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Due to their physiological relevance, cell-based assays using human-induced pluripotent stem cell (iPSC)-derived cells are a promising in vitro pharmacological evaluation system for drug candidates. However, cell-based assays involve complex processes such as long-term culture, real-time and continuous observation of living cells, and detection of many cellular events. Automating multi-sample processing through these assays will enhance reproducibility by limiting human error and reduce researchers' valuable time spent conducting these experiments. Furthermore, this integration enables continuous tracking of morphological changes, which is not possible with the use of stand-alone devices. This report describes a new laboratory automation system called the Screening Station, which uses novel automation control and scheduling software called Green Button Go to integrate various devices. To integrate the above-mentioned processes, we established three workflows in Green Button Go: 1) For long-term cell culture, culture plates and medium containers are transported from the automatic CO 2 incubator and cool incubator, respectively, and the cell culture medium in the microplates is exchanged daily using the Biomek i7 workstation; 2) For time-lapse live-cell imaging, culture plates are automatically transferred between the CQ1 confocal quantitative image cytometer and the SCALE48W automatic CO 2 incubator; 3) For immunofluorescence imaging assays, in addition to the above-mentioned devices, the 405LS microplate washer allows for formalin-fixation and immunostaining of cells. By scheduling various combinations of the three workflows, we successfully automated the culture and medium exchange processes for iPSCs derived from patients with facioscapulohumeral muscular dystrophy, confirmation of their differentiation status by live-cell imaging, and confirmation of the presence of differentiation markers by immunostaining. In addition, deep learning analysis enabled us to quantify the degree of iPSC differentiation from live-cell imaging data. Further, the results of the fully automated experiments could be accessed via the intranet, enabling experiments and analysis to be conducted remotely once the necessary reagents and labware were prepared. We expect that the ability to perform clinically and physiologically relevant cell-based assays from remote locations using the Screening Station will facilitate global research collaboration and accelerate the discovery of new drug candidates., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds., Competing Interests: The authors declare the following competing financial interest(s): Y.H., Y.A., Y.T., K.O., I.N., T.N., and M.O. were employees of Astellas Pharma Inc. at the time this study was being conducted. The company had no role in the design of the study, namely in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. R.Y., N.Y., T.I., D.K.I., K.K., Y.A., and M.S. declare that they have no conflict of interest., (© 2023 The Authors. Published by American Chemical Society.)

Metabolic programs are rewired in cancer cells to support survival and tumor growth. Among these, recent studies have demonstrated that glutamate-oxaloacetate transaminase 1 (GOT1) plays key roles in maintaining redox homeostasis and proliferation of pancreatic ductal adenocarcinomas (PDA). This suggests that small molecule inhibitors of GOT1 could have utility for the treatment of PDA. However, the development of GOT1 inhibitors has been challenging, and no compound has yet demonstrated selectivity for GOT1-dependent cell metabolism or selective growth inhibition of PDA cell lines. In contrast, potent inhibitors that covalently bind to the transaminase cofactor pyridoxal-5'-phosphate (PLP), within the active site of the enzyme, have been reported for kynurenine aminotransferase (KAT) and gamma-aminobutyric acid aminotransferase (GABA-AT). Given the drug discovery successes with these transaminases, we aimed to identify PLP-dependent suicide substrate-type GOT1 inhibitors. Here, we demonstrate that PF-04859989, a known KAT2 inhibitor, has PLP-dependent inhibitory activity against GOT1 and shows selective growth inhibition of PDA cell lines., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

Dihydroorotate dehydrogenase (DHODH) is a flavin-binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three-dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for neglected tropical diseases, especially because these enzymes possess significantly different structural and functional properties from the membrane-associated human enzyme. The existing crystal structures of the flavin mononucleotide (FMN)-bound DHODHs reveal a number of interactions stabilizing FMN. However, to understand the binding mechanism correctly, it is necessary to compare the structures of the FMN-bound and FMN-free forms, because the protein moiety of the former is not necessarily the same as the latter. Here, we prepared the FMN-free DHODH of Trypanosoma brucei using an Escherichia coli overexpression system. Although this apoform lacks enzymatic activity, simple incubation with FMN activated the enzyme. It was stable enough to be crystallized, enabling us to determine its structure by X-ray crystallography at 1.6 Å resolution. We also determined the FMN-bound form at 1.8 Å resolution. Although the two structures have essentially the same scaffold, we observed flipping of a peptide-bond plane in the vicinity of the FMN-binding site, accompanied by an alternative hydrogen-bonding pattern. Comparisons of B factors of the protein main chain revealed that binding of FMN decreased flexibility of most of the residues at the FMN-binding site, but increased flexibility of a lid-like loop structure over the active center. This increase was ascribed to a conformational change in an FMN-contacting residue, Asn195, which induced a rearrangement of a hydrogen-bond network of the residues comprising the lid.

In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.

Chagas disease results from infection by Trypanosoma cruzi and is a neglected tropical disease (NTD). Although some treatment drugs are available, their use is associated with severe problems, including adverse effects and limited effectiveness during the chronic disease phase. To develop a novel anti-Chagas drug, we virtually screened 4.8 million small molecules against spermidine synthase (SpdSyn) as the target protein using our super computer "TSUBAME2.5" and conducted in vitro enzyme assays to determine the half-maximal inhibitory concentration values. We identified four hit compounds that inhibit T. cruzi SpdSyn (TcSpdSyn) by in silico and in vitro screening. We also determined the TcSpdSyn-hit compound complex structure using X-ray crystallography, which shows that the hit compound binds to the putrescine-binding site and interacts with Asp171 through a salt bridge.

Although NMR in fragment-based drug discovery is utilized almost exclusively to evaluate physical binding between molecules, it should be also a powerful tool for biochemical assay, evaluating inhibitory effect of compounds on enzymatic activity. Time-dependent spectral change in real-time monitoring or inhibitor concentration-dependent spectral change after constant-time reaction was processed by factor analysis, by which reaction rate or IC50 value was obtained. Applications to spermidine synthase of Trypanosoma cruzi, which causes Chagas disease, are described.

Neglected tropical diseases (NTDs) are an extremely important issue facing global health care. To improve "access to health" where people are unable to access adequate medical care due to poverty and weak healthcare systems, we have established two consortiums: the NTD drug discovery research consortium, and the pediatric praziquantel consortium. The NTD drug discovery research consortium, which involves six institutions from industry, government, and academia, as well as an international non-profit organization, is committed to developing anti-protozoan active compounds for three NTDs (Leishmaniasis, Chagas disease, and African sleeping sickness). Each participating institute will contribute their efforts to accomplish the following: selection of drug targets based on information technology, and drug discovery by three different approaches (in silico drug discovery, "fragment evolution" which is a unique drug designing method of Astellas Pharma, and phenotypic screening with Astellas' compound library). The consortium has established a brand new database (Integrated Neglected Tropical Disease Database; iNTRODB), and has selected target proteins for the in silico and fragment evolution drug discovery approaches. Thus far, we have identified a number of promising compounds that inhibit the target protein, and we are currently trying to improve the anti-protozoan activity of these compounds. The pediatric praziquantel consortium was founded in July 2012 to develop and register a new praziquantel pediatric formulation for the treatment of schistosomiasis. Astellas Pharma has been a core member in this consortium since its establishment, and has provided expertise and technology in the area of pediatric formulation development and clinical development.

Trypanosoma cruzi causes Chagas disease, a severe disease affecting 8-10 million people in Latin America. While nifurtimox and benznidazole are used to treat this disease, their efficacy is limited and adverse effects are observed. New therapeutic targets and novel drugs are therefore urgently required. Enzymes in the polyamine-trypanothione pathway are promising targets for the treatment of Chagas disease. Spermidine synthase is a key enzyme in this pathway that catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. Fragment-based drug discovery was therefore conducted to identify novel, potent inhibitors of spermidine synthase from T. cruzi (TcSpdSyn). Here, crystal structures of TcSpdSyn in complex with dcSAM, trans-4-methylcyclohexylamine and hit compounds from fragment screening are reported. The structure of dcSAM complexed with TcSpdSyn indicates that dcSAM stabilizes the conformation of the `gatekeeping' loop to form the putrescine-binding pocket. The structures of fragments bound to TcSpdSyn revealed two fragment-binding sites: the putrescine-binding pocket and the dimer interface. The putrescine-binding pocket was extended by an induced-fit mechanism. The crystal structures indicate that the conformation of the dimer interface is required to stabilize the gatekeeping loop and that fragments binding to this interface inhibit TcSpdSyn by disrupting its conformation. These results suggest that utilizing the dynamic structural changes in TcSpdSyn that occur upon inhibitor binding will facilitate the development of more selective and potent inhibitors.

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

As previously reported, triacsin C, a selective inhibitor of acyl-CoA synthetase, inhibited the synthesis of cholesteryl ester and triacylglycerol in mouse peritoneal macrophages, leading to a reduction of lipid droplets. Therefore, the in vivo efficacy was studied. Low-density lipoprotein receptor-knockout (LDLR-/-) mice were fed a high cholesterol diet (0.15%) for two months to measure the atherogenic areas of the hearts and aortas. When triacsin C was orally administered (10 mg/kg/day), the atherosclerotic areas were significantly reduced by 86% in aorta and 36% in hearts. The results strongly suggested that triacsin C shows anti-atherogenic activity by inhibiting acyl-CoA synthetase activity.

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

Fungal beauveriolide III (BeauIII, 1b), a cyclodepsipeptide inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) and showing antiatherogenic activity in mouse models, consists of L-Phe, L-Ala, D-allo-Ile, and 3-hydroxy-4-methyloctanoic acid (HMA) moieties, but the stereochemistry of the HMA part has not until now been fully defined. To determine it, four HMA stereoisomers were synthesized and labeled with (S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethane sulfonate (AP-OTf), a chiral fluorescent reagent. The derivatives were separated by HPLC and compared with the natural HMA derivative, which was thereby identified as (3S,4S)HMA in BeauIII. Furthermore, the four beauveriolide III isomers ((3S,4S)BeauIII (23a), (3R,4R)BeauIII (23b), (3R,4S)BeauIII (23c), and (3S,4R)BeauIII (23d)) were synthesized, and it was shown that all the spectral data for 23a were identical with those for natural 1b. Isomers 23a and 23d showed potent inhibitory activity of lipid droplet accumulation in macrophages, while the other two isomers caused weak inhibition. Thus, the 3S configuration of BeauIII is important for this activity. Furthermore, 23a and 23d showed rather specific inhibition against the ACAT1 isozyme.

We developed a polymeric 2-DE chip system. The chip consisted of an IEF region, an SDS-PAGE region, a valveless connection port, and a sample introduction port. A "junction structure" as a valveless connection port, which allowed separating and connecting the first- and second-dimensional gels, was fabricated between their regions. A "solution inlet" as a sample introduction port was fabricated to perform the liquid and sample introductions without solution leakage. Simultaneous sample monitoring was performed using the on-chip detection system. The performances of the system were demonstrated using commercially available proteins as a standard specimen and tissue-extracted proteins as the real samples. All procedures were employed without any movement of relocation part. This new 2-D separation system realized improved labor-intensive operations and a reduced experimental time.

High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.

During screening for inhibitors of lipid droplet accumulation in mouse peritoneal macrophages, two coumarins identified as decursin and decursinol angelate were isolated from the roots of Angelicae gigantis. The cellular molecular target of these inhibitors in macrophages was studied. Decursin and decursinol angelate inhibited cholesteryl ester (CE) synthesis with IC50 values of 9.7 and 10.1 microM, respectively, whereas they enhanced triacylglycerol (TG) synthesis. Lysosomal metabolism of cholesterol to CE was inhibited by the compounds, indicating that the site of inhibition is one of the steps between the exiting of cholesterol from the lysosomes and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the microsomal fractions prepared from mouse macrophages was studied, and the results showed inhibition of this activity by decursin and decursinol angelate with IC50 values of 43 and 22 microM, respectively. Thus, it was concluded that the compounds inhibit macrophage ACAT activity to decrease CE synthesis, leading to a reduction of lipid droplets in macrophages.

Sespendole was isolated as an inhibitor of lipid droplet formation in macrophages from the culture broth of a fungal strain Pseudobotrytis terrestris FKA-25. The compound inhibited the synthesis of cholesteryl ester and triacylglycerol by mouse macrophages with IC50 values of 4.0 and 3.2 microM, respectively.

Synthesis of beauveriolide III (1b), which is an inhibitor of lipid droplet accumulation in macrophages, was achieved by solid-phase assembly of linear depsipeptide using a 2-chlorotrityl linker followed by solution-phase cyclization. On the basis of this strategy, a combinatorial library of beauveriolide analogues was carried out by radio frequency-encoded combinatorial chemistry. After automated purification using preparative reversed-phase HPLC, the library was tested for inhibitory activity of CE synthesis in macrophages to determine structure-activity relationships of beauveriolides. Among them, we found that diphenyl derivative 7{9,1} is 10 times more potent than 1b.

Four triterpenoids 1-4 isolated from the fruit body of Torametes orientaris were found to inhibit lipid droplet accumulation in macrophages. From the biochemical analysis, compounds 2 and 3 inhibited selectively cholesteryl ester synthesis in macrophages, while compounds 1 and 4 showed inhibition of both cholesteryl ester and triacylglycerol syntheses.

Cancer chemotherapy for hypoxic tumor cells is thought to be an important issue, since hypoxia is related to tumor growth, apoptosis, angiogenesis and metastasis. Here, the bioactivities of xanthohumol (XN), a diacylglycerol acyltransferase inhibitor, against hypoxic cells were investigated. At first, the inhibitory effects of XN on the formation of lipid droplets in the cytoplasm were evaluated in hypoxia. Hypoxia upregulated the synthesis of triglyceride and promoted the formation of lipid droplets in the cytoplasm, however, the treatment of XN downregulated the triglyceride synthesis and completely canceled the appearance of lipid droplets. Second, the effects of XN on the proliferation and the motility of HT-1080 human fibrosarcoma were investigated. The proliferation of HT-1080 was significantly suppressed in the presence of XN only in hypoxic condition but not in normoxic condition. XN also suppressed the motility of HT-1080 that was enhanced by hypoxia. Since, most cells in solid tumor were thought to be in hypoxic condition and acquired malignancy in response to hypoxia, these data suggest that XN may have potent and specific activities against cancerous cells. Furthermore, these data suggested that lipid metabolism may play an important role for hypoxic tumor cells and proposed a new therapeutic target for cancer chemotherapy.

Tetracyclic pyrans (+)-chloropuupehenone (1) and (+)-chloropuupehenol (5) and its C8-R-isomer (+)-3 were synthesized via a one-pot condensation of 1-chloro-2-lithio-3,5,6-tris(tert-butyldimethylsilyloxy)benzene (8) with (4aS,8aS)-3,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethylnaphthalene-1-carboxaldehyde (7). The major condensation product, (4aS,6aR,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (4), after desilylation provided tetracyclic pyran (+)-(4aS,6aR,12bS)-2H-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene-9,10-diol (3). At a dosage of 42 mg/rat over 8 h, pyran diol 3 inhibited the intestinal absorption of cholesterol by 71% in rats. Tetracyclic pyran 4 was also converted to o-quinone 28, which inhibited cholesteryl ester transfer protein (CETP) activity and L1210 leukemic cell viability with IC(50) values of 31 and 2.4 microM, respectively. Diol (+)-5 inhibited CETP activity with an IC(50) value of 16 microM. The minor condensation product, (4aS,6aS,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (6), was transformed into (+)-5 and (+)-1. A stepwise stereoselective synthesis of (+)-1 was also developed utilizing an oxyselenylation ring-closure reaction. The synthetic sequence also produced four biologically active naturally occurring drimanic sesquiterpenes, (+)-drimane-8alpha,11-diol (34), (-)-drimenol (38), (+)-albicanol (39), and (-)-albicanal (31) as intermediates.

Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC(50) values of 0.78 and 0.41 microM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC(50) values of 6.0 and 5.5 microM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents.

Five new beauveriolides were isolated from the acetone extracts of Beauveria sp. FO-6979 mycelia fermented in amino acid-supplemented media. The structures were elucidated by spectroscopic studies including NMR experiments and chemical degradation. All the beauveriolides are cyclodepsipeptides consisting of one 3-hydroxy-4-methyl fatty acid, two L-amino acids and one D-amino acid in common. Beauveriolide VII with the structure of cyclo-[3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl-D-valyl] inhibited lipid droplet formation and cholesteryl ester synthesis in macrophages, but the other beauveriolides showed only slight or almost no effect on lipid droplet formation.

Based on UV spectrum-guided purification, new monordens C, D and E, known monordens A (radicicol) and B and 5-O-methylsclerone were isolated from the fermentation broth of amidepsine-producing Humicola sp. FO-2942 by solvent extraction, silica-gel column chromatography, ODS column chromatography and HPLC. All monordens cause the cell cycle arrest at G1 and G2/M phases in Jurkat cells. But among them, monordens A and E show antifungal activity only against Aspergillus niger.

Beauveriolides I and III, cyclic depsipeptides composed of L-Phe, L-Ala, D-Leu and (3S,4S)-3-hydroxy-4-methyloctanoic acid, and L-Phe, L-Ala, L-allo-Ile and (3S,4S)-3-hydroxy-4-methyloctanoic acid, respectively, were previously isolated from the culture broth of fungal Beauveria sp. FO-6979 as inhibitors of macrophage foam cell formation. To improve the production of these compounds by fermentation, the culture conditions were studied. The production of both beauveriolides was increased five to ten folds by fermentation in the culture media containing tryptone. Further study revealed that addition of L-Leu/L-Ile, but not D-Leu/D-allo-Ile, to the culture medium yielded a high and selective production of beauveriolide I or III. As a result, regardless of their separation difficulty due to the similar physico-chemical properties, a large amount of beauveriolide I or III was prepared from the culture broth obtained from L-Leu- or L-Ile-supplemented fermentation, respectively, by one step purification using silica gel column chromatography.

Leptin-deficient mice (ob/ob) are an excellent murine model for obesity, insulin resistance, and diabetes, all of which are components of a multiple risk factor syndrome that, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis. In the current study, we show an unexpectedly severe hyperlipidemia in ob/ob mice on a background of low density lipoprotein receptor (LDLR) deficiency (-/-). Doubly mutant mice (LDLR-/-;ob/ob) exhibited striking elevations in both total plasma cholesterol (TC) and triglyceride (TG) levels (1715 +/- 87 and 1016 +/- 172 mg/dl, respectively), at age 3-4 months, resulting in extensive atherosclerotic lesions throughout the aorta by 6 months. Lipoprotein analyses revealed the elevated TC and TG levels to be due to a large increase in an apoB-containing broad-beta remnant lipoprotein fraction. While fasting, diet restriction, and low level leptin treatment significantly lowered TG levels, they caused only slight changes in TC levels. Hepatic cholesterol and triglyceride contents as well as mRNA levels of cholesterologenic and lipogenic enzymes suggest that leptin deficiency increased hepatic triglyceride production but did not change cholesterol production in ob/ob mice regardless of their LDLR genotype. These data provide evidence that the hypertriglyceridemia and hypercholesterolemia in the doubly mutant mice are caused by distinct mechanisms and point to the possibility that leptin might have some impact on plasma cholesterol metabolism, possibly through an LDLR-independent pathway. This model will be an excellent tool for future studies on the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis.

Effects of seven cytochalasans including cytochalasins B, D and E and novel phenochalasins A and B were tested on cytosolic lipid droplet formation and neutral lipid synthesis in mouse peritoneal macrophages. Phenochalasin A inhibited lipid droplet formation in a dose-dependent manner at least up to 20 microM without any morphological changes in macrophages. Cytochalasins D and E also inhibited lipid droplet formation only in a narrow range of concentrations, around 1 and 0.1 microM, respectively. At higher concentrations they gave morphological changes in macrophages. The other four cytochalasans only showed severe morphological changes in macrophages. Phenochalasin A and cytochalasins D and E inhibited cholesteryl ester (CE) synthesis specifically with IC50 values of 0.61, 2.4 and 0.20 microM, respectively, while the other cytochalasans inhibited both CE and triacylglycerol syntheses. Thus, among the cytochalasans tested, phenochalasin A showed very specific inhibition of CE synthesis and gave the lowest morphological changes in macrophages, resulting in the best inhibitor of lipid droplet formation in macrophages.

Phomopsis sp. FT-0211, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. Structurally related new compounds designated phenochalasins A and B were isolated from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography and preparative HPLC. Phenochalasin A caused a dose-dependent reduction in the number and size of lipid droplets in macrophages without any cytotoxic effect at least up to 20 microm. On the other hand, phenochalasin B showed inhibition of lipid droplet formation with a severe cytotoxic effect on macrophages.

The structures of phenochalasins A and B were elucidated by spectroscopic studies including various NMR measurements. Phenochalasins A and B have the cytochalasan skeleton of the 21,23-dioxa, 17,22-dione moiety containing unique phenyl and O-methyl phenyl residues at the C-10 position, respectively.

The effect of thirteen different fungal azaphilones, which have a common 6-iso-chromane-like ring, was tested on cholesteryl ester transfer protein (CETP) activity in vitro. Chaetoviridin B showed the most potent inhibitory activity with an IC50 value of < 6.2 microM, followed by sclerotiorin with an IC50 value of 19.4 microM. Rotiorin, chaetoviridin A and rubrorotiorin had moderate inhibitory activity (IC50 ; 30 approximately 40 microM), but others showed very weak or no inhibitory activity. The relationship between the structures and their inhibitory activity indicated that the presence of an electrophilic ketone(s) and/or enone(s) at both C-6 and C-8 positions in the isochromane-like ring is essential for eliciting CETP inhibitory activity. The transfer activity of both CE and TG was inhibited by sclerotiorin to approximately the same extent (IC50: 14.4 and 10.3 microM, respectively). A model of the reaction suggested that sclerotiorin reacts with a primary amine of amino acids such as lysine in the protein to form a covalent bond.

Beauveria sp. FO-6979, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. A new compound beauveriolide III was isolated along with a known compound beauveriolide I from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography, silica gel column chromatography and preparative HPLC. Beauveriolides I and III caused a reduction in the number and size of cytosolic lipid droplets in macrophages at 10 microM without any cytotoxic effect on macrophages.

The structure of fungal beauveriolide III, an inhibitor of lipid droplet formation in mouse macrophages, was elucidated to be cyclo-[(3S,4S)-3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl- D-allo-isoleucyl] by spectral analyses and chemical degradation.

Six new pyripyropenes, M to R, were isolated from the ethyl acetate extracts of the jar fermentation broth of Aspergillus fumigatus FO-1289-2501. Structural elucidation indicated that all the pyripyropenes have the same pyridino-alpha-pyrone sesquiterpene core as pyripyropenes A to L. Among them pyripyropene M showed the most potent inhibition against acyl-CoA : cholesterol acyltransferase activity with an IC50 value of 3.80 microM in rat liver microsomes, but pyripyropenes N to R showed moderate inhibitory activity (IC50 11.0 approximately 78.0 microM).