Discovery of a potent orally available pyrazolopyridone derivative as a novel selective bromodomain and extra-terminal domain (BET)-first bromodomain (BD1) inhibitor.

Clinical studies have shown that inhibitors of bromodomain and extra-terminal domain (BET) proteins, particularly BRD4, have antitumor activity and efficacy. The BET protein has two domains, BD1 and BD2, and we previously focused on BD1 and reported orally bioavailable BD1-selective inhibitors. In this study, we obtained a BD1 inhibitor, a more potent and highly selective pyrazolopyridone derivative 13a, and confirmed its in vivo efficacy.

Discovery of a potent, orally available furopyridine derivative as a novel selective bromodomain and extra-terminal domain (BET)-first bromodomain (BD1) inhibitor.

We explored novel immunosuppressive agents with immune tolerance using a phenotypic drug discovery strategy, focusing on costimulatory molecules in T cells, and obtained triazolothienodiazepine derivatives. Their mechanism of action is to inhibit the bromodomain and extra-terminal domain (BET) family, as we have previously reported. Selective inhibition of the first bromodomain (BD1) of the BET family is expected to exert antitumor and immunosuppressive effects, similar to BET inhibitors. This study identified furopyridine derivatives 7 and 8 with high BD1 inhibitory activity and high selectivity over BD2. Compound 7 was found to be orally bioavailable and exhibited anti-inflammatory activity in a lipopolysaccharide-induced model.

Generation of novel respiratory syncytial virus vaccine candidate antigens that can induce high levels of prefusion-specific antibodies.

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety.

Identifying the Cellular Target of Cordyheptapeptide A and Synthetic Derivatives.

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Drug-Like Properties in Macrocycles above MW 1000: Backbone Rigidity versus Side-Chain Lipophilicity.

Large macrocyclic peptides can achieve surprisingly high membrane permeability, although the properties that govern permeability in this chemical space are only beginning to come into focus. We generated two libraries of cyclic decapeptides with stable cross-ß conformations, and found that peptoid substitutions within the ß-turns of the macrocycle preserved the rigidity of the parent scaffold, whereas peptoid substitutions in the opposing ß-strands led to "chameleonic" species that were rigid in nonpolar media but highly flexible in water. Both rigid and chameleonic compounds showed high permeability over a wide lipophilicity range, with peak permeabilities differing significantly depending on scaffold rigidity. Our findings indicate that modulating lipophilicity can be used to engineer favorable ADME properties into both rigid and flexible macrocyclic peptides, and that scaffold rigidity can be used to tune optimal lipophilicity.

Dynamic conformational changes due to the ATP hydrolysis in the motor domain of myosin: 10-ns molecular dynamics simulations.

Muscle contraction is caused by directed movement of myosin heads along actin filaments. This movement is triggered by ATP hydrolysis, which occurs within the motor domain of myosin. The mechanism for this intramolecular process remains unknown owing to a lack of ways to observe the detailed motions of each atom in the myosin molecule. We carried out 10-ns all-atom molecular dynamics simulations to investigate the types of dynamic conformational changes produced in the motor domain by the energy released from ATP hydrolysis. The results revealed that the thermal fluctuations modulated by perturbation of ATP hydrolysis are biased in one direction that is relevant to directed movement of the myosin head along the actin filament.

Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature.

X-ray crystallography has revealed two similar alpha/beta domains of the aspartate racemase from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3. The active site is located in the cleft between the two domains where two cysteine residues face each other. This arrangement allows the substrate to enter the cleft and enables the two cysteine residues to act synergistically. However, the distance between their thiolates was estimated to be 9.6 angstroms, which is beyond the distance for cooperative action of them. We examined the molecular mechanism for the racemization reaction of this hyperthermophilic aspartate racemase by mutational analyses and molecular dynamics simulations. The mutational analyses revealed that Arg48 and Lys164 were essential for catalysis in addition to the putative catalytic cysteine residues. The molecular dynamics simulations revealed that the distance between the two active gamma-sulfur atoms of cysteine residues oscillate to periodically become shorter than the predicted cooperative distance at high temperature. In addition, the conformation of Tyr160, which is located at the entrance of the cleft and inhibits the entry of a substrate, changes periodically to open the entrance at 375 K. The opening of the gate is likely to be induced by the motion of the adjacent amino acid, Lys164. The entrance of an aspartate molecule was observed by molecular dynamics (MD) simulations driven by the force of the electrostatic interaction with Arg48, Lys164, and also Asp47. These results provide insights into the roles of amino acid residues at the catalytic site and also the activation mechanism of a hyperthermophilic aspartate racemase at high temperature.

Molecular dynamics simulations of evolved collective motions of atoms in the myosin motor domain upon perturbation of the ATPase pocket.

A crucial point for mechanical force generation in actomyosin systems is how the energy released by ATP hydrolysis in the myosin motor domain gives rise to the movement of the myosin head along the actin filament. We assumed the signal of the ATP hydrolysis to be transmitted as modulated atomic vibrations from the nucleotide-binding site throughout the myosin head, and carried out 1-ns all-atom molecular dynamics simulations for that signal transmission. We distributed the released energy to atoms located around the ATPase pocket as kinetic energies and examined how the effect of disturbance extended throughout the motor domain. The result showed that the disturbance signal extended over the motor domain in 150 ps and induced slowly varying collective motions of atoms at the actin-binding site and the junction with the neck, both of which are relevant to the movement of the myosin head along the actin filament. We also performed a principal component analysis of thermal atomic motions for the motor domain, and the first principal component was consistent with the response to the disturbance given to the ATPase pocket.