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There are various flows inside and outside cells in vivo. Nonequilibrium molecular dynamics (NEMD) simulation is a useful tool for understanding the effects of these flows on the dynamics of biomolecules. We propose an NEMD method to generate a Poiseuille-like flow between lipid bilayers. We extended the conventional equilibrium MD method to produce a flow by adding constant external force terms to the water molecules. Using the Lagrange multiplier method, the center of mass of the lipid bilayer is constrained so that the flow does not sweep away the lipid bilayer, but the individual lipid molecules fluctuate. The temperature of the system is controlled properly in the solution and membrane by using the Nosé-Hoover thermostat. We found that the flow velocity increases linearly as the applied external force term increases. It is possible to estimate the appropriate value of acceleration to generate a flow with an arbitrary velocity using this proportional relation once a single short MD simulation is performed. We also found that the flow between two lipid bilayers is slower than the analytical solution of the Navier-Stokes equations between rigid parallel plates due to the interactions between water molecules and the membrane. This method can be applied not only to a flow on lipid membranes but also to a flow on soft surfaces generally.

The reductive conversion of vicinal and geminal dibromoalkenes into the corresponding alkynes with i PrMgCl-LiCl (Turbo Grignard reagent) is described. This reaction proceeded in the presence of various functional groups such as ethers, esters, or carbamates under mild conditions in high yields. Due to the selective reactivity, the easily prepared vic -dibromoalkene is considered to be a protecting group of alkyne toward an electrophile. Although butyl lithium has been widely used for the conversion of gem -dibromoalkenes into alkynes in the Corey-Fuchs alkyne synthesis, we report here alternative mild and chemoselective reaction conditions for alkyne synthesis.

Understanding the dynamic assembly process of amyloid β (Aβ) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer's disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aβ fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aβ antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aβ monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aβ assembly during fibril formation and suggests novel strategies for Alzheimer's therapy aimed at inhibiting the fibril elongation.

Polyglutamine (polyQ) diseases are devastating neurodegenerative disorders characterized by abnormal expansion of glutamine repeats within specific proteins. The aggregation of polyQ proteins is a critical pathological hallmark of these diseases. Arginine was identified as a promising inhibitory compound because it prevents polyQ-protein monomers from forming intra- and intermolecular β-sheet structures and hinders polyQ proteins from aggregating to form oligomers. Such an aggregation inhibitory effect was not observed in other amino acids. However, the underlying molecular mechanism of the aggregation inhibition and the factors that differentiate arginine from other amino acids, in terms of the inhibition of the polyQ-protein aggregation, remain poorly understood. Here, we performed replica-permutation molecular dynamics simulations to elucidate the molecular mechanism by which arginine inhibits the formation of the intramolecular β-sheet structure of a polyQ monomer. We found that the intramolecular β-sheet structure with more than four β-bridges of the polyQ monomer with arginine is more unstable than without any ligand and with lysine. We also found that arginine has 1.6-2.1 times more contact with polyQ than lysine. In addition, we revealed that arginine forms more hydrogen bonds with the main chain of the polyQ monomer than lysine. More hydrogen bonds formed between arginine and polyQ inhibit polyQ from forming the long intramolecular β-sheet structure. It is known that intramolecular β-sheet structure enhances intermolecular β-sheet structure between proteins. These effects are thought to be the reason for the inhibition of polyQ aggregation. This study provides insights into the molecular events underlying arginine's inhibition of polyQ-protein aggregation.

Natural products are important sources of seed compounds for drug discovery. However, it has become difficult in recent years to discover new compounds with valuable pharmacological activities. On the other hand, among the vast number of natural products that have been isolated so far, a considerable number of compounds with specific biological activities are thought to be overlooked in screening that uses biological activity as an index. Therefore, it is conceivable that such overlooked useful compounds may be found by screening compound libraries that have been amassed previously through specific assays. Previously, NPD723, a member of the Natural Products Depository library comprised of a mixture of natural and non-natural products developed at RIKEN, and its metabolite H-006 were found to inhibit growth of various cancer cells at low nanomolar half-maximal inhibitory concentration. Subsequent analysis revealed that H-006 strongly inhibited human dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine biosynthetic pathway. Here, we elucidated the crystal structure of the DHODH-flavin mononucleotide-orotic acid-H-006 complex at 1.7 Å resolution to determine that furocoumavirin, the S-enantiomer of H-006, was the actual inhibitor. The overall mode of interaction of furocoumavirin with the inhibitor binding pocket was similar to that described for previously reported tight-binding inhibitors. However, the structural information together with kinetic characterizations of site-specific mutants identified key unique features that are considered to contribute to the sub-nanomolar inhibition of DHODH by furocoumavirin. Our finding identified new chemical features that could improve the design of human DHODH inhibitors.

The cause of Alzheimer's disease is related to aggregates such as oligomers and amyloid fibrils consisting of amyloid-β (Aβ) peptides. Molecular dynamics (MD) simulation studies have been conducted to understand the molecular mechanism of the formation and disruption of Aβ aggregates. In this Perspective, the MD simulation studies are classified into four categories, focusing on the target systems: aggregation of Aβ peptides in bulk solution, Aβ aggregation at the interface, aggregation inhibitor against Aβ peptides, and nonequilibrium MD simulation of Aβ aggregates. MD simulation studies in these categories are first reviewed. Future perspectives in each category are then presented. Finally, the overall perspective is presented on how MD simulations of Aβ aggregates can be utilized for developing Alzheimer's disease treatment.

Alzheimer's disease (AD) is associated with progressive accumulation of amyloid-β (Aβ) cross-β fibrils in the brain. Aβ species tightly associated with GM1 ganglioside, a glycosphingolipid abundant in neuronal membranes, promote amyloid fibril formation; therefore, they could be attractive clinical targets. However, the active conformational state of Aβ in GM1-containing lipid membranes is still unknown. The present solid-state nuclear magnetic resonance study revealed a nonfibrillar Aβ assemblage characterized by a double-layered antiparallel β-structure specifically formed on GM1 ganglioside clusters. Our data show that this unique assemblage was not transformed into fibrils on GM1-containing membranes but could promote conversion of monomeric Aβ into fibrils, suggesting that a solvent-exposed hydrophobic layer provides a catalytic surface evoking Aβ fibril formation. Our findings offer structural clues for designing drugs targeting catalytically active Aβ conformational species for the development of anti-AD therapeutics.

It is known that oligomers of amyloid-β (Aβ) peptide are associated with Alzheimer's disease. Aβ has two isoforms: Aβ40 and Aβ42. Although the difference between Aβ40 and Aβ42 is only two additional C-terminal residues, Aβ42 aggregates much faster than Aβ40. It is unknown what role the C-terminal two residues play in accelerating aggregation. Since Aβ42 is more toxic than Aβ40, its oligomerization process needs to be clarified. Moreover, clarifying the differences between the oligomerization processes of Aβ40 and Aβ42 is essential to elucidate the key factors of oligomerization. Therefore, to investigate the dimerization process, which is the early oligomerization process, Hamiltonian replica-permutation molecular dynamics simulations were performed for Aβ40 and Aβ42. We identified a key residue, Arg5, for the Aβ42 dimerization. The two additional residues in Aβ42 allow the C-terminus to form contact with Arg5 because of the electrostatic attraction between them, and this contact stabilizes the β-hairpin. This β-hairpin promotes dimer formation through the intermolecular β-bridges. Thus, we examined the effects of amino acid substitutions of Arg5, thereby confirming that the mutations remarkably suppressed the aggregation of Aβ42. Moreover, the mutations of Arg5 suppressed the Aβ40 aggregation. It was found by analyzing the simulations that Arg5 is important for Aβ40 to form intermolecular contacts. Thus, it was clarified that the role of Arg5 in the oligomerization process varies due to the two additional C-terminal residues.

Secretory-abundant heat-soluble (SAHS) proteins, which constitute a protein family unique to tardigrades, are thought to be essential for anhydrobiosis. Our previous study has revealed that one of the SAHS proteins of Ramazzottius varieornatus (RvSAHS1) has a more flexible entrance than a mammalian fatty-acid-binding protein, which has a crystal structure similar to that of RvSAHS1. Recently, SAHS paralogs that are expressed abundantly and specifically in the early embryos of this tardigrade and Hypsibius exemplaris have been identified. Comparing these amino-acid sequences with that of RvSAHS1, we have found characteristic differences as I113F and D146T. In this study, we investigate I113F and D146T mutants' properties of RvSAHS1 using molecular dynamics simulations and compare the structures and fluctuations of their entrances with those of the wild type. The two mutants exhibit different properties at the entrance of the β-barrel structure. The I113F mutant tends to close the entrance more than the wild type due to the enhanced hydrophobic network inside the cavity. The D146T mutant, in contrast to the I113F mutant, tends to open the entrance. The mechanism by which this mutation opens the entrance is also discussed. Even though only a single mutation located far from the entrance is added to the wild type, there is a clear difference in the tendency to open and close the β-barrel entrance. It indicates that the entrance properties of the SAHS protein are sensitive to the amino-acid sequence.

Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the βD and βE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the β-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

Water plays a crucial role in the formation and destruction of biomolecular structures. The mechanism for destroying biomolecular structures was thought to be an active breaking of hydrogen bonds by water molecules. However, using nonequilibrium molecular dynamics simulations, in which an amyloid-β amyloid fibril was destroyed via infrared free-electron laser (IR-FEL) irradiation, we discovered a new mechanism, in which water molecules disrupt protein aggregates. The intermolecular hydrogen bonds formed by C═O and N-H in the fibril are broken at each pulse of laser irradiation. These bonds spontaneously re-form after the irradiation in many cases. However, when a water molecule happens to enter the gap between C═O and N-H, it inhibits the re-formation of the hydrogen bonds. Such sites become defects in the regularly aligned hydrogen bonds, from which all hydrogen bonds in the intermolecular β-sheet are broken as the fraying spreads. This role of water molecules is entirely different from other known mechanisms. This new mechanism can explain the recent experiments showing that the amyloid fibrils are not destroyed by laser irradiation under dry conditions. Additionally, we found that helix structures form more after the amyloid disruption; this is because the resonance frequency is different in a helix structure. Our findings provide a theoretical basis for the application of IR-FEL to the future treatment of amyloidosis.

Aggregates and fibrils of intrinsically disordered α-synuclein are associated with Parkinson's disease. Within a non-amyloid β component (NAC) spanning from the 61st to the 95th residue of α-synuclein, an 11-residue segment called NACore ( 68 GAVVTGVTAVA 78 ) is an essential region for both fibril formation and cytotoxicity. Although NACore peptides alone are known to form aggregates and amyloid fibrils, the mechanisms of aggregation and fibrillation remain unknown. This study investigated the dimerization process of NACore peptides as the initial stage of the aggregation and fibrillation processes. We performed an isothermal-isobaric replica-permutation molecular dynamics simulation, which is one of the efficient sampling methods, for the two NACore peptides in explicit water over 96 μs. The simulation succeeded in sampling a variety of dimer structures. An analysis of secondary structure revealed that most of the NACore dimers form intermolecular β-bridges. In particular, more antiparallel β-bridges were observed than parallel β-bridges. We also found that intramolecular secondary structures such as α-helix and antiparallel β-bridge are stabilized in the pre-dimer state. However, we identified that the intermolecular β-bridges tend to form directly between residues with no specific structure rather than via the intramolecular β-bridges. This is because the NACore peptides still have a low propensity to form the intramolecular secondary structures even though they are stabilized in the pre-dimer state.

Oligomer formation of amyloid-β peptides (Aβ) is accelerated at a hydrophilic/hydrophobic interface. However, details of the acceleration mechanism have not been elucidated. To understand the effects of the interface on oligomerization at the atomic level, we performed molecular dynamics simulations for an Aβ40 monomer in the presence and absence of the hydrophilic/hydrophobic interface. Nuclear magnetic resonance experiments of Aβ40 peptides with gangliosidic micelles were also carried out. In the simulations and experiments, the hydrophobic residues of Aβ40 bound to the interface stably. Moreover, we found that Aβ40 formed a hairpin structure at the interface more readily than in bulk water. From these results, we discussed the acceleration mechanism of the oligomer formation at the interface.

Oligomers of amyloid-β peptides (Aβ) are formed during the early stage of the amyloidogenesis process and exhibit neurotoxicity. The oligomer formation process of Aβ and even that of Aβ fragments are still poorly understood, though understanding of these processes is essential for remedying Alzheimer's disease. In order to better understand the oligomerization process of the C-terminal Aβ fragment Aβ(29-42) at the atomic level, we performed the Hamiltonian replica-permutation molecular dynamics simulation with Aβ(29-42) molecules using the explicit water solvent model. We observed that oligomers increased in size through the sequential addition of monomers to the oligomer, rather than through the assembly of small oligomers. Moreover, solvent effects played an important role in this oligomerization process.

The amyloid-β peptides form amyloid fibrils which are associated with Alzheimer's disease. Amyloid-β(29-42) is its C-terminal fragment and a critical determinant of the amyloid formation rate. This fragment forms the amyloid fibril by itself. However, the fragment conformation in the fibril has yet to be determined. The oligomerization process including the dimerization process is also still unknown. The dimerization process corresponds to an early process of the amyloidogenesis. In order to investigate the dimerization process and conformations, we applied the Hamiltonian replica-permutation method, which is a better alternative to the Hamiltonian replica-exchange method, to two amyloid-β(29-42) molecules in explicit water solvent. At the first step of the dimerization process, two amyloid-β(29-42) molecules came close to each other and had intermolecular side chain contacts. When two molecules had the intermolecular side chain contacts, the amyloid-β(29-42) tended to have intramolecular secondary structures, especially β-hairpin structures. The two molecules had intermolecular β-bridge structures by coming much closer at the second step of the dimerization process. Formation of these intermolecular β-bridge structures was induced by the β-hairpin structures. The intermolecular β-sheet structures elongated at the final step. Structures of the amyloid-β(29-42) in the monomer and dimer states are also shown with the free-energy landscapes, which were obtained by performing efficient sampling in the conformational space in our simulations.

We describe the disruption of amyloid fibrils of Alzheimer's amyloid-β peptides by ultrasonic cavitation. For this purpose, we performed nonequilibrium all-atom molecular dynamics simulations with sinusoidal pressure and visualized the process with movies. When the pressure is negative, a bubble is formed, usually at hydrophobic residues in the transmembrane region. Most β-strands maintain their secondary structures in the bubble. When the pressure becomes positive, the bubble collapses, and water molecules crash against the hydrophilic residues in the nontransmembrane region to disrupt the amyloid. Shorter amyloids require longer sonication times for disruption because they do not have enough hydrophobic residues to serve as a nucleus to form a bubble. These results agree with experiments in which monodispersed amyloid fibrils were obtained by ultrasonication.

It is known experimentally that an AK16 peptide forms more α-helix structures with increasing pressure while proteins unfold in general. In order to understand this abnormality, molecular dynamics (MD) simulations with the simulated tempering method for the isobaric-isothermal ensemble were performed in a wide pressure range from 1.0 × 10(-4) GPa to 1.4 GPa. From the results of the simulations, it is found that the fraction of the folded state decreases once and increases after that with increasing pressure. The partial molar volume change from the folded state to unfolded state increases monotonically from a negative value to a positive value with pressure. The behavior under high pressure conditions is consistent with the experimental results. The radius of gyration of highly helical structures decreases with increasing pressure, which indicates that the helix structure shrinks with pressure. This is the reason why the fraction of the folded state increases as pressure increases.

Screening of Si-based flux materials for solution growth of SiC single crystals was demonstrated using a thin film composition-spread technique. The reactivity and diffusion of carbon in a composition spread of the flux was investigated by secondary ion mass spectroscopy depth profiling of the annealed flux thin film spread on a graphite substrate. The composition dependence of the chemical interaction between a seed crystal and flux materials was revealed by high-temperature thermal behavior observation of the flux and the subsequent morphological study of the surface after removing the flux using atomic force microscopy. Our new screening approach is shown to be an efficient process for understanding flux materials for SiC solution growth.

We propose a new method for molecular dynamics and Monte Carlo simulations, which is referred to as the replica-permutation method (RPM), to realize more efficient sampling than the replica-exchange method (REM). In RPM, not only exchanges between two replicas but also permutations among more than two replicas are performed. Furthermore, instead of the Metropolis algorithm, the Suwa-Todo algorithm is employed for replica-permutation trials to minimize its rejection ratio. We applied RPM to particles in a double-well potential energy, Met-enkephalin in a vacuum, and a C-peptide analog of ribonuclease A in explicit water. For comparison purposes, replica-exchange molecular dynamics simulations were also performed. As a result, RPM sampled not only the temperature space but also the conformational space more efficiently than REM for all systems. From our simulations of C-peptide, we obtained the α-helix structure with salt bridges between Gly2 and Arg10, which is known in experiments. Calculating its free-energy landscape, the folding pathway was revealed from an extended structure to the α-helix structure with the salt bridges. We found that the folding pathway consists of the two steps: The first step is the "salt-bridge formation step," and the second step is the "α-helix formation step."

We applied the multibaric-multithermal (MUBATH) molecular dynamics (MD) algorithm to an alanine dipeptide in explicit water. The MUBATH MD simulation covered a wide range of conformational space and sampled the states of PII, C5, alphaR, alphaP, alphaL, and C7(ax). On the other hand, the conventional isobaric-isothermal simulation was trapped in local-minimum free-energy states and sampled only a few of them. We calculated the partial molar enthalpy difference DeltaH and partial molar volume difference DeltaV among these states by the MUBATH simulation using the AMBER parm99 and AMBER parm96 force fields and two sets of initial conditions. We compared these results with those from Raman spectroscopy experiments. The Raman spectroscopy data of DeltaH for the C5 state against the PII state agreed with both MUBATH data with the AMBER parm96 and parm99 force fields. The partial molar enthalpy difference DeltaH for the alphaR state and the partial molar volume difference DeltaV for the C5 state by the Raman spectroscopy agreed with those for the AMBER parm96 force field. On the other hand, DeltaV for the alphaR state by the Raman spectroscopy was consistent with our AMBER-parm99 force-field result. All the experimental results fall between those of simulations using AMBER parm96 and parm99 force fields, suggesting that the ideal force-field parameters lie between those of AMBER parm96 and parm99.

The effectiveness of the ensemble perturbation method, in which many pairs of perturbed and unperturbed molecular dynamics simulations are executed for the ensemble average, has been demonstrated by calculating the subtle anisotropic structural change of carbonmonoxy myoglobin (MbCO) triggered by ligand photolysis. The results show that Mb largely expands in the direction perpendicular to the heme plane and slightly contracts in the horizontal one. This agrees well with the report in the transient grating experiment. In addition, it is suggested that the expansion contributes strongly to the fast energy-transfer process to the water solvent because it is undergone almost within several picoseconds. The mechanical work done on the solvent by the expansion within 1 ps was thermodynamically estimated to be 4.8 kcal/mol.

In clinical, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions involving transporters can often directly affect the therapeutic safety and efficacy of many important drugs. However, there have been few studies on food-drug interactions involving transporters. Dietary polyphenols have been widely assumed to be beneficial for human health. Polyphenols are found ubiquitously, and they are commercially prepared and used as functional foods. We have reported that ferulic acid, which is one of the most well-known polyphenols and is used as a functional food, affected the transport of nateglinide, an antidiabetic drug, by Caco-2 cells. In this study, we investigated the effects of other polyphenols on the nateglinide/H(+) transport system. We report here that caffeic acid and p-coumaric acid have a different inhibitory manner on the uptake of nateglinide. The results of this study are useful to identify the substrate specificity of the nateglinide/H(+) cotransporter.

We disclose optimization efforts based on the novel non-nucleoside adenosine deaminase (ADA) inhibitor, 4 (K(i) = 680 nM). Structure-based drug design utilizing the crystal structure of the 4/ADA complex led to discovery of 5 (K(i) = 11 nM, BA = 30% in rats). Furthermore, from metabolic considerations, we discovered two inhibitors with improved oral bioavailability [6 (K(i) = 13 nM, BA = 44%) and 7 (K(i) = 9.8 nM, BA = 42%)]. 6 demonstrated in vivo efficacy in models of inflammation and lymphoma.

[reaction: see text] An efficient, three-step iterative synthesis of polypyrrolinones has been achieved on solid support, setting the stage for the construction of a wide variety of libraries based on the pyrrolinone scaffold. Central to the approach is an effective end-game sequence featuring pyrrolinone ring construction with traceless release from the solid support.