Discovery of small molecule mechanistic target of rapamycin inhibitors as anti-aging and anti-cancer therapeutics

Abstract

To date, the most studied drug in anti-aging research is the mTOR inhibitor - rapamycin. Despite its almost perfect anti-aging profile, rapamycin exerts one significant limitation - inappropriate physicochemical properties. Therefore, we have decided to utilize virtual high-throughput screening and fragment-based design in search of novel mTOR inhibiting scaffolds with suitable physicochemical parameters. Seven lead compounds were selected from the list of obtained hits that were commercially available (4, 5, and 7) or their synthesis was feasible (1, 2, 3, and 6) and evaluated in vitro and subsequently in vivo. Of all these substances, only compound 3 demonstrated a significant cytotoxic, senolytic, and senomorphic effect on normal and cancerous cells. Further, it has been confirmed that compound 3 is a direct mTORC1 inhibitor. Last but not least, compound 3 was found to exhibit anti-SASP activity concurrently being relatively safe within the test of in vivo tolerability. All these outstanding results highlight compound 3 as a scaffold worthy of further investigation.

Keywords: SASP phenotype; aging; anti-aging therapy; cancer; mTOR.

FIGURE 1

Chemical structure of rapamycin.

FIGURE 2

Binding mode of compound 1 (left panel) and compound 3 (right panel) within the ATP binding site of mTOR. Magnesium ions – yellow spheres. Selected interactions between ligands and residues colored as yellow dashes.

FIGURE 3

Binding modes of compound 5 (left panel) and compound 6 (right panel) within the rapamycin binding site of mTOR. FRB domain – grey cartoon, water ions – red spheres. Selected interactions between ligands and residues colored as yellow dashes.

SCHEME 1

Chemical synthesis of compounds 1 and 2. Reagents and conditions: (a) 170°C, (b) HBr (47%), reflux.

SCHEME 2

Synthetic route to compound 3. Reagents and conditions: (a) (Boc)₂O, DMAP, DCM, 0°C to RT, (b) Pd(PPh₃)₄, TEA, HBpin, 1,4-dioxane, 80°C, (c) 3-Iodoaniline, Cs₂Co₃, MeOH, 100°C, (d) (Boc)₂O, TEA, 1,4-dioxane/water (2:1), 0°C to RT, (e) HATU, DIPEA, DMF, 0°C to RT, and (f) TFA, DCM, 0°C.

SCHEME 3

Chemical synthesis of compounds 6. Reagents and conditions: (a) Pd(dppf)CL₂.DCM, sat. sol. Na₂CO₃, 1,4-dioxane, 100°C.

FIGURE 4

Screening of new compounds on proliferation of normal and cancerous cells. Normal human dermal fibroblasts BJ (A,B), human immortalized retinal pigment epithelium RPE-1 (C,D), and human glioblastoma U373 (E,F) cells were exposed to compounds 1–7 in the concentration range of 0–100 μM for 24 h and assayed by crystal violet (A,C,E) and resazurin (B,D,F) assays. Torkinib served as a reference compound. The experiment was done in triplicate. Data were normalized to untreated cells and plotted as mean ± SEM. Two-tailed Student’s t-test, ^****p < 0.0001; ^***p < 0.001; ^**p < 0.01; *p < 0.05.

FIGURE 5

The cytotoxic effect of new compound 3 on glioblastoma cells. Human glioblastoma T98 (A,B), A172 (C,D), and U87 (E,F) cells were exposed to compound 3 in the concentration range of 0–100 μM for 24 h and assayed by crystal violet (A,C,E) and resazurin (B,D,F) assays. The experiment was done in duplicate. Data were normalized to untreated cells and plotted as mean ± SEM. Two-tailed Student’s t-test, ^****p < 0.0001; ^***p < 0.001. (G) Cytotoxic effect of torkinib and compound 3 (concentration range of 0–100 μM) demonstrated by time-lapse microscopy (72 h) using Incucyte SX1 platform. End-point images (72 h) are presented. Red color is annexin V staining. Bar, 100 μm.

FIGURE 6

Screening of new compounds on senescent cells. Ionizing radiation (IR)-induced RPE-1 (A,B) and docetaxel (DTX)-induced BJ (C,D) cells were exposed to compounds 1–7 in the concentration range of 0–100 μM for 24 h. Torkinib served as a reference compound. Temozolomide (TMZ)-induced glioblastoma U87 cells (E,F) were exposed to compound 3 in the concentration range of 0–100 μM for 24 h. The senescent cell response was detected by crystal violet (A,C,E) and resazurin (B,D,F) assays. The experiment was performed in triplicate. Data were normalized to untreated cells and plotted as mean ± SEM. Two-tailed Student’s t-test, ^****p < 0.0001; ^***p < 0.001; ^**p < 0.01; *p < 0.05.

FIGURE 7

Time-lapse microscopy of torkinib and compound 3 using Incucyte SX1 platform. γ-irradiated RPE-1 (A) and TMZ-induced U87 (B) senescent cells were exposed to torkinib and compound 3 in a concentration range of 0–100 μM for 72 h. Images were acquired every 60 min. Note that the first image was acquired 30 min (corresponding to 0 h in graph plots) after adding compounds. The graphs plot changes in cell confluence during the treatment. Average cell confluence and standard error from four images are shown. End-point images (72 h) are presented. Red color is annexin V staining. Bar, 400 μm.

FIGURE 8

Inhibitory effect of new compounds on mTORC1 kinase substrate p70 S6 kinase in BJ cells. All compounds (1–7) and control compound torkinib were tested at a concentration of 100 μM for 1- and 5-h-long exposure. Whole-cell lysates were subjected to SDS-PAGE/immunoblotting analysis and probed for phospho-p70 S6K (Thr389) and total p70 S6K. GAPDH was used as a loading control. Note that the robustly elevated level of GAPDH after 1 h exposure to torkinib observed in all experimental replicates is not a loading artifact. Quantitative analysis of immunoblots was done by Image J 1.48v program with GAPDH as the internal control.

FIGURE 9

Compound 3 is the direct inhibitor of mTORC1 kinase. (A) In vitro mTOR kinase enzyme assay of compound 3 tested in the concentration range of 0–100 μM. The percentage of mTOR kinase activity inhibition is plotted. (B) Inhibition of mTORC1 kinase activity assayed in BJ cells after 1- and 5-h-long exposure to compound 3 (0–100 μM) by determination of phosphorylation level of mTORC1 substrate p70 S6K with anti-Thr389 p70 S6K and total p70 S6K antibodies. GAPDH was used as a loading control. Quantitative analysis of immunoblots was done by Image J 1.48v program with GAPDH as the internal control.

FIGURE 10

Inhibitory effect of new compound 3 on mTORC1 kinase and its substrate S6 kinase in various cell lines. Human dermal fibroblasts BJ, immortalized retinal pigment epithelium RPE-1, glioblastoma U87, U373, T98, A172, breast adenocarcinoma MDA-MB-231, lung cancer H1299, prostate cancer DU-145 cells, ionizing radiation (IR)-induced RPE-1, docetaxel (DTX)-induced BJ, and temozolomide (TMZ)-induced U87 were exposed to compound 3 (100 μM) for 1- and 5-h-long exposure. Whole-cell lysates were subjected to SDS-PAGE/immunoblotting analysis and probed for phospho-p70 S6K (Thr389) and total p70 S6K. GAPDH was used as a loading control. Quantitative analysis of immunoblots was done by Image J 1.48v program with GAPDH as the internal control.

FIGURE 11

Inhibitory effect of new compound 3 on mTORC2 kinase and its substrate AKT kinase in various cell lines. Human fibroblasts BJ, immortalized retinal pigment epithelium RPE-1, glioblastoma U87, U373, T98, and A172, breast adenocarcinoma MDA-MB-231, lung cancer H1299, and prostate cancer DU-145 cells were exposed to compound 3 (100 μM) for 1 and 5 h. Whole-cell lysates were subjected to SDS-PAGE/immunoblotting and probed for phospho-AKT (Ser473) and total AKT with specific antibodies. GAPDH was used as a loading control. Quantitative analysis of immunoblots was done by Image J 1.48v program with GAPDH as the internal control.

FIGURE 12

Secretion of pro-inflammatory cytokines by senescent cells after exposure to compound 3 and torkinib. Determination of 11 pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12p70, IL-27, MCP-1, IFNγ, TNFα, and IP-10, and IP-10 in docetaxel-(DIS-BJ), ionizing-radiation- (IR-RPE-1), and temozolomide-induced (TMZ-U87) senescent cells exposed to compound 3 and torkinib (50 μM) for 24 h. Conditioned media were collected 24 h after treatment and analyzed for the level of cytokines by FACS analysis. The experiment was done in triplicate (TMZ-U87 in duplicate). Data were normalized to untreated cells as fold induction to control (set as 1, red line) and plotted as mean ± SEM. ND, not detected. Two-tailed Student’s t-test, *p < 0.05.

FIGURE 13

Mice weights after repeated administration of increasing doses (5, 25, and 50 mg/kg body weight) of compound 3 (33) and torkinib (T).

FIGURE 14

Group 3/25 – kidneys (HE, 200×): narrowing of Bowman’s space (arrow), focal tubular atrophy (asterisk).

FIGURE 15

Group 3/50 – kidneys (HE, 200×): multifocal mild tubular damage (arrow).

FIGURE 16

Group torkinib/5 – kidneys (HE, 200×): focal interstitial fibrosis (arrow), mild tubular damage, hyaline droplets.