Hydroxytakakiamide and Other Constituents from a Marine Sponge-Associated Fungus Aspergillus fischeri MMERU23, and Antinociceptive Activity of Ergosterol Acetate, Acetylaszonalenin and Helvolic Acid

Abstract

An unreported prenylated indole derivative hydroxytakakiamide (4) was isolated, together with the previously described ergosterol (1), ergosterol acetate (2), and (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (3), from the column fractions of the crude ethyl acetate extract of the culture of a marine sponge-associated fungus, Aspergillus fischeri MMERU 23. The structure of 4 was elucidated by the interpretation of 1D and 2D NMR spectral data and high-resolution mass spectrum. The absolute configuration of the stereogenic carbon in 3 was proposed to be the same as those of the co-occurring congeners on the basis of their biogenetic consideration and was supported by the comparison of its sign of optical rotation with those of its steroisomers. The crude ethyl acetate extract and 2 were evaluated, together with acetylaszonalenin (5) and helvolic acid (6), which were previously isolated from the same extract, for the in vivo antinociceptive activity in the mice model. The crude ethyl acetate extract exhibited antinociceptive activity in the acetic acid-induced writhing and formalin tests, while 2, 5, and 6 displayed the effects in the late phase of the formalin test. On the other hand, neither the crude ethyl acetate extract nor 2, 5, and 6 affected the motor performance of mice in both open-field and rotarod tests. Additionally, docking studies of 2, 5, and 6 were performed with 5-lipoxygenase (5-LOX) and phosphodiesterase (PDE) enzymes, PDE4 and PDE7, which are directly related to pain and inflammatory processes. Molecular docking showed that 6 has low affinity energy to PDE4 and PDE7 targets while retaining high affinity to 5-LOX. On the other hand, while 2 did not display any hydrogen bond interactions in any of its complexes, it achieved overall better energy values than 6 on the three antinociceptive targets. On the other hand, 5 has the best energy profile of all the docked compounds and was able to reproduce the crystallographic interactions of the 5-LOX complex.

Keywords: Aspergillaceae; Aspergillus fischeri; acetylaszonalenin; antinociceptive activity; ergosterol acetate; helvolic acid; hydroxytakakiamide; marine sponge-associated fungus.

Figure 1

Structures of ergosterol (1), ergosterol acetate (2), (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (3), hydroxytakakiamide (4), acetylasznalenin (5), and helvolic acid (6).

Figure 2

Effects of oral administration of the crude ethyl acetate extract of A. fischeri MMERU 23 (Ext) in acetic acid-induced writhing. Mice were divided in three groups, 6 mice per group (n = 6). The treatment group received 50, 100, and 200 mg/kg of Ext, p.o., the control group received a vehicle 60 min before intraperitoneal injection with 0.8% acetic acid (injected at time zero), and the third group received a reference drug, morphine (Mor, 5 mg/kg, s.c.), 40 min before intraperitoneal injection with 0.8% acetic acid. Data are expressed as mean ± SEM. ** Significantly different from control group (p < 0.05). *** Significantly different from control group (p < 0.0001) as determined by ANOVA and followed by Bonferroni’s test.

Figure 3

Effects of treatment with the crude extract of A. fischeri MMERU 23 (Ext) in the formalin test. Panels (A) and (B) represent the effects of Ext in the early and late phases of the formalin test in mice, respectively. Mice were orally administered with 50, 100, and 200 mg/kg of Ext or vehicle (control group) 60 min before a formalin injection (injected at time zero). Morphine (Mor, 5 mg/kg, s.c.) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. * Significantly different from the control group (p < 0.05), as determined by ANOVA and followed by Bonferroni´s test.

Figure 4

Effects of treatment with 2 in the formalin test. Panels (A) and (B) represent the effects of 2 on the early and late phases of the formalin test in mice, respectively. Mice were treated with 2 (30, 60, and 90 mg/kg) or vehicle (control group) via the oral route 60 min before a formalin injection (injected at time zero). Morphine (Mor, 5 mg/kg) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. *** Significantly different from the control group (p < 0.0001) as determined by ANOVA and followed by Bonferroni´s test.

Figure 5

Effects of treatment with 5 in the formalin test. Panels (A) and (B) represent effects of 5 on the early and late phases of the formalin test in mice, respectively. Mice were treated with 5 (30, 60, and 90 mg/kg) or vehicle (control group) via the oral route 60 min before a formalin injection (injected at time zero). Morphine (Mor, 5 mg/kg) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. *** Significantly different from the control group (p < 0.0001) as determined by ANOVA and followed by Bonferroni´s test.

Figure 6

Effects of treatment with 6 in the formalin test. Panels (A) and (B) represent the effects of 6 on the early and late phases of the formalin test in mice, respectively. Mice were orally administered with 6 (1, 5, and 10 mg/kg) or vehicle (control group) before a formalin injection. Morphine (Mor, 5 mg/kg, s.c.) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. ** Significantly different from the control group (p < 0.05). *** Significantly different from the control group (p < 0.0001) as determined by ANOVA followed by Bonferroni´s test.

Figure 7

Effects of Ext in the open field (A) and rotarod (B) tests. Mice were orally administered with 200 mg/kg of Ext or vehicle (control group) 60 min before the evaluation. Diazepam (DZP, 10 mg/kg) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. * Significantly different from the control group (p < 0.05), as determined by ANOVA and followed by Bonferroni’s test.

Figure 8

Effects of 2 in the open field (A) and rotarod (B) tests. Mice were treated with 2 (90 mg/kg) or vehicle (control group) by oral route 60 min before the evaluation. Diazepam (DZP; 10 mg/kg) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. *** Significantly different from the control group (p < 0.0001) as determined by ANOVA and followed by Bonferroni´s test.

Figure 9

Effect of 5 in the open-field (A) and rota rod (B) tests. Mice were treated with 5 (90 mg/kg) or vehicle (control group) by oral route 60 min before the evaluation. Diazepam (DZP, 10 mg/kg) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. *** Significantly different from the control group (p < 0.0001) as determined by ANOVA and followed by Bonferroni´s test.

Figure 10

Effect of 6 in the open field (A) and rotarod (B) tests. Mice were treated with 6 (10 mg/kg) or vehicle (control group) by oral route 60 min before the evaluation. Diazepam (DZP, 10 mg/kg) was used as a reference drug. Data are expressed as means ± S.E.M.; n = 6 mice per group. *** Significantly different from the control group (p < 0.0001) as determined by ANOVA and followed by Bonferroni´s test.

Figure 11

Re-docking of the ligands of 5-LOX [A] (PDB ID: 6N2W; RMSD = 1.79 Å), PDE4 [B] (PDB ID: 4KP6; RMSD = 1.95 Å), and PDE7 [C] (PDB ID: 1ZKL; RMSD = 0.94 Å) on Autodock Vina software. The crystallographic ligand poses are shown by green sticks while the re-docked poses are shown by blue sticks.

Figure 12

Comparison of the interaction patterns for the crystallographic ligands versus 2, 5, and 6 on their active sites. [A1] 5-LOX ligand (PDB: 6N2W) on green sticks, [B1] PDE4 ligand (PDB: 4KP6) on violet sticks, and [C1] PDE7 ligand (PDB: 1ZKL) on grey sticks. The compounds are paired with the most favorable pose of 6 (orange-colored sticks), 2 (salmon-colored sticks), and 5 (olive green-colored sticks) and on each target as follows: [A2] 6 on 5-LOX (−8.1 kcal/mol), [B2] 6 on PDE4 (−3.3 kcal/mol), and [C2] 6 on PDE7 (−3.0 kcal/mol); [A3] 2 on 5-LOX (−8.7 kcal/mol), [B3] 2 on PDE4 (−5.5 kcal/mol), and [C3] 2 on PDE7 (−6.8 kcal/mol); [A4] 5 on 5-LOX (−9.0 kcal/mol), [B4] 5 on PDE4 (−7.8 kcal/mol), and [C4] 5 on PDE7 (−8.6 kcal/mol). Protein residues are shown in blue; hydrogen bonds are shown in navy blue lines and hydrophobic interactions are shown in black dashed lines.