Anti-barnacle activity of novel simple alkyl isocyanides derived from citronellol

Twenty novel simple alkyl isocyanides derived from citronellol were synthesized and evaluated for their antifouling activity and toxicity against cypris larvae of the barnacle, Balanus amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC₅₀ range of 0.08–1.49 μg ml^?1. Simple isocyanides containing a benzoate and chloro group showed the most potent anti-barnacle activity. In addition, none of the synthesized compounds showed significant toxicity and LC₅₀ values were <10 μg ml^?1. The LC₅₀/EC₅₀ ratios of almost all of the synthesized compounds were >10². The results indicate that these simple isocyanides are promising low-toxicity antifouling agents.     

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.     

Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited k_cat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.     

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.     

Design and synthesis of novel pyrimido[4,5-b]azepine derivatives as HER2/EGFR dual inhibitors

A novel 7,6 fused bicyclic scaffold, pyrimido[4,5-b]azepine was designed to fit into the ATP binding site of the HER2/EGFR proteins. The synthesis of this scaffold was accomplished by an intramolecular Claisen-type condensation. As the results of optimization lead us to 4-anilino and 6-functional groups, we discovered 6-substituted amide derivative 19b, which has a 1-benzothiophen-4-yloxy group attached to the 4-anilino group. An X-ray co-crystal structure of 19b with EGFR demonstrated that the N-1 and N-3 nitrogens of the pyrimido[4,5-b]azepine scaffold make hydrogen-bonding interactions with the main chain NH of Met793 and the side chain of Thr854 via a water-mediated hydrogen bond network, respectively. In addition, the NH proton at the 9-position makes an additional hydrogen bond with the carbonyl group of Met793, as we expected. Compound 19b revealed potent HER2/EGFR kinase (IC₅₀: 24/36 nM) and BT474 cell growth (GI₅₀: 18 nM) inhibitory activities based on its pseudo-irreversible (PI) profile.     

Phytuberol,from Japanese Domestic Tobacco,Nicotiana tabacum cv. Suifu

Carcinogenesis is believed to be induced through the oxidative damage of DNA, and antioxidants are expected to suppress it. So, the polyphenolic antioxidants in daily foods were investigated to see whether they protect against genetic damage by active oxygen. In the evaluation, we used a bioassay and a chemical determination, a Salmonella mutagenicity test for mutation by a N-hydroxyl radical from one of the dietary carcinogens 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the formation of 8-hydroxyl (8-OHdG) from 2′-deoxyguanosine (2′-dG) in a Fenton OH-radical generating system. Thirty-one antioxidants including flavonoids were compared in terms of radical-trapping activity with bacterial DNA and 2′-dG. Antioxidants inhibited the mutation but the IC₅₀ values were in the mM order. Against 8-OHdG formation, only α-tocopherol had a suppressive effect with an IC₅₀ of 1.5 μM. Thus, except α-tocopherol, the dietary antioxidants did not scavenge the biological radicals faster than bacterial DNA and intact 2′-dG, indicating that they failed to prevent oxidative gene damage and probably carcinogenesis.     

Purification of 7-Keto-8-aminopelargonic Acid-7,8-Diaminopelargonic Acid Aminotransferase,an Enzym Involved in Biotin Biosynthesis,from Brevibacterium divaricatum

Vitamin B₆ is synthesized by green Cytisus scoparius callus and green Phellodendron amurense callus cultured on Linsmaier and Skoog Agar-medium with 10^?5m of ±-naphthaleneacetic acid (NAA) and 10^?6 m of 6-benzyladenine (BA). Even when thiamine and inositol were omitted from this medium, the growth and vitamin B₆ content of Cytisus scoparius callus did not change. Vitamin B₆ contents of clones of the calluses varied and were unstable during long-term subculture. Clonal selection was repeated to obtain stable strains with high vitamin B₆ content, and the vitamin B₆ content of one strain of green Cytisus scoparius callus became 4-times higher than that of the green leaves.     

Isolation of New Tobacco Constituents, 8,9-Dihydro-8,9-dihydroxymegastigmatrienone,from Japanese Domestic Suifu Tobacco

The tetradecapeptide of a renin substrate, DRVYIHPFHLLVYS, was used as a substrate for assaying several fungal aspartic and acidic proteinases in the acidic pH range. Aspartic and acidic proteinases froll) Phycomycetes, Mucor and Rhizopus, and Deuteromycotina, Aspergillus and Penicillium, cleaved the tetradecapeptide at its tyrosyl⁴-isoleucyl⁵ (Y⁴-I⁵),histidyI⁶-proly⁷ (H⁶_P⁷) and leucyl¹¹-valyl¹² (L¹¹-V¹²) bonds in the acidic pH range, while acidic proteinases type B and type A-I from Scytalidium lignicolumn, and those from Cladosporium and Basidiomycetes, Pycnoporus sanguineus, and the yeast, Rhodotorula glutinis; showed slightly different specificities towards the tetradecapeptide. Pepsin primarily cleaved the valy³-tyrosyl⁴ (V³-Y⁴) and leucyl¹⁰-leucyl¹¹ (L¹⁰-L¹¹) bonds. All of the aspartic and acidic proteinases of fungal origin tested in the present study have different specificities from that of pepsin.     

Hydrolysis of Proteins by Successive Incubation with Proteinase and Aminopeptidases Mixture

1. A trial test was attempted of complete hydrolysis of peptides and proteins into amino acids by enzymes. “Neutral proteinase” of Bacillus subtilis or “Alkalophilic proteinase” of a Streptomyces sp. was used for preliminary digestion of substrate, and a mixture of three aminopeptidases of Bacillus subtilis was employed for subsequent hydrolysis of proteinase digest.

2. The oxidized insulin B chain was hydrolyzed completely by the method. Several proteins including enzymes which contained no or less cystine and cysteine were also hydrolyzed almost completely.

3. On the other hand, certain glycoproteins were hydrolyzed to leave a few glycopeptides in which all glycomoieties of the proteins were retained. The implications of the results are discussed.