Published papers

In multicomponent drugs, such as Kampo (traditional Japanese medicine) medicines, compound–compound interactions, including synergistic effects, inhibitory effects, and complex formations, should be considered. Orengedokuto is a Kampo formula, which is used for the treatment of various diseases, including inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. During the decoction process in the production of Orengedokuto, yellow precipitates are obtained from berberine–baicalin complexation; these precipitates enhance the in vivo coprecipitation and coabsorption of both compounds. Scutellaria root contains various baicalin analogs, such as wogonoside, oroxyloside, and scutellarin; however, the compound–compound interactions between berberine and flavonoid glycosides have not been investigated. Herein, we performed precipitation assays for berberine and the crude extract of Scutellaria root to detect and quantify compounds that interact with berberine by HPLC analysis. Furthermore, we performed precipitation assays and NMR experiments to identify chemical structures that affect the formation of precipitates; specifically, we analyzed all combinations of berberine and identified flavonoids to provide insights into associated compound–compound interactions. The NMR experiments indicated that C-8 modification affected the pi–pi interaction between berberine and flavonoids. This information enables the synthesis of more effective molecules and provides insights into their functions, such as drug delivery. The investigation of the interaction mechanisms of berberine and baicalin analogs in Kampo medicines can provide comprehensive insights regarding multicomponent drugs.

Objective: Dictyostelium differentiation-inducing factors 1 and 3 [DIF-1 (1) and DIF-3 (2), respectively], along with their derivatives, such as Ph-DIF-1 (3) and Bu-DIF-3 (4), demonstrate antibacterial activity in vitro against Gram-positive bacteria, including methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-sensitive Enterococcus faecalis (VSE), and vancomycin-resistant Enterococcus faecium [VRE (VanA)]. This study investigates the therapeutic potential of DIF compounds against these Gram-positive bacteria. Results: In vitro tests revealed that the antibacterial activity of 3 and 4 was lost in the presence of human serum albumin (HSA), suggesting that HSA might inhibit their effectiveness. Further evaluation of less hydrophobic derivatives, DIF-1-NH2 (5) and NH2-Bu-DIF-3 (6), showed no antibacterial activity, even in the absence of HSA. However, ester derivatives Ph-DIF-1(AHA) (7) and Bu-DIF-3(2Ac) (8) exhibited antibacterial activity against the target bacteria in vitro, although this activity was also lost in the presence of HSA. We hypothesize that these ester derivatives may function as prodrugs, with their antibacterial activity possibly restored by hydrolysis through bacterial esterases. The results suggest that suitable ester modifications could enhance the in vivo antibacterial potential of DIF compounds, particularly if they can bypass HSA binding and be activated by bacterial enzymes.

Our previous studies demonstrated that the ethyl acetate extract of the juice of jabara (JEx), a Japanese citrus fruit, inhibited cytochrome P450 (CYP) 3A4 activity in a time-dependent manner, but does not inhibit CYP2C19 activity. Its components, 3,3′,4′,5,6,7,8-heptamethoxyflavone (HpMF) and 3,3′,4′,5,6,7-hexamethoxyflavone (HxMF), have been shown to be responsible for this CYP3A4 inhibition. This study aimed to investigate the nature of CYP2C9 inhibition by JEx, HpMF, and HxMF. The time-dependent inhibition kinetics of JEx against human recombinant CYP2C9 were investigated in vitro using warfarin as a substrate. The time-independent inhibition kinetics of JEx, HpMF, and HxMF against CYP2C9 were also determined. JEx did not cause time-dependent inhibition of CYP2C9, while JEx, HpMF, and HxMF inhibited CYP2C9 activity in a time-independent manner with IC50 values of 2.65 % equivalent, 16.2 µM, and 57.8 µM, respectively. HpMF and HxMF were considered to be primarily responsible for CYP2C9 inhibition by JEx. The ingestion of 250 mL of jabara juice was estimated to reduce intestinal CYP2C9 activity to 3 % of the control value assuming of dilution with 210 mL of intestinal fluid, suggesting that jabara juice may cause food-drug interactions via intestinal CYP2C9 inhibition.

Drug development for regulating the innate immune system is important for the prevention and treatment of autoinflammatory and autoimmune diseases. In this context, we investigated the effect of resveratrol derivatives on the inflammatory reactions in the brain. Resveratrol, which can be found in Vitis plants in the form of oligomers, exhibits neuroprotective effects; however, its regulatory effects on innate immunity are still unclear. We examined the effects of (+)-hopeaphenol, a resveratrol tetramer, and its derivatives on the polyinosinic-polycytidylic acid (poly IC)-induced production of interferon (IFN)-β and C-X-C motif chemokine 10 (CXCL10) in the cultured human cerebral microvascular endothelial cell line hCMEC/D3. (+)-Hopeaphenol (1-10 μM) inhibited the poly IC-induced production of not only CXCL10 but also retinoic acid-inducible gene-I in a dose-dependent manner and significantly reduced the poly IC-induced IFN-β gene expression and protein release from hCMEC/D3 cells by inhibiting the phosphorylation of p65 but not that of the interferon regulatory transcription factor IRF3. A docking study indicated a high affinity of (+)-hopeaphenol for p65. These results suggest that (+)-hopeaphenol can regulate the innate immune system by inhibiting the poly IC/IFN-β/CXCL10 signaling axis via suppression of the phosphorylation of the transcription factor NF-ĸB.

Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation–inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum.

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography–tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10–40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2–10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10–40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2–20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

For the treatment and prevention of autoinflammatory diseases, it is essential to develop the drug, regulating the innate immune system. Although differentiation-inducing factor (DIF) derivatives, extracted from the cellular slime mold, Dictyostelium discoideum, exhibit immunomodulatory effects, their effects on the regulation of innate immunity in brain are unknown. In this study, we used the human cerebral microvascular endothelial cell line, hCMEC/D3, to investigate the effects of DIF derivatives on the generation of C-X-C motif chemokine (CXCL) 10 and interferon (IFN)-β induced by polyinosinic-polycytidylic acid (poly IC). DIF-3 (1-10 μM), but not DIF-1 and DIF-2, dose-dependently inhibited the biosynthesis of not only CXCL10 but also CXCL16 and C-C motif chemokine 2 induced by poly IC. DIF-3 also strongly decreased IFN-β mRNA expression and protein release from the cells induced by poly IC through the prohibition of p65, a subtype of NF-ĸB, not interferon regulatory transcription factor 3 phosphorylation. In the docking simulation study, we confirmed that DIF-3 had a high affinity to p65. These results suggest that DIF-3 regulates the innate immune system by inhibiting TLR3/IFN-β signaling axis through the NF-ĸB phosphorylation inhibition.

Baicalin and berberine are biologically active constituents of the crude drugs Scutellaria root and Coptis rhizome/Phellodendron bark, respectively. Baicalin and berberine are reported to combine together as a 1:1 complex that forms yellow precipitates by electrostatic interaction in decoctions of Kampo formulae containing these crude drugs. However, the structural basis and mechanism for the precipitate formation of this compound–compound interaction in aqueous solution remains unclarified. Herein, we searched for berberine derivatives in the Coptis rhizome that interact with baicalin and identified the chemical structures involved in the precipitation formation. Precipitation assays showed that baicalin formed precipitates with berberine and coptisine but not with palmatine and epiberberine. Thus, the 2,3-methylenedioxy structure may be crucial to the formation of the precipitates, and electrostatic interaction is necessary but is not sufficient. In this multicomponent system experiment, palmatine formed a dissociable complex with baicalin and may competitively inhibit the formation of berberine and coptisine precipitation with baicalin. Therefore, the precipitation formed by berberine and baicalin was considered to be caused by the aggregation of the berberine–baicalin complex, and the 2,3-methylenedioxy structure is likely crucial to the aggregation of the complex. Graphic abstract: (Figure presented.)

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

The construction of a chemical library based on natural products is a promising method for the synthesis of natural product-like compounds. In this study, we synthesized a terpenoid alkaloid-like compound library based on the humulene skeleton. Our strategy, which enables access to diverse ring systems such as 11-membered monocyclic, oxabicyclic, and medium-sized aza ring-containing scaffolds, involves the introduction of a nitrogen atom, an intermolecular C-O bond formation via Lewis acid-mediated epoxide-opening transannulation, and a ring-reconstruction strategy based on olefin metathesis. A cheminformatics analysis based on their structural and physicochemical properties revealed that the synthesized compounds have high three-dimensionality and high natural product likeness scores but with structural novelty. The usefulness of the terpenoid alkaloid-like compound library for drug discovery and the accessibility to structure-activity relationship studies were validated by performing an assay for osteoclast-specific tartrate-resistant acid phosphatase activity, resulting in the identification of a lead compound for bone-resorptive diseases such as osteoporosis.

The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50–100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20–30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment.

Some citrus fruits are known to cause clinically significant drug interactions by inhibiting intestinal cytochrome P450 (CYP) enzymes. This in vitro study aimed to investigate the kinetics of the inhibition of CYP3A4 and CYP2C19 by the juice of jabara, a Japanese citrus fruit that does not contain furanocoumarins such as 6′,7′-dihydroxybergamottin, and to identify the inhibitory compound(s). CYP3A4 and CYP2C19 activity levels were determined in vitro using recombinant CYP preparations and their respective substrates. The ethyl acetate extract (EAE) of jabara juice was separated to isolate and identify the compound(s) that inhibited CYP3A4. Then, the time-dependent kinetics of the inhibition of CYP3A4 and CYP2C19 by the EAE and its inhibitory compound(s) were analyzed. The EAE of jabara juice was found to inhibit CYP3A4 in a time-dependent manner. Two flavonoids, 3,3′,4′,5,6,7,8-heptamethoxyflavone (HpMF) and 3,3′,4′,5,6,7-hexamethoxyflavone (HxMF), were identified as the responsible compounds. HpMF and HxMF inhibited CYP3A4 activity in a concentration- and time-dependent manner, with inhibition constants (KI) of 10.0 and 7.90 µM and maximal inactivation rate constants (kinact,max) of 0.00856 and 0.0134 min−1, respectively. The EAE did not inhibit CYP2C19, even when preincubation was employed. These findings imply that jabara juice may cause food-drug interactions via time-dependent inhibition of intestinal CYP3A4.

Natural products and their derivatives have been very useful in the search for biologically active compounds and for the development of new drugs because of their structural diversity However, to retain their usefulness in the future, new approaches to increase the chemical diversity of such natural products must be developed. Diversity-oriented synthesis has recently emerged as an efficient methodology to construct complex and diverse compounds from simple and similar precursors. Thus, through the combination of natural products chemistry and diversity-oriented synthesis, we propose a new approach, diversity-enhanced extracts, for increasing the diversity of natural product-like compounds. Diversity-enhanced extracts are prepared from chemical reactions that remodel molecular scaffolds directly on extracts of natural resources. The subsequent isolation of each compound produced from such reactions affords a diverse natural product like library. We applied this method of diversification on several medicinal plants. From diversity enhanced extracts of the medicinal plants, we obtained new and diverse sesquiterpenoids, indole alkaloids, meroterpenoids, and biaryls with some containing new molecular skeletons.

An unnatural terpenoid scaffold containing a bicyclo[5.4.0]undecane moiety, as well as a salvialane skeleton based on an intramolecular C-C bond formation strategy were synthesized. Such a strategy was made possible by the removal of strained E-olefin conformations of the humulene skeleton. Some compounds were identified to show PPAR alpha antagonist activity.

Metarhizin C, a stereoisomer of BR-050 was isolated from a fungus Tolypocladium album RK17-F0007 through a screening program to search for new antitumor compounds. A structure of the isomer was determined by spectroscopic methods including detailed analysis of NOESY correlation and mass spectrometry, and found to be identical to that of 3-desacylmetarhizin A with the absolute structure. Previously, it had been isolated by Kikuchi et al and proposed as BR-050 including the stereo-structure. However, detailed analysis for the newly isolated isomer confirmed that 3-desacylmetarhizin A was not identical to BR-050. Therefore, we assigned it metarhizin C as a new BR-050 isomer. Metarhizin C showed selective cytotoxicity against osteosarcoma MG-63 cells in a glucose independent condition with IC50 value of 0.79 µg/ml, while > 30 µg/ml of IC50 value in a normal condition, and inhibited a mitochondrial respiration.

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.