Marinoquinolones and Marinobactoic Acid: Antimicrobial and Cytotoxic ortho-Dialkylbenzene-Class Metabolites Produced by a Marine Obligate Gammaproteobacterium of the Genus Marinobacterium.

Chemical investigation of the culture extract of a marine obligate proteobacterium, Marinobacterium sp. C17-8, isolated from scleractinian coral Euphyllia sp., led to the discovery of three new o-dialkylbenzene-class metabolites, designated marinoquinolones A (1) and B (2) and marinobactoic acid (3). Spectroscopic analysis using MS and NMR revealed the structures of 1 and 2 to be 4-quinolones with an o-dialkylbenzene-containing side chain at C3 and 3 to be a fatty acid bearing an o-dialkylbenzene substructure. The 4-quinolone form of 1 and 2 was unequivocally determined by comparison of the 1H, 13C, and 15N chemical shifts of 1 with those predicted for 2-methyl-4-quinolone A and its tautomer 2-methyl-4-quinolinol B by quantum chemical calculation. Compound 1 was proven to be racemic by X-ray crystallographic analysis and chiral-phase HPLC analysis of its chemical degradation product. Compounds 1-3 exhibited antimicrobial activity against bacteria and filamentous fungi at MIC of 6.3-50 µg/mL. In addition, all compounds showed cytotoxicity against P388 murine leukemia cells at micromolar ranges.

Kumemicinones A-G, Cytotoxic Angucyclinones from a Deep Sea-Derived Actinomycete of the Genus Actinomadura.

Chemical investigation of the fermentation products of a deep sea water-derived actinomycete, Actinomadura sp. KD439, identified seven new angucyclinones, designated as kumemicinones A-G (1-7), together with the known SF2315B and miaosporone E. NMR and MS spectroscopic analyses, combined with X-ray crystallography and quantum chemical calculations of NMR chemical shifts and electronic circular dichroism (ECD) spectra, uncovered the structures of new angucyclinones as regioisomers of SF2315B at the allyl alcohol unit (1 and 2), an epoxy ring-opened γ-hydroxy enone isomer (3), a B/C-ring-rearranged product (4), or dimers with a new mode of bridging (5-7), adding new structural variation to this antibiotic group. The absolute configuration of SF2315B was also determined by comparison of ECD spectra with those of 1 and 2. All the angucyclinones exhibited cytotoxicity against P388 murine leukemia cells, with IC50 values ranging from 1.8 to 53 µM.

Structural study of the recognition mechanism of tau antibody Tau2r3 with the key sequence (VQIINK) in tau aggregation.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.

Carapanins A-C: new limonoids from andiroba (Carapa guianensis) fruit oil.

Carapanins A-C (1-3) were isolated from the fruit oil of Carapa guianensis. Compounds 1 and 2 are limonoids with unique structures. Namely, compound 1 is an andirobin-type limonoid with a C-15/C-30 γ-lactone instead of the δ-lactone of the D-ring, and compound 2 is a mexicanolide-type limonoid with a C-16/C-30 δ-lactone ring. The absolute structures of 1 and 2 were determined using X-ray crystallography, whereas the structure of 3 was established mainly via NMR and mass spectroscopy. The inhibitory effects of 1-3 on nitric oxide production were evaluated, and it was revealed that 2 and 3 were potent nitric oxide inhibitors.

Strophasterols E and F: Rearranged ergostane-type sterols from Pleurotus eryngii.

Strophasterols E (1) and F (2) were isolated from the fruiting bodies of Pleurotus eryngii, together with four new ergostane-type sterols (3-6). Single-crystal X-ray diffraction analysis performed on the tris-p-bromobenzoate derivatives of compounds 1 and 2 allowed these two compounds to be identified as the structurally rare (22S,23R)- and (22S,23S)-5α,6α-epoxy-3ß,7ß,23-trihydroxy-15(14 → 22)-abeo-ergost-8-en-14-one, respectively. The inhibitory effects on nitric oxide production of the six new steroids thus isolated from the fruiting bodies of P. eryngii were also evaluated.

Silyl Group-Directed 6-exo-dig Iodocyclization of Homopropargylic Carbamates and Amides.

Iodocyclization of silyl group-substituted homopropargylic carbamates and amides proceeded via 6-exo-dig mode to afford 6-vinylene-4,5-dihydro-1,3-oxazines in moderate to quantitative yields. This is the first report for silyl group-solely directed iodocyclization of alkynes utilizing the ß-silyl effect. Under these mild reaction conditions, various functionalities such as secondary alcohol, acetal, urea, and sulfide were tolerated.

Pleurocins A and B: Unusual 11(9 → 7)-abeo-Ergostanes and Eringiacetal B: A 13,14-seco-13,14-Epoxyergostane from Fruiting Bodies of Pleurotus eryngii and Their Inhibitory Effects on Nitric Oxide Production.

Two novel 11(9 → 7)-abeo-ergostane-type steroids, named pleurocins A (1) and B (2), a 13,14-seco-13,14-epoxy ergostane, named eringiacetal B (3), and an ergostane steroid (4) were isolated from the fruiting bodies of Pleurotus eryngii (Pleurotaceae). Their structures were determined by spectroscopic data and X-ray crystallography. A possible biogenesis pathway for 1-3 was also described. Compounds 1-3 exhibited inhibitory activities against NO production with almost no cytotoxicity at concentrations lower than 30 µM.

Identification of key amino acids responsible for the distinct aggregation properties of microtubule-associated protein 2 and tau.

The carboxyl-terminal sequence of tau composes the framework for its intracellular inclusions that appear in diverse neurodegenerative disorders known as tauopathies. However, microtubule-associated protein 2 (MAP2), which contains a homologous carboxyl-terminal sequence of tau, is undetectable in the mature tau inclusions. The mechanisms underlying this phenomenon have remained largely unknown. Here, we show that tau and MAP2 have different aggregation properties: tau aggregates to form filaments but MAP2 remains to be granules. Exchanging (221) YKPV(224) of tau (0N3R) near the PHF6 motif for (340) TKKI(343) of MAP2c profoundly changed aggregation properties, suggesting that the YKPV motif is important for filament formation, whereas the TKKI motif is for granule formation. Thus, these minimal sequences may determine the different fates of tau and MAP2 in the formation of inclusions in tauopathies. Tau and microtubule-associated protein 2 (MAP2) are homologous microtubule-associated proteins in neurons. So far, it is largely unknown why tau but not MAP2 is selectively involved in the filamentous inclusions (neurofibrillary tangles, NFT) formation in tauopathies, including Alzheimer's disease. In this study, we found that the difference of only two amino acids in tau and MAP2 sequences may determine their different fates in tauopathies. These results may lead to the elucidation of tau deregulation in pathological conditions.

CH-π interaction in VQIVYK sequence elucidated by NMR spectroscopy is essential for PHF formation of tau.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. Investigation of the mechanism of tau aggregation is important for the clarifying the cause of AD and the development of therapeutic drugs. The microtubule-binding domain, which consists of repeats of similar amino acids (R1-R4) is thought to form the core component of paired helical filament (PHF). The hexapeptide(306) VQIVYK(311) of R3 has been shown to take a key role of promoting tau aggregation and assumed that its CH-π interaction between the side chains of Ile308 and Tyr310 would contribute in stabilizing the filament. In this work, we investigated a short isoform of tau (4RTau), R3, VQIVYK peptide and their mutants by thioflavin S (ThS) fluorescence, and NMR measurements, and proved for the first time that this CH-π interaction stabilizes the filament at the atomic level. In addition, by molecular modeling, we revealed that this interaction further supports an extended amphipathic structure for molecular self-association during the process of PHF formation of tau protein. The present work indicates new approach that inhibits the CH-π interaction for developing a therapeutic agent for AD.

A conserved motif within the flexible C-terminus of the translational regulator 4E-BP is required for tight binding to the mRNA cap-binding protein eIF4E.

Although the central α-helical Y(X)4LΦ motif (X, variable amino acid; Φ, hydrophobic amino acid) of the translational regulator 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role for the C-terminal region in such an interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1-3) subtypes were investigated by SPR (surface plasmon resonance) analysis and ITC (isothermal titration calorimetry). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with eIF4E by approximately 1000-fold in the presence of this conserved region compared with that in the absence of this region. The sequential deletion of this conserved region in 4E-BP1 showed that deletion of Val81 leads to a considerable decrease in the binding ability of 4E-BP. Molecular dynamics simulation suggested that the conserved PGVTS/T region functions as a kind of paste, adhering the root of both the eIF4E N-terminal and 4E-BP C-terminal flexible regions through a hydrophobic interaction, where valine is located at the crossing position of both flexible regions. It is concluded that the conserved PGVTS/T motif within the flexible C-terminus of 4E-BP plays an auxiliary, but indispensable, role in strengthening the binding of eIF4E to the core Y(X)4LΦ motif.

Botryorhodines K and L, two new cytotoxic depsidones from a fungus of the genus Arcopilus.

Botryorhodines K (1) and L (2), two new depsidone derivatives, along with one known metabolite, 4-O-demethylbarbatic acid (3), were isolated from the culture extract of a fungus of the genus Arcopilus. The structures of 1‒3 were determined by the analysis of NMR and MS spectral data and the absolute configuration of 1 was established by single-crystal X-ray diffraction analysis. Compounds 1 and 2 showed antimicrobial activity against Gram-positive bacteria and cytotoxicity against murine leukemia P388 cells.

Identification and function of the second eIF4E-binding region in N-terminal domain of eIF4G: comparison with eIF4E-binding protein.

The eukaryotic initiation factor 4E (eIF4E) serves as a master switch that controls mRNA translation through the promotive binding to eIF4G and the regulative binding with the endogenous inhibitor 4E-BP. Although the bindings of eIF4G and 4E-BP to eIF4E proceed through the common eIF4E recognition Y(X)(4)Lφ motif (X: variable, φ: hydrophobic) (first binding site), the relationship between their eIF4E binding mode and the functional difference is hardly known. Recently, we have clarified the existence and function of the second eIF4E binding site in 4E-BP. Surface plasmon resonance (SPR) analysis based on the sequential comparison between 4E-BP and eIF4GI clarified that eIF4G has the second binding site at the periphery of the (597)SDVVL(601) sequence and that it plays an auxiliary but indispensable function in stabilizing the binding of the first binding sequence (572)YDREFLL(578). The kinetic parameters of the interactions of the eIF4GI and 4E-BP2 fragment peptides with eIF4E showed that the association (ka) and dissociation (kd) rates of the former peptide are about three and two orders of magnitude lower than those of the latter peptide, respectively. This means that eIF4G has a potent resistive property for release from eIF4E, although its rate of binding to eIF4E is not as high as that of 4E-BP, that is, 4E-BP is apt to bind to and be released from eIF4E, as compared with eIF4G. Isothermal titration calorimetry (ITC) showed the opposite behavior between the second binding sites of eIF4GI and 4E-BP for the interaction with eIF4E. This clearly indicates the importance of the second binding region for the difference in function between eIF4G and 4E-BP for eIF4E translation.

Structural scaffold for eIF4E binding selectivity of 4E-BP isoforms: crystal structure of eIF4E binding region of 4E-BP2 and its comparison with that of 4E-BP1.

To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E-binding protein 2 (4E-BP2) than of 4E-BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E-BP2 in complex with m(7) GTP-bound human eIF4E has been determined by X-ray diffraction analysis and compared with that of 4E-BP1. The crystal structure revealed that the Pro47-Ser65 moiety of 4E-BP2 adopts a L-shaped conformation involving extended and α-helical structures and extends over the N-terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E-BP1. Although the pattern of the overall interaction of 4E-BP2 with eIF4E was similar to that of 4E-BP1, a notable difference was observed for the 60-63 sequence in relation to the conformation and binding selectivity of the 4E-BP isoform, i.e. Met-Glu-Cys-Arg for 4E-BP1 and Leu-Asp-Arg-Arg for 4E-BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E-BP2 over 4E-BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59-Leu60 sequence of 4E-BP2.

Nyuzenamides A and B: Bicyclic Peptides with Antifungal and Cytotoxic Activity from a Marine-Derived Streptomyces sp.

Two bicyclic peptides, nyuzenamides A (1) and B (2), were discovered from Streptomyces isolated from suspended matter in deep sea water collected in the Sea of Japan. Their structures were determined through nuclear magnetic resonance and mass spectrometry analyses in combination with X-ray crystallography and the chiral-phase gas chromatography-mass spectrometry method to comprise ten amino acid residues containing four unusual amino acids along with aromatic acyl units. Both compounds displayed antifungal activity against pathogenic fungi and cytotoxicity against P388 murine leukemia cells.

Crystal structure of the human tau PHF core domain VQIINK complexed with the Fab domain of monoclonal antibody Tau2r3.

Neurofibrillary tangles formed by abnormally aggregated tau protein are a histopathological feature of tauopathies. A tau aggregation inhibitor is a potential therapeutic agent for tauopathies. In this study, we prepared a monoclonal antibody for tau, monoclonal antibody to tau protein (Tau2r3), using as epitope the 272 GGKVQIINKKLD283 peptide in the microtubule-binding domain of tau, the key region mediating tau aggregation. We show that Tau2r3 clearly inhibits tau aggregation. To analyze the inhibition mechanism of Tau2r3, we solved the crystal structure of the Fab domain of Tau2r3 (Fab2r3) in complex with the VQIINK peptide. In the Fab2r3-VQIINK structure, the second and sixth polar residues and the fourth hydrophobic residue of VQIINK are crucial for binding to Fab2r3. The structural data for the Fab2r3-VQIINK complex could contribute to the design of new tau aggregation inhibitors.

Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin.

Parkin is the gene product identified as the major cause of autosomal recessive juvenile Parkinsonism (AR-JP). Parkin, a ubiquitin ligase E3, contains a unique ubiquitin-like domain in its N-terminus designated Uld which is assumed to be a interaction domain with the Rpn 10 subunit of 26S proteasome. To elucidate the structural and functional role of Uld in parkin at the atomic level, the X-ray crystal structure of murine Uld was determined and a molecular dynamics simulation of wild Uld and its five mutants (K27N, R33Q, R42P, K48A and V56E) identified from AR-JP patients was performed. Murine Uld consists of two alpha helices [Ile23-Arg33 (alpha1) and Val56-Gln57 (alpha2)] and five beta strands [Met1-Phe7 (beta1), Tyr11-Asp18 (beta2), Leu41-Phe45 (beta3), Lys48-Pro51 (beta4) and Ser65-Arg72 (beta5)] and its overall structure is essentially the same as that of human ubiquitin with a 1.22 A rmsd for the backbone atoms of residues 1-76; however, the sequential identity and similarity between both molecules are 32% and 63%, respectively. This close resemblance is due to the core structure built by same hydrogen bond formations between and within the backbone chains of alpha1 and beta1/2/5 secondary structure elements and by nearly the same hydrophobic interactions formed between the nonpolar amino acids of their secondary structures. The side chain NetaH of Lys27 on the alpha1 helix was crucial to the stabilization of the spatial orientations of beta3 and beta4 strands, possible binding region with Rpn 10 subunit, through three hydrogen bonds. The MD simulations showed the K27N and R33Q mutations increase the structural fluctuation of these beta strands including the alpha1 helix. Reversely, the V56E mutant restricted the spatial flexibility at the periphery of the short alpha2 helix by the interactions between the polar atoms of Glu56 and Ser19 residues. However, a large fluctuation of beta4 strand with respect to beta5 strand was induced in the R42P mutant, because of the impossibility of forming paired hydrogen bonds of Pro for Arg42 in wild Uld. The X-ray structure showed that the side chains of Asp39, Gln40 and Arg42 at the N-terminal periphery of beta3 strand protrude from the molecular surface of Uld and participate in hydrogen bonds with the polar residues of neighboring Ulds. Thus, the MD simulation suggests that the mutation substitution of Pro for Arg42 not only causes the large fluctuation of beta3 strand in the Uld but also leads to the loss of the ability of Uld to trap the Rpn 10 subunit. In contrast, the MD simulation of K48A mutant showed little influence on the beta3-beta4 loop structure, but a large fluctuation of Lys48 side chain, suggesting the importance of flexibility of this side chain for the interaction with the Rpn 10 subunit. The present results would be important in elucidating the impaired proteasomal binding mechanism of parkin in AR-JP.

Three-/four-repeat-dependent aggregation profile of tau microtubule-binding domain clarified by dynamic light scattering analysis.

The analysis of the self-assembly mechanism of the tau microtubule-binding domain (MBD) could provide the information needed to develop an effective method for the inhibition of the tau filament formation because of its core region that forms the filament. The MBD domain in the living body consists of similar three or four 31- to 32-residue repeats, namely 3RMBD (R134) and 4RMBD (R1234), respectively. The filament formation of the MBD has been mainly investigated by fluorescence spectroscopy utilizing the beta-sheet structure-binding signal sensor thioflavin. This method observes the aggregation indirectly, and provides no information on the time-dependent change in aggregation size or volume. Thus, to determine the structure necessary for initiating MBD self-association, the dynamic light scattering (DLS) method was applied to the analysis of the aggregations of 3RMBD, 4RMBD and their component single repeats and shown to be a powerful tool for directly analyzing filament formation. DLS analysis clearly showed that the building unit for initiating the aggregation is the intermolecular R3-R3 disulfide-bonded dimer for 3RMBD and the intramolecular R2-R3 disulfide-bonded monomer for 4RMBD, and their aggregation processes under physiological condition differ from each other, which has not been clearly revealed by the conventional fluorescence method. The repeat-number-dependent aggregation model of MBD, together with the function of each repeat, reported in this paper should help to devise a method of preventing tau PHF formation.

Cancer chemopreventive activity of oleanane-type triterpenoids from the stem bark of Betula ermanii.

In search for cancer chemopreventive agents from natural sources, three oleanane- and four known lupane-type triterpenoids, and sitosterol from the stem bark of Betula ermanii were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among them, 3beta-acetoxy-12alpha-hydroxyoleanan-13beta,28-olide (1) and 3beta-acetoxy-11alpha,12alpha-epoxyoleanan-13beta,28-olide (2) were investigated for the inhibitory effect in a two-stage carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. 3beta-Acetoxy-11alpha,12alpha-epoxyoleanan-13beta,28-olide (2) was found to exhibit the potent antitumor promoting activity in the in vivo carcinogenesis test.

Dataset on synthesis and crystallographic structure of phenyl(TMP)iodonium(III) acetate.

The data in this article are related to research article ''Efficient N-arylation of azole compounds utilizing selective aryl-transfer TMP-iodonium (III) reagents (Koseki et al., 2019). For the title compound, phenyl(2,4,6-trimethoxyphenyl)iodonium(III) acetate (Ph(TMP)IOAc), the single-crystal X-ray diffraction measurement together with NMR analysis, like also the method of synthesis and crystallization are presented. The X-ray structure analysis has revealed that the two types of geometries regarding the acetate anion attached to phenyl (TMP)iodonium (III) cation are found in the crystal states.

Importance of C-terminal flexible region of 4E-binding protein in binding with eukaryotic initiation factor 4E.

Although the alpha-helical Y(X)4Lvarphi containing region of eIF4E-binding protein (4EBP) is the major binding region with eukaryotic initiation factor 4E (eIF4E), the roles of its N- and C-terminal regions in the binding are hardly known. To clarify the roles of these flexible regions in the interaction, the binding features of the sequentially N-, C-, or both-terminal-residue-deleted 4EBP2 mutants were investigated by surface plasmon resonance (SPR) analysis. It was shown that the C-terminal His74-Glu89 sequence has an auxiliary, but indispensable, function in stabilizing the binding to eIF4E. The possible interaction with eIF4E was estimated by molecular dynamics simulation. This is the first report on the importance of the C-terminal flexible region in the eIF4E-binding regulation of 4EBP.