biostudies-literatureArtoni FEuropean Research Councile13888https://www.ebi.ac.uk/biostudies/studies/S-EPMC10410055biostudies-literatureUnknown22(8)Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogues (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice hinted that rapamycin may be also acting independently from mTOR to influence various cellular processes. Here, we generated a gene-edited cell line that expresses a rapamycin-resistant mTOR mutant (mTOR<sup>RR</supAging cellUnbiased evaluation of rapamycin's specificity as an mTOR inhibitor.PMC10410055757729Grutzmacher NArtoni FDemetriades CfalseUnbiased evaluation of rapamycin's specificity as an mTOR inhibitor.Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogues (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice hinted that rapamycin may be also acting independently from mTOR to influence various cellular processes. Here, we generated a gene-edited cell line that expresses a rapamycin-resistant mTOR mutant (mTOR<sup>RR</sup2023-01-01T00:00:00Z2023 Aug2026-05-29T05:55:08.153Z2024-11-20T19:17:41.356ZS-EPMC104100553722202010.1111/acel.13888