title: An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR.
abstract: Mammalian target of rapamycin (mTOR), a large multidomain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein-drug interactions at the domain level, while NMR gave insights into the protein-drug interactions at the residue level. The use of the FKBP12-FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.
authors: Analysis and Pharmacokinetics Research Labs, Department of Drug Discovery, Astellas Pharma Inc., Tsukuba 350-8585,  Japan.
authors: Mitsuhiro
authors: M
authors: Sekiguchi
authors: Yoshihiro
authors: Y
authors: Kobashigawa
authors: Masashi
authors: M
authors: Kawasaki
authors: Masashi
authors: M
authors: Yokochi
authors: Tetsuo
authors: T
authors: Kiso
authors: Ken-ichi
authors: K
authors: Suzumura
authors: Keitaro
authors: K
authors: Mori
authors: Toshio
authors: T
authors: Teramura
authors: Fuyuhiko
authors: F
authors: Inagaki
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D011993:Recombinant Fusion Proteins; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A