Activity difference of three labdane diterpenoids on human constitutive androstane receptor.

The constitutive androstane receptor (CAR) regulates enzyme transcription related to drug metabolism; therefore, natural compound clarification in food that interacts with CAR is significant for drug development. We revealed that 13-epimanool, which is a compound found in the common sage, is bound to hCAR based on differential scanning fluorometry (DSF) measurements using recombinant hCAR protein. Similar labdane diterpenoids were examined, which revealed that manool and sclareol, which were both natural compounds contained in herbs, are bound to hCAR. They exhibited different effects for CAR activity in the luciferase assay despite the structural similarity. Manool was a partial agonist, 13-epimanool was a weak partial agonist, and sclareol was an antagonist. The activity of hCAR may be regulated by slight differences in the bound compound.

Three new meroterpenoids from Sargassum macrocarpum and their inhibitory activity against amyloid ß aggregation.

Amyloid ß (Aß) is thought to be involved in the pathogenesis of Alzheimer's disease (AD). Aß aggregation in the brain is considered the cause of AD. Therefore, inhibiting Aß aggregation and degrading existing Aß aggregates is a promising approach for the treatment and prevention of the disease. In searching for inhibitors of Aß42 aggregation, we found that meroterpenoids isolated from Sargassum macrocarpum possess potent inhibitory activities. Therefore, we searched for active compounds from this brown alga and isolated 16 meroterpenoids, which contain three new compounds. The structures of these new compounds were elucidated using two-dimensional nuclear magnetic resonance techniques. Thioflavin-T assay and transmission electron microscopy were used to reveal the inhibitory activity of these compounds against Aß42 aggregation. All the isolated meroterpenoids were found to be active, and compounds with a hydroquinone structure tended to have stronger activity than those with a quinone structure.

Estimation of the Viscosity of an Antibody Solution from the Diffusion Interaction Parameter.

Understanding a monoclonal antibody's (MAb) physicochemical properties early in drug discovery is important for determining developability. Viscosity is important because antibodies with high viscosity have limited administration routes. Predicting the viscosity of highly concentrated MAb solutions is therefore essential for assessing developability. Here, we measured the viscosity and diffusion interaction coefficient (kDiff) of 3 MAbs under 15 different formulation conditions (pH and salt) and evaluated correlations between parameters. We also used a computational approach to identify the key factors underlying differences in concentration-dependent curves for viscosity among the MAbs and formulation conditions. Results showed that viscosity increased exponentially at high concentrations, and that this concentration-dependency could be predicted from kDiff. Attempts to set viscosity criterion for use by subcutaneous (SC) and intramuscular (IM) administration suggested that solutions with kDiff greater than -20 mL/g may be candidates. Computational analysis suggested that the presence of a large negative charge in the complementarity determining region (CDR) is a major factor underlying the difference in concentration-dependency among the three MAbs under different formulation conditions. Because it is possible to predict the administration form of antibody solutions, determination of kDiff at the early discovery stage may be essential for effective antibody development.

Structure-activity relationship in advanced glycation end products formation inhibitory activity of phlorotannins.

The structure and inhibitory activity of advanced glycation end products (AGEs) formation were studied using six model compounds and seven phlorotannins isolated from brown alga Ecklonia stolonifera. As a result, it was inferred that AGEs formation inhibitory activity was stronger when electron-rich groups were present because of the addition of many oxygen atoms to the phlorotannins.

Expression, Purification and Characterization of CAR/NCOA-1 Tethered Protein in E. coli Using Maltose-Binding Protein Fusion Tag and Gelatinized Corn Starch.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

Macrocarquinoids A-C, new meroterpenoids from Sargassum macrocarpum.

The production and accumulation of advanced glycation end products (AGEs) have been implicated in diabetes and diabetic complication. This study was conducted as a search for an AGE inhibitor from brown alga, Sargassum macrocarpum. Separation and purification were performed using AGEs inhibitory activity as an index, yielding isolation of 11 meroterpenoids, of which 3 were new compounds: macrocarquinoids A (1), B (6), and C (9). Their structures were elucidated using NMR spectral analysis with 2D techniques. All tested compounds showed AGEs inhibitory activity. Particularly, macrocarquinoid C (9) possessed the strongest activity (IC50: 1.0 mM) of isolated compounds. This activity was stronger than that of aminoguanidine (positive control).

Thermodynamic evaluation of the binding of bisphosphonates to human farnesyl pyrophosphate synthase.

Bisphosphonates (BPs) are the drug of choice for treating bone diseases such as osteoporosis, Paget's disease, and metastatic bone disease. BPs with nitrogen-containing side chains (N-BPs) are known to act as inhibitors for farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. In this study, we evaluated the effect of different side chains on the binding affinity of BPs to human FPPS using calorimetric techniques. Differential scanning calorimetry (DSC) was used to determine the thermal unfolding of FPPS in the presence of BPs. The addition of a series of clinically available BPs increased the structural stability of human FPPS by preferential binding, as indicated by an increase in the FPPS unfolding temperature. The magnitude of the increase was correlated with in vivo antiresorptive efficacy, suggesting that the stabilization of FPPS underlies the inhibitory effect of the BPs. Isothermal titration calorimetry (ITC) experiments were performed to evaluate the binding thermodynamics of BPs against human FPPS. Analysis of the binding energetics revealed that over 30 years of optimization practiced by different pharmaceutical companies has enhanced the enthalpic contribution as well as binding affinity of BPs. The larger enthalpic contribution observed for newer, more potent BPs derives from both improved hydrogen bonding interactions and shape complementarity based on comparisons of our results with available structure information.

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 (ΔT (m)) 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 EC(50) (r(2) = 0.84) than with ΔT m (r(2) = 0.71). The correlation of ΔT m measured using differential scanning calorimetry (DSC) with EC(50) (r(2) = 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.

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.

Absorption, metabolism and excretion of [(14)C]mirabegron (YM178), a potent and selective ß(3)-adrenoceptor agonist, after oral administration to healthy male volunteers.

The mass balance and metabolite profiles of 2-(2-amino-1,3-thiazol-4-yl)-N-[4-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)[U-(14)C]phenyl]acetamide ([(14)C]mirabegron, YM178), a ß(3)-adrenoceptor agonist for the treatment of overactive bladder, were characterized in four young, healthy, fasted male subjects after a single oral dose of [(14)C]mirabegron (160 mg, 1.85 MBq) in a solution. [(14)C]Mirabegron was rapidly absorbed with a plasma t(max) for mirabegron and total radioactivity of 1.0 and 2.3 h postdose, respectively. Unchanged mirabegron was the most abundant component of radioactivity, accounting for approximately 22% of circulating radioactivity in plasma. Mean recovery in urine and feces amounted to 55 and 34%, respectively. No radioactivity was detected in expired air. The main component of radioactivity in urine was unchanged mirabegron, which accounted for 45% of the excreted radioactivity. A total of 10 metabolites were found in urine. On the basis of the metabolites found in urine, major primary metabolic reactions of mirabegron were estimated to be amide hydrolysis (M5, M16, and M17), accounting for 48% of the identified metabolites in urine, followed by glucuronidation (M11, M12, M13, and M14) and N-dealkylation or oxidation of the secondary amine (M8, M9, and M15), accounting for 34 and 18% of the identified metabolites, respectively. In feces, the radioactivity was recovered almost entirely as the unchanged form. Eight of the metabolites characterized in urine were also observed in plasma. These findings indicate that mirabegron, administered as a solution, is rapidly absorbed after oral administration, circulates in plasma as the unchanged form and metabolites, and is recovered in urine and feces mainly as the unchanged form.

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.

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.

Carinatumins A-C, new alkaloids from Lycopodium carinatum inhibiting acetylcholinesterase.

Three new Lycopodium alkaloids, carinatumins A-C (1-3), have been isolated from the club moss Lycopodium carinatum. Structures and stereochemistry of 1-3 were elucidated on the basis of 2D NMR correlations. Carinatumins A (1) and B (2) exhibited a potent inhibitory activity against acetylcholinesterase.

Agesamides A and B, bromopyrrole alkaloids from sponge Agelas species: application of DOSY for chemical screening of new metabolites.

Diffusion-ordered NMR spectroscopy (DOSY) is a versatile and powerful NMR technique and a noninvasive analytical method for mixture analysis that does not require prior physical separation of the analytes. In our search for new metabolites from natural resources, DOSY was applied for constituent analysis of crude bromopyrrole fractions separated from an Okinawan marine sponge Agelas sp. so that two new bromopyrrole alkaloids, agesamides A (1) and B (2), have been isolated. The structures and relative stereochemistry of 1 and 2 were elucidated from spectroscopic data.

Structure of a new cyclic nonapeptide, segetalin F, and vasorelaxant activity of segetalins from Vaccaria segetalis.

A new cyclic nonapeptide, segetalin F, has been isolated from the seeds of Vaccaria segetalis and the structure including absolute stereochemistry was elucidated by using 2D NMR and chemical means. A series of segetalins showed a vasorelaxant activity against norepinephrine (NE)-induced contractions of rat aorta.

Perinadine A, a novel tetracyclic alkaloid from marine-derived fungus Penicillium citrinum.

[structure: see text] A novel tetracyclic alkaloid, perinadine A (1), was isolated from the cultured broth of the fungus Penicillium citrinum, which was separated from the gastrointestine of a marine fish, and the structure was elucidated on the basis of spectroscopic data including 2D NMR spectra. Biogenetically, perinadine A (1) may be derived from citrinin (4), a well-known mycotoxin, and a scalusamide A-type pyrrolidine alkaloid.

Oxylipins arabidopsides C and D from Arabidopsis thaliana.

Two new oxylipins, arabidopsides C (1) and D (2), were isolated from the aerial parts of Arabidopsis thaliana, and the structures of 1 and 2 were elucidated using spectroscopic data, primarily NMR and MS, and chemical means. Arabidopsides C (1) and D (2) are rare digalactosyl diacylglycerides containing 12-oxophytodienoic acid and/or dinor-oxophytodienoic acid. Arabidopside D (2) and arabidopsides A (3) and B (4), which were also isolated from this plant, exhibited inhibitory effects on the growth of the root of cress (Lepidium sativum) seedlings at 5 x 10(-5) mol/L.

YM-254890 analogues, novel cyclic depsipeptides with Galpha(q/11) inhibitory activity from Chromobacterium sp. QS3666.

The structure elucidation and biological activity of novel YM-254890 (1) analogues and semi-synthetic derivatives are described. Three natural analogues, YM-254891 (2), YM-254892 (3), and YM-280193 (4), were isolated from the fermentation broth of Chromobacterium sp. QS3666, and two hydrogenated derivatives, YM-385780 (5) and YM-385781 (6), were synthesized from YM-254890. Their structures were determined by one- and two-dimensional NMR studies and mass spectrometry. Among these compounds, two natural analogues 2-3 which possessed acyl groups at beta-HyLeu-1 and one derivative 6 whose conformation was similar to that of 1 showed comparable Galpha(q/11) inhibitory activity to that of 1. This indicates that the acyl beta-HyLeu residue plays an important role in activity and also that the alpha,beta-unsaturated carbonyl group of the N-MeDha residue is not critical to activity. The other hydrogenated derivative 5 had significantly less activity, which could be attributed to conformational differences.

Subincanadines A-C, novel quaternary indole alkaloids from Aspidosperma subincanum.

Three novel quaternary indole alkaloids with an unprecedented 1-azoniatricyclo[4.3.3.0(1,5)]undecane moiety, subincanadines A-C (1-3), as well as two new indole alkaloids with a 1-azabicyclo[5.2.2]undecane moiety, subincanadines D (4) and E (5), and a new indole alkaloid with a 1-azabicyclo[4.3.1]decane moiety, subincanadine F (6), have been isolated from the barks of Aspidosperma subincanum Mart, and the structures of 1-6 and the stereochemistry of 1-3 were elucidated by spectroscopic data and chemical means.