Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K.

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11-Pbs2-Hog1 MAPK cascade and the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr-518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser-514 and Thr-518 under optimal osmostress conditions. Mono-phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2-Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in the absence of osmostress. We also report that the rapid-and-transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

Repetitive DNA symmetry elements negatively regulate gene expression in embryonic stem cells.

Transcription factor (TF) binding to genomic DNA elements constitutes one of the key mechanisms that regulates gene expression program in cells. Both consensus and nonconsensus DNA sequence elements influence the recognition specificity of TFs. Based on the analysis of experimentally determined c-Myc binding preferences to genomic DNA, here we statistically predict that certain repetitive, nonconsensus DNA symmetry elements can relatively reduce TF-DNA binding preferences. This is in contrast to a different set of repetitive, nonconsensus symmetry elements that can increase the strength of TF-DNA binding. Using c-Myc enhancer reporter system containing consensus motif flanked by nonconsensus sequences in embryonic stem cells, we directly demonstrate that the enrichment in such negatively regulating repetitive symmetry elements is sufficient to reduce the gene expression level compared with native genomic sequences. Negatively regulating repetitive symmetry elements around consensus c-Myc motif and DNA sequences containing consensus c-Myc motif flanked by entirely randomized sequences show similar expression baseline. A possible explanation for this observation is that rather than complete repression, negatively regulating repetitive symmetry elements play a regulatory role in fine-tuning the reduction of gene expression, most probably by binding TFs other than c-Myc.

Four- and two-armed hetero porphyrin dimers: their specific recognition and self-sorting behaviours.

In this study we self-assembled the four-armed porphyrin hetero dimer capsule Cap4, stabilized through amidinium-carboxylate salt bridges, in CH2Cl2 and CHCl3. The dimer capsule Cap4 was kinetically and thermodynamically more stable than the corresponding two-armed dimer Cap2. The number of arms strongly influenced their recognition behaviour; guests possessing small aromatic faces (e.g., 1,3,5-trinitrobenzene) preferred residing in the cavity of the two-armed capsule Cap2, rather than in Cap4, both thermodynamically and kinetically; in contrast, large aromatic guests (e.g., 9,10-dibromoanthracene) were encapsulated predominantly by Cap4 because of favourable entropic effects. The number of arms enabled self-sorting behaviour of the dimer formation; complexation studies using an equimolar mixture of the four porphyrin constituents of the two capsules revealed the quantitative formation of the corresponding dimers Cap2 and Cap4. Furthermore, we examined the specific molecular recognition of Cap2 and Cap4; NMR experiments of mixtures of Cap2 and Cap4 in the presence of favourable guests for Cap2 and Cap4 revealed that these guest molecules were encapsulated selectively by their preferred hosts.

Monitoring and Predicting the Size of Fine Particles Prepared in a Fluidized-Bed Granulator Using a Handheld-Type Raman Spectrometer.

We prepared two kinds of fine particles by treating lactose monohydrate (Lac) with the same formulation in a fluidized-bed granulator, which differed in the spraying air pressure. Raman intensities of treated Lac during processing were measured using a handheld-type Raman spectrometer and plotted against particle size. As particle size increased, Raman intensity decreased in both operating conditions. A linear relationship between them was observed, and regression lines were obtained with good correlation coefficients as calibration curves in both operating conditions. We also prepared two other types of fine particles by treating Lac with the same formulation in a fluidized-bed granulator, which differed in the scale or processing mechanism. The particle size could be successfully predicted using the calibration curve obtained taking powder porosity into consideration. Based on this study, we present a new at-line approach in process analytical technology to monitor and predict particle size using a handheld-type Raman spectrometer.

Development of Low Molecular Weight Ligands for Integrin αvß3.

We designed and synthesized non-peptide organic molecular ligands for integrin αvß3. Candidate ligands featured amidino analog and carboxy groups as binding sites on either side of a spacer, which consisted of benzophenone or an analog, such as diphenyl sulfide, diphenyl sulfoxide, diphenyl sulfone, or diphenyl ether. Competitive binding assays to integrin αvß3 with respect to [125I]echistatin were used to determine inhibitory activity of the synthetic ligands. Ligands bearing 2-aminobenzimidazoyl and glycyl groups separated by a benzophenone spacer demonstrated more potent binding than did a linear Arg-Gly-Asp (RGD) tripeptide that represents the native integrin αvß3 binding motif. Ligands possessing 2-aminobenzimidazoyl and carboxy groups and diphenyl sulfoxide or diphenyl ether spacers inhibited binding of [125I]echistatin with IC50 values similar to that of the linear RGD tripeptide.

Nonthermal excitation effects mediated by sub-terahertz radiation on hydrogen exchange in ubiquitin.

Water dynamics in the hydration layers of biomolecules play crucial roles in a wide range of biological functions. A hydrated protein contains multiple components of diffusional and vibrational dynamics of water and protein, which may be coupled at ∼0.1-THz frequency (10-ps timescale) at room temperature. However, the microscopic description of biomolecular functions based on various modes of protein-water-coupled motions remains elusive. A novel approach for perturbing the hydration dynamics in the subterahertz frequency range and probing them at the atomic level is therefore warranted. In this study, we investigated the effect of klystron-based, intense 0.1-THz excitation on the slow dynamics of ubiquitin using NMR-based measurements of hydrogen-deuterium exchange. We demonstrated that the subterahertz irradiation accelerated the hydrogen-deuterium exchange of the amides located in the interior of the protein and hydrophobic surfaces while decelerating this exchange in the amides located in the surface loop and short 310 helix regions. This subterahertz-radiation-induced effect was qualitatively contradictory to the increased-temperature-induced effect. Our results suggest that the heterogeneous water dynamics occurring at the protein-water interface include components that are nonthermally excited by the subterahertz radiation. Such subterahertz-excited components may be linked to the slow function-related dynamics of the protein.

Quantitative Difference in Solubility of Diastereomeric (2H/1H)-Isotopomers.

Many achiral organic compounds become chiral by an isotopic substitution of one of the enantiotopic moieties in their structures. Although spectroscopic methods can recognize the molecular chirality due to an isotopic substitution, the effects of isotopically chiral compounds in enantioselective reactions have remained unsolved because the small chirality arises only from the difference between the number of neutrons in the atomic nuclei. The difference between the diastereomeric isotopomers of reactive sources should be the key to these effects. However, the energy difference between them is difficult to calculate, even using present computational methods, and differences in physical properties have not yet been reported. Here, we demonstrate that the small energy difference between the diastereomeric isotopomers at the molecular level can be enhanced to appear as a solubility difference between the diastereomeric (2H/1H) isotopomers of α-aminonitriles, synthesized from an isotopically chiral amine, achiral aldehyde, and HCN. This small, but measurable, difference induces the chiral (d/l) imbalance in the suspended α-aminonitrile; therefore, a second enhancement in the solid-state chirality proceeds to afford a highly stereoimproved aminonitrile (>99% selectivity) whose handedness arises completely from the excess enantiomer of isotopically chiral amine, even in a low enantiomeric excess and low deuterium-labeling ratio. Because α-aminonitriles can be hydrolyzed to chiral α-amino acids with the removal of an isotope-labeling moiety, the current sequence of reactions represents a highly enantioselective Strecker amino acid synthesis induced by the chiral hydrogen (2H/1H) isotopomer. Thus, hydrogen isotopic chirality links directly with the homochirality of α-amino acids via a double enhancement of α-aminonitrile, the chiral intermediate of a proposed prebiotic mechanism.

Conformational Plasticity of Cyclic Ras-Inhibitor Peptides Defines Cell Permeabilization Activity.

Cyclorasins 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies.

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.

Spotlight on the Ballet of Proteins: The Structural Dynamic Properties of Proteins Illuminated by Solution NMR.

Solution NMR spectroscopy is a unique and powerful technique that has the ability to directly connect the structural dynamics of proteins in physiological conditions to their activity and function. Here, we summarize recent studies in which solution NMR contributed to the discovery of relationships between key dynamic properties of proteins and functional mechanisms in important biological systems. The capacity of NMR to quantify the dynamics of proteins over a range of time scales and to detect lowly populated protein conformations plays a critical role in its power to unveil functional protein dynamics. This analysis of dynamics is not only important for the understanding of biological function, but also in the design of specific ligands for pharmacologically important proteins. Thus, the dynamic view of structure provided by NMR is of importance in both basic and applied biology.

Absolute asymmetric Strecker synthesis in a mixed aqueous medium: reliable access to enantioenriched α-aminonitrile.

Without using chiral sources, the Strecker reaction of achiral hydrogen cyanide, p-tolualdehyde and benzhydrylamine gave enantioenriched l- or d-N-benzhydryl-α-(p-tolyl)glycine nitriles with up to >99% ee in a mixed solvent of water and methanol. Therefore, total spontaneous resolution of α-aminonitriles could occur through a prebiotic mechanism of α-amino acid synthesis. Moreover, it was demonstrated that the repetition of partial dissolution and crystallization of a suspended conglomerate of aminonitrile under solution-phase racemization could generate the enantiomeric imbalance to afford, in combination with the amplification of chirality, an enantioenriched product in every case. Among the 73 experiments that were carried out, d- and l-enriched isomers occurred 36 and 37 times, respectively. This stochastic behavior, under achiral or racemic starting conditions, meets the requirements of the spontaneous absolute asymmetric Strecker synthesis. The implications of the present results for the origin of chirality of α-amino acids are discussed.

Highly Stereoselective Strecker Synthesis Induced by a Slight Modification of Benzhydrylamine from Achiral to Chiral.

2-Methylbenzhydrylamine is a chiral variant of achiral benzhydrylamine; however, the chirality formed from the small difference between the phenyl and o-tolyl groups is not expected to induce sufficient stereoselectivity in conventional homogeneous reactions. Initiated by the spontaneous formation and asymmetric amplification of the enantioenriched N-benzhydryl-α-aminonitrile forming conglomerate, we here report that (S)- and (R)-configured title amine, upon the Strecker reaction with achiral aldehydes and HCN, afford the corresponding α-aminonitriles with up to >99.5 % diastereomeric excess, in conjunction with an enhancement of chirality in the solid state. l-Alanine with 98 % ee was synthesized from the (S)-amine by using the method discussed here. Achiral aromatic and heteroaromatic aldehydes could also be successfully utilized to afford chiral α-aminonitriles in a highly stereoselective manner. The stereodivergent synthesis of styrylglycine nitriles has also been accomplished by using racemic and enantioenriched 2-methylbenzhydrylamine. Thus, accompanied with a small rearrangement of the common substrate from achiral toward chiral, the present reactions induce an enhancement of chirality, and expands the concept of stereoselective synthesis to increase the opportunity to access highly enantioenriched compounds such as α-amino acids.

Differences in Powder Properties of Two 1,4-Dihydropyridine-Type Compounds Evaluated through Thermal Analysis.

The powder properties of two 1,4-dihydropyridine type compounds, manidipine dihydrochloride (Man) and benidipine hydrochloride (Ben), which possess similar physicochemical properties, were compared through thermal and mechanical analyses. Man and Ben were compressed with lactose monohydrate (Lac) and magnesium stearate (Mgst) at different compression forces. As an index, we focused on the onset temperatures of Lac dehydration during thermal analysis and plotted them against compression forces to evaluate the differences in powder properties between Man and Ben. To discuss in detail, the Lac ratio was selected as a formulation factor and compression speed as a process factor, which would be influenced to the onset temperature or its profile. It could be represented that Man was more adherent than Ben through thermal analysis by changing these critical factors, which were consistent with the results obtained through mechanical analysis.

Forbidden Coherence Transfer of 19F Nuclei to Quantitatively Measure the Dynamics of a CF3-Containing Ligand in Receptor-Bound States.

The dynamic property of a ligand in the receptor-bound state is an important metric to characterize the interactions in the ligand-receptor interface, and the development of an experimental strategy to quantify the amplitude of motions in the bound state is of importance to introduce the dynamic aspect into structure-guided drug development (SGDD). Fluorine modifications are frequently introduced at the hit-to-lead optimization stage to enhance the binding potency and other characteristics of a ligand. However, the effects of fluorine modifications are generally difficult to predict, owing to the pleiotropic nature of the interactions. In this study, we report an NMR-based approach to experimentally evaluate the local dynamics of trifluoromethyl (CF3)-containing ligands in the receptor-bound states. For this purpose, the forbidden coherence transfer (FCT) analysis, which has been used to study the dynamics of methyl moieties in proteins, was extended to the 19F nuclei of CF3-containing ligands. By applying this CF3-FCT analysis to a model interaction system consisting of a ligand, AST-487, and a receptor, p38α, we successfully quantified the amplitude of the CF3 dynamics in the p38α-bound state. The strategy would bring the CF3-containing ligands within the scope of dynamic SGDD to improve the affinity and specificity for the drug-target receptors.

Asymmetric Strecker Reaction Arising from the Molecular Orientation of an Achiral Imine at the Single-Crystal Face: Enantioenriched l- and d-Amino Acids.

Strecker synthesis has long been considered one of the prebiotic reactions for the synthesis of α-amino acids. However, the correlation between the origin of chirality and highly enantioenriched α-amino acids through this method remains a puzzle. In the reaction, it may be conceivable that the handedness of amino acids has been determined at the formation stage of the chiral intermediate α-aminonitrile, that is, the enantioselective addition of hydrogen cyanide to an imine. Herein, an enantiotopic crystal surface of an achiral imine acted as an origin of chirality for the enantioselective formation of α-aminonitriles by the addition of HCN. In conjunction with the amplification of the enantiomeric excess and multiplication of enantioenriched aminonitrile, a large amount of near enantiopure α-amino acids, with the l- and d-handedness corresponding to the molecular orientation of the imine, is reported.

Preparation of Fine Particles with Improved Solubility Using a Complex Fluidized-Bed Granulator Equipped with a Particle-Sizing Mechanism.

A new type of fluidized-bed granulator equipped with a particle-sizing mechanism was used for the preparation of fine particles that improved the solubility of a poorly water-soluble drug substance. Cefteram pivoxyl (CEF) was selected as a model drug substance, and its solution with a hydrophilic polymer, hydroxypropyl cellulose (HPC-L), was sprayed on granulation grade lactose monohydrate (Lac). Three types of treated particles were prepared under different conditions focused on the spraying air pressure and the amount of HPC-L. When the amount of HPC-L was changed, the size of the obtained particles was similar. However, particle size distribution was dependent on the amount of HPC-L. Its distribution became more homogenous with greater amounts of HPC-L, but the particle size distribution obtained by decreasing the spraying air pressure was not acceptable. By processing CEF with HPC-L using a complex fluidized-bed granulator equipped with a particle-sizing mechanism, the dissolution ratio was elevated by approximately 40% compared to that of unprocessed CEF. Moreover, in the dissolution profile of treated CEF, the initial burst was suppressed, and nearly zero order release was observed up to approximately 60% in the dissolution profile. This technique may represent a method with which to design fine particles of approximately 100 µm in size with a narrow distribution, which can improve the solubility of a drug substance with low solubility.

Improvement of Ligand Affinity and Thermodynamic Properties by NMR-Based Evaluation of Local Dynamics and Surface Complementarity in the Receptor-Bound State.

The thermodynamic properties of a ligand in the bound state affect its binding specificity. Strict binding specificity can be achieved by introducing multiple spatially defined interactions, such as hydrogen bonds and van der Waals interactions, into the ligand-receptor interface. These introduced interactions are characterized by restricted local dynamics and improved surface complementarity in the bound state. In this study, we experimentally evaluated the local dynamics and the surface complementarity of weak-affinity ligands in the receptor-bound state by forbidden coherence transfer analysis in free-bound exchange systems (Ex-FCT), using the interaction between a ligand, a myocyte-enhancer factor 2A (MEF2A) docking peptide, and a receptor, p38α, as a model system. The Ex-FCT analyses successfully provided information for the rational design of a ligand with higher affinity and preferable thermodynamic properties for p38α.

Rotaxane Formation from Borate Ion-Containing Crown Ether and Ammonium Ion: Enhancement of Their Association through Ion-Pairing.

We synthesized [2]rotaxanes featuring a catechol borate ion-containing crown ether and secondary ammonium ions. These rotaxane components show both ion-ion interactions and hydrogen bonds. X-ray crystallography and NMR spectroscopy allowed elucidation of the rotaxane structure. Moreover, 1H NMR spectroscopy revealed the rotaxane synthesis can be thermodynamically controlled. The binding affinity between the borate-containing crown ether and ammonium ions is enhanced by ion-pairing.

Acid-base responsive molecular switching of a [2]rotaxane incorporating two different stations in an axle component.

Interlocked compounds such as rotaxanes and catenanes exhibit unique kinetic properties in response to external chemical or physical stimuli and are therefore expected to be applied to molecular machines and molecular sensors. To develop a novel rotaxane for this application, an isophthalamide macrocycle and a neutral phenanthroline axle were used. Stable pseudorotaxanes are known to be formed using hydrogen bonds and π-π interactions. In this study, we designed a non-symmetric axial molecule and synthesized a [2]rotaxane with the aim of introducing two different stations; a phenanthroline and a secondary amine/ammonium unit. Furthermore, 1H NMR measurements demonstrated that the obtained rotaxane acts as a molecular switch upon application of external acid/base stimuli.

Affinity-directed substrate/H+-antiport by a MATE transporter.

Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-µM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.