The allosteric mechanism of mTOR activation can inform bitopic inhibitor optimization

mTOR serine/threonine kinase is a cornerstone in the PI3K/AKT/mTOR pathway. Yet, the detailed mechanism of activation of its catalytic core is still unresolved, likely due to mTOR complexes' complexity. Its dysregulation was implicated in cancer and neurodevelopmental disorders. Using extensive molecular dynamics (MD) simulations and compiled published experimental data, we determine exactly how mTOR's inherent motifs can control the conformational changes in the kinase domain, thus kinase activity. We also chronicle the critical regulation by the unstructured negative regulator domain (NRD). When positioned inside the catalytic cleft (NRD IN state), mTOR tends to adopt a deep and closed catalytic cleft. This is primarily due to the direct interaction with the FKBP-rapamycin binding (FRB) domain which restricts it, preventing substrate access. Conversely, when outside the catalytic cleft (NRD OUT state), mTOR favors an open conformation, exposing the substrate-binding site on the FRB domain. We further show how an oncogenic mutation (L2427R) promotes shifting the mTOR ensemble toward the catalysis-favored state. Collectively, we extend mTOR's "active-site restriction" mechanism and clarify mutation action. In particular, our mechanism suggests that RMC-5552 (RMC-6272) bitopic inhibitors may benefit from adjustment of the (PEG₈) linker length when targeting certain mTOR variants. In the cryo-EM mTOR/RMC-5552 structure, the distance between the allosteric and orthosteric inhibitors is ∼22.7 Å. With a closed catalytic cleft, this linker bridges the sites. However, in our activation mechanism, in the open cleft it expands to ∼24.7 Å, offering what we believe to be the first direct example of how discovering an activation mechanism can potentially increase the affinity of inhibitors targeting mutants.