course of 4 years, 30% of subjects in one trial of rapalogs for TSC developed hypercholesterolemia;59 in a second study 5 of 18 (27%) participants were hospitalized for pneumonia over the course of 4 years60. One clinical trial of a rapalog for TSC had a particularly high incidence of side effects, with 72% of the subjects developing hypercholesterolemia, 66% developing hyperlipidemia, and 22% of subjects developing hyperglycemia; in that trial one subject died as a result of bacterial sepsis13. Thus, TSC represents a disease in which minimizing the side effect of rapamycin may be particularly important.

As a proof of principle, we compared the effect of rapamycin and DL001 on mouse embryonic fibroblasts (MEFs) lacking Tsc1. We observed that both rapamycin and DL001 effectively suppressed the hyperactive mTORC1 activity of Tsc1−/− MEFs to below wild-type levels (Fig. 5a). MEFs lacking a functional TSC have increased expression of a number of genes involved in metabolic pathways, including glycolysis, sterol, and lipid biogenesis, which can be reversed by treatment with rapamycin61. We find that MEFs lacking Tsc1 have increased expression of Pdk1, Pfkp, Mvk, Sc5d, Ascl3, and Scd1, and that the expression of these genes can be repressed to normal levels by treatment with either 100 nM rapamycin or 100 nM DL001 (Fig. 5b–d). Importantly, rapamycin and DL001 are equally efficient at suppressing hyperactive mTORC1. Finally, as many of the symptoms of TSC1 such as seizures originate in the brain, we realized it was critically important to determine if DL001 can regulate mTORC1 signaling in the brain. We find that rapamycin and DL001 are both capable of efficiently inhibiting mTORC1 in the brain (Fig. 5e).