Mitsuhiro Sekiguchi

Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein–protein complex structure determination

Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

Hyrtioseragamines A and B, new alkaloids from the sponge Hyrtios species.

Two novel alkaloids with a furo[2,3-b]pyrazin-2(1H)-one moiety and a guanidino group, hyrtioseragamines A (1) and B (2), have been isolated from an Okinawan marine sponge Hyrtios species. The structures of 1 and 2 were elucidated on the basis of spectroscopic data and chemical conversions. Compounds 1 and 2 are the first natural products possessing a furo[2,3-b]pyrazine-related moiety.

Echinophyllins A and B, novel nitrogen-containing clerodane diterpenoids from Echinodorus macrophyllus

Two novel nitrogen-containing clerodane diterpenoids, echinophyllins A (1) and B (2), were isolated from the leaves of the Brazilian medicinal plant Echinodorus macrophyllus (‘Chapeu-de-couro’), and their structures and relative stereochemistry were elucidated by spectroscopic data. Echinophyllins A (1) and B (2) possess a unique α,β-unsaturated γ-lactam ring consisting of a clerodane diterpene and a tyramine unit or N-feruloyl putrescine unit, respectively.

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

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.

High-Throughput Evaluation Method for Drug Association with Pregnane X Receptor (PXR) Using Differential Scanning Fluorometry

The pregnane xenobiotic receptor (PXR) is a key transcriptional regulator of cytochrome P450 (CYP) 3A, a crucial enzyme in the metabolism and detoxification of xenobiotics and endobiotics. PXR is activated by a wide variety of chemicals and serves as a master regulator of detoxification in mammals. Here, we report a fast evaluation method for PXR-drug interactions using differential scanning fluorometry (DSF). DSF analysis revealed that PXR associates with a fluorescence dye in the native state as well as in the unfolded state, which prevented precise evaluation of any shift in the transition midpoint (ΔTm) due to association with a drug. Hence, we defined a new parameter, (dF/dT)50, where F is fluorescence intensity and T is temperature, to describe the ligand concentration. (dF/dT)50 exhibited better correlation with EC50 (r2 = 0.84) than with ΔTm (r2 = 0.71). The correlation of ΔTm measured using differential scanning calorimetry (DSC) with EC50 (r2 = 0.86) was similar to the above (dF/dT)50 correlation. Therefore, the use of (dF/dT)50 enables DSF to be used for the rapid evaluation of PXR-drug interactions and could provide prescreening to narrow down the collection of candidate ligands that most likely result in transcriptional activation of CYP3A4.