Structural and Computational Analyses of the Unique Interactions of Opicapone in the Binding Pocket of Catechol O-Methyltransferase: A Crystallographic Study and Fragment Molecular Orbital Analyses.

A third-generation inhibitor of catechol O-methyltransferase (COMT), opicapone (1), has the 3-nitrocatechol scaffold as do the second-generation inhibitors such as entacapone (2) and tolcapone (3), but only 1 can sustainably inhibit COMT activity making it suitable for a once-daily regimen. These improvements should be attributed to the optimized sidechain moiety (oxidopyridyloxadiazolyl group) of 1 substituted at the 5-position of the 3-nitrocatechol ring. We analyzed the role of the sidechain moiety by solving the crystal structures of COMT/S-adenosylmethionine (SAM)/Mg/1 and COMT/S-adenosylhomocysteine (SAH)/Mg/1 complexes. Fragment molecular orbital (FMO) calculations elucidated that the dispersion interaction between the sidechains of Leu 198 and Met 201 on the ß6ß7-loop and the oxidopyridine ring of 1 were unique and important in both complexes. In contrast, the catechol binding site made a remarkable difference in the sidechain conformation of Lys 144. The ε-amino group of Lys 144 was outside of the catalytic pocket and was replaced by a water molecule in the COMT/SAH/Mg/1 complex. No nitrocatechol inhibitor has ever been reported to make a complex with COMT and SAH. Thus, the conformational change of Lys 144 found in the COMT/SAH/Mg/1 complex is the first crystallographic evidence that supports the role of Lys 144 as a catalytic base to take out a proton ion from the reaction site to the outside of the enzyme. The fact that 1 generated a complex with SAH and COMT also suggests that 1 could inhibit COMT twofold, as a typical substrate mimic competitive inhibitor and as a product-inhibition enhancer.

Crystal Structure of Catechol O-Methyltransferase Complexed with Nitecapone.

Catechol O-methyltransferase (COMT) is known as an important drug-target protein in the field of Parkinson's disease. All clinically approved COMT inhibitors bring a 5-substituted-3-nitrocatechol ring as a pharmacophore, and they bind to COMT with S-adenosylmethionine (SAM) and an Mg2+ ion to form a quaternary complex (COMT/SAM/Mg2+/inhibitor). However, structural information about such quaternary complexes is only available for a few inhibitors. Here, a new crystal structure of COMT complexed with nitecapone (5), SAM and Mg2+ is revealed. Comparison of the structures of these complexes indicates that conformation of the catechol binding pocket is almost constant regardless of structure of the inhibitors. The only restriction of the side chain of inhibitors (i.e., the substituent at the 5-position of 3-nitrocatechol) seems to be that it does not make steric repulsion with COMT. However, recent crystallographic and biochemical studies suggest that COMT is a flexible protein, and its conformational flexibility seems crucial for its catalytic process. Based on this information, implications of these quaternary inhibitor complexes were investigated. Met 40 in the α2α3-loop makes atomic contacts with SAM or S-adenosylhomocysteine and the 3-position of the catechol inhibitor. This interaction seems to play a critical role in the affinity of the inhibitor and to stabilize the COMT/SAM/Mg2+/nitrocatechol inhibitor complex by fixing the flexible α2α3-loop.

Evaluation of irradiation effects of near-infrared free-electron-laser of silver alloy for dental application.

In the application of lasers in dentistry, there is a delicate balance between the benefits gained from laser treatment and the heat-related damage arising from laser irradiation. Hence, it is necessary to understand the different processes associated with the irradiation of lasers on dental materials. To obtain insight for the development of a safe and general-purpose laser for dentistry, the present study examines the physical effects associated with the irradiation of a near-infrared free-electron laser (FEL) on the surface of a commonly used silver dental alloy. The irradiation experiments using a 2900-nm FEL confirmed the formation of a pit in the dental alloy. The pit was formed with one macro-pulse of FEL irradiation, therefore, suggesting the possibility of efficient material processing with an FEL. Additionally, there was only a slight increase in the silver alloy temperature (less than 0.9 °C) despite the long duration of FEL irradiation, thus inferring that fixed prostheses in the oral cavity can be processed by FEL without thermal damage to the surrounding tissue. These results indicate that dental hard tissues and dental materials in the oral cavity can be safely and efficiently processed by the irradiation of a laser, which has the high repetition rate of a femtosecond laser pulse with a wavelength around 2900 nm.

Whole transcriptome analysis using next-generation sequencing of sterile-cultured Eisenia andrei for immune system research.

Recently, earthworms have become a useful model for research into the immune system, and it is expected that results obtained using this model will shed light on the sophisticated vertebrate immune system and the evolution of the immune response, and additionally help identify new biomolecules with therapeutic applications. However, for earthworms to be used as a genetic model of the invertebrate immune system, basic molecular and genetic resources, such as an expressed sequence tag (EST) database, must be developed for this organism. Next-generation sequencing technologies have generated EST libraries by RNA-seq in many model species. In this study, we used Illumina RNA-sequence technology to perform a comprehensive transcriptome analysis using an RNA sample pooled from sterile-cultured Eisenia andrei. All clean reads were assembled de novo into 41,423 unigenes using the Trinity program. Using this transcriptome data, we performed BLAST analysis against the GenBank non-redundant (NR) database and obtained a total of 12,285 significant BLAST hits. Furthermore, gene ontology (GO) analysis assigned 78 unigenes to 24 immune class GO terms. In addition, we detected a unigene with high similarity to beta-1,3-glucuronyltransferase 1 (GlcAT-P), which mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1 (human natural killer-1, also known as CD57), a marker of NK cells. The identified transcripts will be used to facilitate future research into the immune system using E. andrei.