Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, San Francisco, CA 94158, USA.
Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
Cell Rep. 2013 Dec 26;5(6):1725-36. doi: 10.1016/j.celrep.2013.11.040. Epub 2013 Dec 19.
Current approaches for identifying synergistic targets use cell culture models to see if the combined effect of clinically available drugs is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that may be synergistic targets. Here, we created a tool to screen and identify molecular targets that may synergize with new inhibitors of target of rapamycin (TOR), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. Although clinical results from TOR complex 1 (TORC1)-specific inhibition using rapamycin analogs have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand this increased therapeutic efficacy and to discover secondary targets for combination therapy, we engineered Tor2 in S. cerevisiae to accept an orthogonal inhibitor. We used this tool to create a chemical epistasis miniarray profile (ChE-MAP) by measuring interactions between the chemically inhibited Tor2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1- and TORC2-dependent functions. The results showed a TORC2-specific interaction with the pentose phosphate pathway, a previously unappreciated TORC2 function that suggests a role for the complex in balancing the high energy demand required for ribosome biogenesis.
目前用于鉴定协同靶点的方法是使用细胞培养模型，观察联合使用临床上可用的药物是否比其单独疗效预测的效果更好。需要新的技术来系统地和理性地鉴定可能是协同靶点的靶点和途径。在这里，我们创建了一个工具来筛选和鉴定可能与雷帕霉素（TOR）的新抑制剂协同的分子靶点，TOR 是一种保守的蛋白质，是营养信号通路中细胞增殖信号的主要整合因子。尽管使用雷帕霉素类似物抑制 TOR 复合物 1（TORC1）的临床结果令人失望，但使用也靶向 TORC2 的抑制剂的试验却很有希望。为了了解这种治疗效果的提高，并发现联合治疗的次要靶点，我们在酿酒酵母中对 Tor2 进行了工程改造，使其能够接受正交抑制剂。我们使用该工具通过测量化学抑制的 Tor2 激酶与多样化的缺失突变体文库之间的相互作用，创建了化学遗传微型数组图谱（ChE-MAP）。ChE-MAP 鉴定了已知的 TOR 成分，并区分了 TORC1 和 TORC2 依赖性功能。结果显示与戊糖磷酸途径的 TORC2 特异性相互作用，这是 TORC2 的一个以前未被重视的功能，表明该复合物在平衡核糖体生物发生所需的高能量需求方面发挥作用。
