During our screening program for microbial inhibitors of triacylglycerol synthesis in mammalian cells, four structurally related new compounds, dinapinones A1 (1) and A2 (2) and monapinones A (3) and B (4), were isolated from the culture broth of Penicillium pinophilum FKI-3864. Compounds 3 and 4 were produced by the fungus only when fermented in seawatersupplemented medium. The structures of 1 to 4 were elucidated by spectroscopic studies including various NMR experiments. Compounds 1 and 2 were atropisomers consisting of two monomers with the same planar structure of dihydronaphthopyranone as 3. The absolute stereochemistry of 3 was elucidated by NOE experiment and circular dichroism spectra. Furthermore, the stereochemistry of 1 and 2 was elucidated by in vitro conversion from the structure-defined 3 to its dimers 1 and 2. Keywords: atropisomer; dinapinone; monapinone; Penicillium pinophilum; seawater-supplemented medium; triacylglycerol biosynthesis INTRODUCTION Triacylglycerol (TG) synthesis is important in many metabolic processes in mammals, including lactation, energy storage in fat and muscle, fat absorption in the intestine and the assembly of lipoprotein particles in the liver and small intestine; however, excess accumulation of TG in certain organs and tissues causes fatty liver, obesity and hypertriglyceridemia. [2][3][4] Recently, much attention has been paid to lifestyle-related metabolic syndrome as a high-risk condition leading to severe diseases such as atherosclerosis, hypertension and diabetes. For example, accumulation of TG in the liver has been thought to trigger the inflammation, apoptosis, and fibrosis that characterize the progression of hepatic steatosis to nonalcoholic steatohepatitis and cirrhosis. 5 Against this background, we established a cell-based assay system using Raji cells 6 and Chinese hamster ovary-K1 (CHO-K1) cells 7 to screen microbial metabolites for inhibitors of TG synthesis. Through our continuous screening efforts, we recently discovered a series of new dihydronaphthopyranone-containing compounds named dinapinones A1 (1) and A2 (2) (Figure 1) and monapinones A (3) and B (4) in the culture of Penicillium pinophilum FKI-3864. Study of the culture conditions indicated that monapinones were produced by the fungus, when the strain was fermented in the original dinapinone production medium supplemented with seawater. 7,8 In this study, we demonstrated that 1

Two isozymes for human acyl-coenzyme A:diacylglycerol acyltransferase (DGAT), DGAT1 and DGAT2, were independently expressed in DGAT-deficient Saccharomyces cerevisiae to establish DGAT1-and DGAT2-S. cerevisiae. The selectivity of DGAT inhibitors of natural origin towards the isozymes was assessed in enzyme assays using the microsomal fractions prepared from DGAT1-and DGAT2-S. cerevisiae. Amidepsines and xanthohumol inhibited DGAT1 and DGAT2 with similar potency, whereas roselipins were found to inhibit DGAT2 selectively. Keywords: acyl-CoA:diacylglycerol acyltransferase; amidepsine; DGAT; isozyme; roselipin; triacylglycerol; xanthohumol INTRODUCTION Triacylglycerol (TG) is the major energy-storage form of long-chain fatty acids in animals. 1,2 TG synthesis is important in many biological processes, including intestinal fat absorption, fat storage in adipocytes and energy metabolism in muscle, but excessive accumulation of TG in adipocytes as a result of a fat-rich diet or sedentary lifestyle causes obesity.Acyl-coenzyme A (CoA):diacylglycerol acyltransferase (DGAT, EC2.3.1.20) is a membrane-bound enzyme that catalyzes TG formation by acyl esterifications of diacylglycerol. Two biological pathways for TG synthesis, the glycerol phosphate pathway and the monoacylglycerol pathway, have been reported. These pathways form diacylglycerol (DG), which in turn is acylated by DGAT to form TG. 3 Recent molecular biological studies have revealed the existence of two different DGAT isozymes, DGAT1 and DGAT2, 4-6 in mammals, and extensive studies including biological experiments and knockout mice have shown that these isozymes have different functions in mammals. 7-13 Increased DGAT2 activity in the liver causes hepatic steatosis, whereas DGAT1 plays a role in very low-density lipoprotein (VLDL) synthesis in the liver and increases plasma VLDL concentration. Furthermore, newborn DGAT2-deficient mice die within hours of birth, whereas DGAT1-deficient mice are viable and have a modest reduction in tissue TG. Therefore, it is important to determine the selectivity of inhibitors towards the two DGAT isozymes for developing them as pharmaceutical drugs.Our research group conducted an enzyme assay involving rat liver microsomes to discover several DGAT inhibitors from natural sources,

Five new monapinones, including a dinapinone monomer, were isolated from the culture broth of the dinapinone-producing Penicillium pinophilum FKI-3864 in a medium modified to contain seawater. The structures of these monapinones were elucidated by various NMR experiments. Monapinones possessed the same dihydronaphthopyranone skeleton as the dinapinones, with different hydroxyalkyl chains: monapinone A was identified as the monomeric portion of the atropisomer dinapinones A1 and A2, and monapinones A and B showed weak inhibition of triacylglycerol (TG) synthesis in intact mammalian cells, whereas the others showed almost no effect on TG synthesis. The Journal of Antibiotics (2011) Keywords: dinapinone monomer; monapinone; Penicillium pinophilum; seawater-containing medium; triacylglycerol biosynthesis INTRODUCTION During our screening program to discover inhibitors of triacylglycerol (TG) synthesis using intact Chinese hamster ovary-K1 (CHO-K1) cells, dinapinones A1 (6) and A2 (7) were isolated from the culture broth of Penicillium pinophilum FKI-3864. 1 These are atropisomers formed from two monomers with the same dihydronaphthopyranone structure. 1,2 Interestingly, the mixture (1:1) of 6 and 7 showed full inhibitory activity against TG synthesis in CHO-K1 cells (IC 50 , 0.054 mM), although 6 or 7 alone showed less potent activity (IC 50 , 0.65 or 412 mM, respectively).Through our study of the culture conditions for dinapinone production, we found that five new compounds containing the dinapinone monomeric unit-designated monapinones A (1) to E (5) (Figure 1) were produced when the dinapinone-producing fungus was cultured in the original medium supplemented with seawater. Interestingly, compounds 1-5 were produced by the fungus only in the original production medium supplemented with seawater, whereas 6 and 7 were produced very slightly in the same medium. The structure elucidation of 1 and 2, including their absolute stereochemistry, will be described in detail elsewhere. 2 In this study, the fermentation, isolation, structure elucidation of 3-5 and TG inhibitory activity of monapinones are described.

Production of Monapinones by Fermentation of the Dinapinone-Producing Fungus Penicillium pinophilum FKI-3864 in a Seawater-Containing Medium. -Monapinones A-E (Ia)-(Ie) are isolated, and compounds (Ia) and (Ib) are shown to inhibit triacylglycerol synthesis weakly. -(KAWAMOTO, K.; YAMAZAKI, H.; OHTE, S.; MASUMA, R.; UCHIDA, R.; TOMODA*, H.; J. Antibiot. 64 (2011) 7, 503-508, http://dx.doi.org/10.1038/ja.2011.33 ; Grad. Sch. Pharm. Sci., Kitasato Univ., Minato, Tokyo 108, Japan; Eng.) -R. Langenstrassen 47-212