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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.

SUBMITTER: Sekiguchi M

PROVIDER: S-EPMC3196870 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR.

Sekiguchi Mitsuhiro M Kobashigawa Yoshihiro Y Kawasaki Masashi M Yokochi Masashi M Kiso Tetsuo T Suzumura Ken-ichi K Mori Keitaro K Teramura Toshio T Inagaki Fuyuhiko F

Protein engineering, design & selection : PEDS 20110906 11

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 ...[more]

PMID: 21900305

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Project description:UnlabelledMammalian target of rapamycin (mTOR) is a key regulator of cell growth, proliferation and angiogenesis. mTOR signaling is frequently hyper activated in a broad spectrum of human cancers thereby making it a potential drug target. The current drugs available have been successful in inhibiting the mTOR signaling, nevertheless, show low oral bioavailability and suboptimal solubility. Considering the narrow therapeutic window of the available inhibitors, through computational approaches, the present study pursues to identify a compound with optimal oral bioavailability and better solubility properties in addition ensuing high affinity between FKBP12 and FRB domain of mTOR. Current mTOR inhibitors; Everolimus, Temsirolimus Deforolimus and Echinomycin served as parent molecules for similarity search with a threshold of 95%. The query molecules and respective similar molecules were docked at the binding cleft of FKBP12 protein. Aided by MolDock algorithm, high affinity compounds against FKBP12 were retrieved. Patch Dock supervised protein-protein interactions were established between FRB domain of mTOR and ligand (query and similar) bound and free states of FKBP12. All the similar compounds thus retrieved showed better solubility properties and enabled better complex formation of mTOR and FKBP12. In particular Everolimus similar compound PubChem ID: 57284959 showed appreciable drugs like properties bestowed with better solubility higher oral bioavailability. In addition this compound brought about enhanced interaction between FKBP12 and FRB domain of mTOR. In the study, we report Everolimus similar compound PubChem ID: 57284959 to be potential inhibitor for mTOR pathway which can overcome the affinity and solubility concerns of current mTOR drugs.AbbreviationsmTOR - Mammalian Target of Rapamycin, FRB domain - FKBP12-rapamycin associated protein, FKBP12 - FK506-binding protein 12, OPLS - Optimized Potentials for Liquid Simulations, Akt - RAC-alpha serine/threonine-protein kinase, PI3K - phosphatidylinositide 3-kinases.

Project description:BackgroundDysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells.FindingsMouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera.Conclusions/significanceThese findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.

Dataset Information

An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR.

Publications

An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR.

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