Breen Anna K, Thomas Sarah, Beckett David, Agsalud Matthew, Gingras Graham, Williams Judd, Wasko Brian M
Department of Biomedical Sciences, Western University of Health Sciences, Lebanon, OR, 97355, USA.
Geroscience. 2025 Jan 30. doi: 10.1007/s11357-025-01534-8.
Inhibition of the target of rapamycin (TOR/mTOR) protein kinase by the drug rapamycin extends lifespan and health span across diverse species. However, rapamycin has potential off-target and side effects that warrant the discovery of additional TOR inhibitors. TOR was initially discovered in Saccharomyces cerevisiae (yeast) which contains two TOR paralogs, TOR1 and TOR2. Yeast lacking functional Tor1 are viable but are hypersensitive to growth inhibition by TORC1 inhibitors, which is a property of yeast that can be exploited to identify TOR inhibitors. Additionally, yeast lacking FK506-sensitive proline rotamase (FPR1) or containing a tor1-1 allele (a mutation in the Fpr1-rapamycin binding domain of Tor1) are robustly and selectively resistant to rapamycin and analogs that allosterically inhibit TOR activity via an FPR1-dependent mechanism. To facilitate the identification of TOR inhibitors, we generated a panel of yeast strains with mutations in TOR pathway genes combined with the removal of 12 additional genes involved in drug efflux. This creates a drug-sensitive strain background that can sensitively and effectively identify TOR inhibitors. In a wild-type yeast strain background, 25 µM of Torin1 and 100 µM of GSK2126458 (omipalisib) are necessary to observe TOR1-dependent growth inhibition by these known TOR inhibitors. In contrast, 100 nM Torin1 and 500 nM GSK2126458 (omipalisib) are sufficient to identify TOR1-dependent growth inhibition in the drug-sensitized background. This represents a 200-fold and 250-fold increase in detection sensitivity for Torin1 and GSK2126458, respectively. Additionally, for the TOR inhibitor AZD8055, the drug-sensitive system resolves that the compound results in TOR1-dependent growth sensitivity at 100 µM, whereas no growth inhibition is observed in a wild-type yeast strain background. Our platform also identifies the caffeine analog aminophylline as a TOR1-dependent growth inhibitor via selective tor1 growth sensitivity. We also tested nebivolol, isoliquiritigenin, canagliflozin, withaferin A, ganoderic acid A, and taurine and found no evidence for TOR inhibition using our yeast growth-based model. Our results demonstrate that this system is highly effective at identifying compounds that inhibit the TOR pathway. It offers a rapid, cost-efficient, and sensitive tool for drug discovery, with the potential to expedite the identification of new TOR inhibitors that could serve as geroprotective and/or anti-cancer agents.
雷帕霉素这种药物对雷帕霉素靶蛋白（TOR/mTOR）激酶的抑制作用可延长多种物种的寿命和健康期。然而，雷帕霉素存在潜在的脱靶效应和副作用，这使得发现其他TOR抑制剂成为必要。TOR最初是在酿酒酵母（酵母）中发现的，酵母中含有两个TOR旁系同源物TOR1和TOR2。缺乏功能性Tor1的酵母能够存活，但对TORC1抑制剂的生长抑制作用高度敏感，这是酵母的一种特性，可用于鉴定TOR抑制剂。此外，缺乏FK506敏感脯氨酸旋转异构酶（FPR1）或含有tor1-1等位基因（Tor1的Fpr1-雷帕霉素结合结构域中的突变）的酵母对雷帕霉素和通过FPR1依赖性机制变构抑制TOR活性的类似物具有强大且选择性的抗性。为便于鉴定TOR抑制剂，我们构建了一组TOR途径基因发生突变并去除另外12个参与药物外排的基因的酵母菌株。这创造了一种药物敏感的菌株背景，能够灵敏且有效地鉴定TOR抑制剂。在野生型酵母菌株背景中，25 μM的Torin1和100 μM的GSK2126458（奥米帕利西布）是观察这些已知TOR抑制剂对TOR1依赖性生长抑制所必需的。相比之下，100 nM的Torin1和500 nM的GSK2126458（奥米帕利西布）就足以在药物敏感背景中鉴定出TOR1依赖性生长抑制。这分别代表Torin1和GSK2126458的检测灵敏度提高了200倍和250倍。此外，对于TOR抑制剂AZD8055，药物敏感系统显示该化合物在100 μM时会导致TOR1依赖性生长敏感，而在野生型酵母菌株背景中未观察到生长抑制。我们的平台还通过选择性的tor1生长敏感性将咖啡因类似物氨茶碱鉴定为TOR1依赖性生长抑制剂。我们还测试了奈必洛尔、异甘草素、卡格列净、牛膝甾酮、灵芝酸A和牛磺酸，使用我们基于酵母生长的模型未发现它们有TOR抑制的证据。我们的结果表明，该系统在鉴定抑制TOR途径的化合物方面非常有效。它为药物发现提供了一种快速、经济高效且灵敏的工具，有可能加速新型TOR抑制剂的鉴定，这些抑制剂可作为老年保护剂和/或抗癌药物。
