Leone Marilisa, Crowell Kevin J, Chen Jinhua, Jung Dawoon, Chiang Gary G, Sareth Sina, Abraham Robert T, Pellecchia Maurizio
Cancer Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2006 Aug 29;45(34):10294-302. doi: 10.1021/bi060976+.
The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.
雷帕霉素的哺乳动物靶点（mTOR）是一种复杂地参与控制细胞生长信号通路的蛋白质。雷帕霉素是一种天然产物，它通过与mTOR的FKBP-雷帕霉素结合（FRB）结构域相互作用来结合并抑制mTOR功能。在此，我们报告FRB的核磁共振溶液结构以及旨在鉴定和表征靶向雷帕霉素结合口袋的新型配体的进一步研究。通过测定mTOR的激酶活性来研究配体以及在没有FKBP12时雷帕霉素的生物活性。虽然我们发现雷帕霉素即使在没有FKBP12的情况下（在低微摩尔范围内）也能结合FRB结构域并抑制mTOR的激酶活性，但我们最有效的配体以相似的结合亲和力与FRB结合，但在高得多的浓度下才抑制mTOR的激酶活性。然而，我们也鉴定出一种低亲和力化合物，它也能够抑制mTOR。因此，我们已经鉴定出能够直接模拟雷帕霉素或能够使FRB结合与mTOR催化活性抑制解离的化合物。这样，这些配体可用于解读细胞中mTOR的复杂调控，并验证FRB结构域作为新型治疗化合物开发的可能靶点。
