Rapamycin passes the torch: a new generation of mTOR inhibitors

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12–rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochemistry shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin–FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity.

 

Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion

mTOR kinase activity is essential for cell adhesion. Serum-starved Rh30 and/or HeLa cells, infected with Ad-mTOR-T, Ad-mTOR-TE, or Ad-GFP (for control), or with lentiviral shRNAs to mTOR or GFP, were treated with or without rapamycin (Rapa, 100 ng/ml) for 2 h, followed by stimulation with or without IGF-1 (10 ng/ml) for 1 h. (A and C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B and D) Adherent cells were determined using CN IV-coated cell adhesion assay. (A) Western blot analysis showed stable expression of FLAG-tagged mutants of mTOR in Rh30 cells infected with Ad-mTOR-T and Ad-mTOR-TE, but not in the control cells infected with Ad-GFP. Expression of mTOR-T, but not mTOR-TE or GFP, prevented rapamycin inhibition of the basal or IGF-1-stimulated phosphorylation of 4E-BP1 (Thr70) in Rh30 cells. (B) Ectopic expression of mTOR-T strongly increased cell adhesion and conferred high resistance to rapamycin, whereas expression of mTOR-TE remained sensitive to rapamycin. (C) Lentiviral shRNA to mTOR, but not GFP, downregulated mTOR in Rh30 cells. (D) Downregulation of mTOR inhibited the basal and IGF-1-stimulated adhesion in Rh30 and HeLa cells. Results are means ± SE ( n = 12). * P < 0.05, ** P < 0.01, difference versus control group. ## P < 0.01, difference versus IGF-1 group. $$ P < 0.01, Ad-mTOR-T group versus Ad-GFP group, or mTOR shRNA group versus GFP shRNA group.

Rapamycin inhibits the basal or IGF-1-stimulated cell adhesion. Rh30, Rh1, HT29 and HeLa cells were treated with or without rapamycin (Rapa, 100 ng/ml) in the presence or absence of IGF-1 (10 ng/ml) for 1.5 h following pre-incubation with rapamycin for 2 h, respectively. (A) Adherent cells were determined using CN IV-, fibronectin- or laminin-coated cell adhesion assay, and (B) cell viability was evaluated by MTS assay, as described in Materials and Methods. Results are means ± SE ( n = 4–12). * P < 0.05, ** P < 0.01, difference versus control group. ## P < 0.01, difference versus IGF-1 group.