title: Biochemical and pharmacological inhibition of mTOR by rapamycin and an ATP-competitive mTOR inhibitor.
abstract: The mammalian target of rapamycin (mTOR) is the catalytic subunit of two multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). Clinically used rapamycin and rapalogs are FKBP12-dependent allosteric inhibitors of mTORC1. The recently discovered WYE-125132 and related drugs represent a new generation of ATP competitive and highly specific inhibitors targeting mTOR globally. As mTORC1 and mTORC2 mediate diverse sets of both redundant and distinctive cellular pathways of growth, nutrient and energy homeostasis, rapamycin and WYE-125132 elicit both overlapping and distinctive pharmacological properties with important implications in treating cancer, metabolic, and age-related degenerative diseases. Detailed methods are described for the determination of mTOR inhibition by rapamycin and WYE-125132 in assays of recombinant mTOR enzyme, immunprecipitated native mTOR complexes, growth factor- and amino acid-induced cellular phosphorylation cascades as well as the PI3K/AKT/mTOR hyperactive breast tumor model in vitro and in vivo. The methods have been particularly useful in discovery and biochemical characterization of mTOR inhibitors, cellular and in vivo mTOR substrate phosphorylation analysis, and in deciphering novel biomarkers of mTORC1 and mTORC2 signaling pathways.
authors: Cancer Pharmacology, Fudan University, Shanghai, PR, China. yuker26@gmail.com
authors: Ker
authors: K
authors: Yu
authors: Lourdes
authors: L
authors: Toral-Barza
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D010671:Phenylurea Compounds / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000494:pharmacology; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; C493245:CRTC2 protein, human; D046912:Multiprotein Complexes; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011506:Proteins; D011720:Pyrazoles; D014157:Transcription Factors; C547581:WYE 125132; D000255:Adenosine Triphosphate; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus