Development of actin dimerization inducers inspired by actin-depolymerizing macrolides.

Several natural cytotoxic C2-symmetric bis-lactones, such as swinholide A and rhizopodin, sequester actin dimer from the actin network and potently inhibit actin dynamics. To develop new protein-protein interaction (PPI) modulators, we synthesized structurally simplified actin-binding side-chain dimers of antitumor macrolide aplyronine A. By fixing the two side-chains closer than those of rhizopodin, the C4 linker analog depolymerized filamentous actin more potently than natural aplyronines. Cross-link experiments revealed that actin dimer was formed by treatment with the C4 linker analog. Molecular dynamics simulations showed that this analog significantly changed the interaction and spatial arrangement of the two actins compared to those in rhizopodin to provide a highly distorted and twisted orientation in the complex. Our study may promote the development of PPI-based anticancer and other drug leads related to cytoskeletal dynamics.

Globunoids A-D, undescribed bichalconoid and biflavanoids with α-glucosidase and α-amylase inhibitory activities from Knema globularia stems.

A bichalconoid, globunoid A (1) and three biflavanones, globunoids B-D (2-4), previously undescribed, were isolated from the stems of Knema globularia, along with fourteen known analogues 5-18. The chemical structures of 1-4 were elucidated by the comprehensive spectroscopic analysis including UV, IR, HRESIMS, and NMR; the absolute configurations were determined based on their NOESY data, DP4+ statistical analysis, and ECD calculation. Up to now, compounds 2 and 3 represent the first 3,3″-linked biflavanone structures. Among the isolated compounds, 2, 3, and 2,3-dihydrocalodenin B (6) potently inhibited α-glucosidase and α-amylase activities, with IC50 values in the range 1.1-7.5 µM. Furthermore, the most active compound 6 was found to be a non-competitive inhibitor against these two enzymes.

Identification and α-glucosidase inhibitory activity evaluation of two new coumarins derived from Mansonia gagei J. R. Drumm.

Five coumarins were isolated from the heartwood of Mansonia gagei, which included two newly discovered compounds, namely 11-hydroxypopulene E (1) and mansorin D (2), along with three previously identified compounds. The structures were determined through the utilisation of comprehensive spectroscopic data, ECD calculations, and a thorough comparison with existing literature data. The α-glucosidase inhibitory activities of all isolated compounds were assessed in yeast. Out of the compounds tested, compound 2 exhibited the most significant activity, displaying a percentage inhibition of 34.33% at a concentration of 200 µM.

Eriodictyol and thymonin act as GPR35 agonists.

Although herbs and spices have been used in traditional medicine for more than a century owing to their health benefits, the associated underlying mechanism is still not clear. Since the G protein-coupled receptor 35 (GPR35) has been linked to exert various antioxidant and anti-inflammatory effects, we screened 19 different herbs and spices for possible GPR35 agonist(s) to understand the GPR35-dependent functions of herbs and spices. Among the screened extracts, the ethyl acetate extract of thyme exhibited a remarkable GPR35 agonistic activity. Activity-guided separations allowed us to identify 2 polyphenolic phytochemicals, eriodictyol and thymonin, acting as GPR35 agonists. Both eriodictyol and thymonin showed a potent and specific agonist activity toward GPR35 with half maximal effective concentration values of 5.48 and 8.41 µm, respectively. These findings indicate that these phytochemicals may have beneficial health effects upon GPR35 activation.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

α-Glucosidase inhibition of sesquiterpenoids from the heartwood of Mansonia gagei.

Nine undescribed sesquiterpenoids, along with ten known compounds, were isolated from the ethyl acetate extract of Mansonia gagei heartwood. Their structures were determined by spectroscopic data analysis (FTIR, 1D, 2D NMR, and HRESIMS), and their absolute configurations were established by ECD calculation. The isolated compounds were evaluated for their inhibitory effect against α-glucosidase from yeast. The results showed that mansonone U, mansonialactam, heliclactone and mansonone S exhibited exceptionally potent activities when compared to the positive control, acarbose, with IC50 values of 12.38 ± 0.71, 0.20 ± 0.05, 13.12 ± 2.85, and 12.05 ± 1.91 µM, respectively. Among them, mansonialactam possessed the most potent inhibitory activity against yeast α-glucosidase, and it showed an uncompetitive inhibition mode.

The Asymmetric Total Synthesis and Configuration Confirmation of Aplysiaenal and Nhatrangin A, Truncated Derivatives of Aplysiatoxin and Oscillatoxin.

Asymmetric total syntheses of aplysiaenal (1) and nhatrangin A (2), truncated derivatives of the aplysiatoxin/oscillatoxin family of marine natural products, from configurationally defined intermediates are described. NMR spectra of our synthesized nhatrangin A did not match with either those obtained from authentic samples of the natural product or material obtained via two other total syntheses, but were similar to that obtained from a sample obtained in a third total synthesis. By independently synthesizing the fragments used in its total syntheses, we were able to confirm the configuration of nhatrangin A and clarified that the discrepancy in the spectroscopic data is due to salt formation of the carboxylic acid moiety.

A new neolignan and a new phenolic acid from the heartwood of Mansonia gagei J.R. Drumm.

One new neolignan (1) and one new phenolic compound (2), together with four known compounds (3-6) were isolated from the heartwood of Mansonia gagei. Their structures were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR and HRESIMS. The absolute configuration of 2 was established based on the DP4+ protocol and by comparison of experimental and calculated ECD spectra. All isolated compounds were evaluated by DPPH assay for antioxidant activity, while compounds 3-6 were assayed using the MTT-based colorimetric assay for cytotoxicity against lung cancer cell line A549. In terms of antioxidant activity, 1 and 3 exhibited stronger activity (IC50 14.91 ± 1.10 and 17.46 ± 0.16 µM, respectively) than the positive control, ascorbic acid (IC50 30.20 ± 0.47 µM). Among the compounds tested for cytotoxicity, compound 3 showed the highest activity, with an IC50 value of 26.04 ± 2.95 µM.

Ligand-Dissociation-Type N,N-Dimethylaminopyrene Probes for In-Situ Site-Specific Protein Labeling.

To develop practical methods for in-situ labeling of target proteins and to analyze their binding modes with bioactive ligands, 6-N,N-dimethylaminopyrene-N-acyl-N-alkylsulfonamide-4,8-diazacyclononyne (dmpy-NASA-DACN) tags were synthesized. Strain-promoted azide-alkyne cyclization with azide-conjugated ligands (biotin and sulfonamide) gave ligand-dissociation-type dmpy probes. With these probes, specific labeling of avidin and human carboxylase 1 (hCA1) proceeded even in the presence of cell lysate proteins in ca. 10% RIPA buffer. Affinity purification, in-gel tryptic digestion on polystyrene gel, and MALDI MS analysis established the dmpy-labeled positions of target proteins. Molecular modeling studies also supported why the dmpy-labeling reactions proceeded site-specifically near ligand-binding sites on the target proteins. Our findings might contribute to the development of chemical probes that specifically label various biomacromolecules in cells.

The C29-C34 parts of antitumor macrolide aplyronine A serve as versatile actin-affinity tags.

Anticancer drug development inspired by natural products based on protein-protein interactions (PPI) is a promising strategy. We developed structurally-simplified C29-C34 side-chain analogs of aplyronine A (ApA), an antitumor marine macrolide. Among them, the analog possessing the C23 acyloxy group, the C29 N,N-dimethyl-L-alanine ester and the C34 N-methyl enamide showed potent actin-depolymerizing activity. Binding kinetics, molecular docking, and affinity-purification experiments revealed that they are versatile actin-affinity tags to accelerate studies on the mode of action related to cytoskeletal dynamics and the development of PPI-based drug leads.

6-Paradol Acts as a Potential Anti-obesity Vanilloid from Grains of Paradise.

SCOPE: Grains of Paradise (GOP), the seeds of Aframomum melegueta, has anti-obesity effects. However, the mechanisms underlying the effects remain unclear. METHODS AND RESULTS: This study sets up to study the anti-obesity impact and homeostatic effects of 6-paradol, a major vanilloid found in GOP, and investigates the physiological outputs and the lipometabolism-related gene in fat and liver in high-fat-induced obese mice with a comparison with structurally similar vanilloids (6-gingerol and 6-shogaol). The vanilloids are synthesized in adequate quantities for performing animal experiments and orally administered to 6-week-old male mice over 2 weeks. This study found that 6-paradol decreased body weight gain and visceral and subcutaneous fats in 2 weeks, whereas 6-gingerol and 6-shogaol have no effect. Additionally, 6-paradol suppresses the hepatic cholesterol and triglyceride and significantly decreases the gene expression related to fatty acid synthesis, lipid transportation, and adipocyte differentiation in both liver and adipose tissue. Moreover, phosphorylation of AMP-activated protein kinase (AMPK) that greatly contributes to lipometabolism is promoted by 6-gingerol but not 6-paradol. CONCLUSION: These results suggest that 6-paradol regulates several obesity-related genes in an AMPK-independent manner. Therefore, it could be the principal active vanilloid in GOP giving it anti-obesity properties with a different mechanism.

Total synthesis and biological evaluation of oscillatoxins D, E, and F.

Oscillatoxins (OTXs) and aplysiatoxins are biosynthetically related polyketides produced by marine cyanobacteria. We previously developed a synthetic route to phenolic O-methyl analogs of OTX-D and 30-methyl-OTX-D during collective synthesis of these natural products. According to our synthetic strategy, we achieved total synthesis of OTX-D, 30-methyl-OTX-D, OTX-E, and OTX-F by deprotecting the O-methyl group in an earlier intermediate, and determined their biological activities. Although OTX-D and 30-methyl-OTX-D have been reported to show antileukemic activity against L1210 cell line, we found that their cytotoxicity in vitro against this cell line is relatively weak (IC50: 29-52 µm). In contrast, OTX-F demonstrated cell line-selective antiproliferative activity against DMS-114 lung cancer cells, which implies that OTXs target as yet unknown target molecules as part of this unique activity.

A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.

N,N-Dimethylaminopyrene as a fluorescent affinity mass tag for ligand-binding mode analysis.

Elucidation of the binding mode of protein-ligand interactions provides insights for the design of new pharmacological tools and drug leads. Specific labeling of target proteins with chemical probes, in which the ligands are conjugated with reacting and detecting groups, can establish the binding positions of ligands. Label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) is a promising detection method to selectively detect labeled molecules. However, previous LDI MS tags, such as nitrogen-substituted pyrenes, had problems with low sensitivity and stability. Here we show 6-N,N-dimethylaminopyrene (dmpy) as a versatile mass tag, which was detected at an amount of 0.1 fmol by LA-LDI MS and applicable for MS/MS analysis. By using ligand-dissociation-type dmpy probes and affinity purification with a polystyrene gel, we demonstrated that dmpy-labeled peptides were predominantly detected by MALDI MS. Our dmpy-probe-labeling method might be highly useful for determining the target biomacromolecules of various ligands and their binding sites.

Acyl-CoA dehydrogenase long chain (ACADL) is a target protein of stylissatin A, an anti-inflammatory cyclic heptapeptide.

Stylissatin A (SA) is a cyclic heptapeptide isolated from the marine sponge Stylissa massa. SA shows anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells, but the detailed mechanism of action remains unclear. Here we report that D-Tyr1-tBuSA, a more potent SA derivative, inhibited production of the proinflammatory cytokines Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in LPS-stimulated RAW264.7 cells (EC50 = 1.4 and 5.9 µM, respectively). This compound also inhibited the LPS-stimulated expression of inducible nitric oxide synthase (iNOS) at 20 µM. Using a biotin derivative of SA, acyl-CoA dehydrogenase long chain (ACADL) was identified as a target protein of SA and its derivatives. It is proposed that SA and its derivatives might suppress the ß-oxidation of fatty acids by ACADL, and the accumulation of fatty acids on macrophages would inhibit the nuclear factor-kappa B (NF-κB) signaling pathway and iNOS expression to show anti-inflammatory activity. Our research might provide a new mechanism of inflammation in macrophages, and contribute to the development of treatments for inflammatory diseases.

Post K-Pg diversification of the mammalian order Eulipotyphla as suggested by phylogenomic analyses of ultra-conserved elements.

The origin of the mammalian order Eulipotyphla has been debated intensively with arguments around whether they began diversifying before or after the Cretaceous-Palaeogene (K-Pg) boundary at 66 Ma. Here, we used an in-solution nucleotide capture method and next generation DNA sequencing to determine the sequence of hundreds of ultra-conserved elements (UCEs), and conducted phylogenomic and molecular dating analyses for the four extant eulipotyphlan lineages-Erinaceidae, Solenodontidae, Soricidae, and Talpidae. Concatenated maximum-likelihood analyses with single or partitioned models and a coalescent species-tree analysis showed that divergences among the four major eulipotyphlan lineages occurred within a short period of evolutionary time, but did not resolve the interrelationships among them. Alternative suboptimal phylogenetic hypotheses received consistently the same amount of support from different UCE loci, and were not significantly different from the maximum likelihood tree topology, suggesting the prevalence of stochastic lineage sorting. Molecular dating analyses that incorporated among-lineage evolutionary rate differences supported a scenario where the four eulipotyphlan families diversified between 57.8 and 63.2 Ma. Given short branch lengths with low support values, traces of rampant genome-wide stochastic lineage sorting, and post K-Pg diversification, we concluded that the crown eulipotyphlan lineages arose through a rapid diversification after the K-Pg boundary when novel niches were created by the mass extinction of species.

Synthesis and Biological Activities of Aplyronine A Analogues toward the Development of Antitumor Protein-Protein Interaction Inducers between Actin and Tubulin: Conjugation of the C1-C9 Macrolactone Part and the C24-C34 Side Chain.

Aplyronine A (ApA) is an antitumor marine macrolide that induces an protein-protein interaction (PPI) between actin and tubulin. The C1-C9 macrolactone part including the C7 N,N,O-trimethylserine (TMSer) ester is important for its highly potent activities. To develop new antitumor PPI inducers, four aplyronine analogues were synthesized, which bear the C1-C9 macrolactone part with 0-2 TMSer ester(s) and the C24-C34 actin-binding side chain. Despite exhibiting potent actin-depolymerizing activity comparable to that of ApA, these analogues did not show potent cytotoxicity or depolymerize microtubules. Molecular modeling studies suggested that the whole macrolactone moiety of aplyronines was important to fix the conformation of the C7 TMSer ester moiety, while the linear C1-C9 part was insufficient. Still, our study newly proposed that fixed conformations of the C7 or C9 TMSer esters in aplyronines that protrude from the actin surface are important for binding to tubulin and inhibit microtubule dynamics.

Anti-inflammatory marine cyclic peptide stylissatin A and its derivatives inhibit differentiation of murine preadipocytes.

Stylissatin A, an anti-inflammatory cyclic heptapeptide, and its derivatives potently inhibited the differentiation of preadipocytes and reduced triglyceride accumulation in mature adipocytes, with little cytotoxicity. Our studies might contribute to the development of leads for new anti-inflammatory and anti-obesity agents.

Specific protein-labeling and ligand-binding position analysis with amidopyrene probes as LDI MS tags.

To readily analyze the binding mode of protein-ligand interactions, we developed ligand-bound-type and ligand-dissociation-type probes having 6-amidopyrene (apy) as a detecting group. Matrix- and label-assisted laser desorption/ionization mass spectrometry (MALDI and LA-LDI MS) analyses and a covalent docking simulation using these probes precisely determined the binding position of the ligand biotin on the target protein avidin (RMSD = 0.786 and 0.332 Å). Our apy-probe-labeling method may be useful for determining the unknown ligand-binding sites of various target proteins.

Sydowianumols A, B, and C, Three New Compounds from Discomycete Poculum pseudosydowianum.

Three new compounds, sydowianumols A (1), B (2), and C (3), were isolated from culture broth and mycelial extracts of Poculum pseudosydowianum (TNS-F-57853), an endophytic fungus isolated from fresh leaves of Quercus crispula. The structures of new compounds 1-3 were elucidated from spectroscopic data. Sydowianumols A (1) and B (2) exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with 90% minimum inhibitory concentration (MIC90) values of 12.5 µg/mL.