Prenylcoumarin with Rev-export inhibitory activity from Cnidii Monnieris Fructus

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 12, 2010, Pages 3717-3720

  Tamura, Satoru ^a   Fujitani, Toshiaki ^a   Kaneko, Masafumi ^b   Murakami, Nobutoshi ^a,b  

^a OSAKA UNIVERSITY   (Japan)

^b JAPAN SCIENCE AND TECHNOLOGY AGENCY   (Japan)

Author keywords

Anti HIV; Cnidium monnieri Cusson; Osthol; Prenylcoumarin; Rev export inhibitor

Indexed keywords

ANTI HUMAN IMMUNODEFICIENCY VIRUS AGENT; CARBONYL DERIVATIVE; CNIDIUM MONNIERI EXTRACT; COUMARIN DERIVATIVE; OSTHOLE; PHENYLCOUMARIN; REV PROTEIN; UNCLASSIFIED DRUG;

ARTICLE; CELL NUCLEUS; CONTROLLED STUDY; CYTOPLASM; DRUG SYNTHESIS; FLUORESCENT ANTIBODY TECHNIQUE; HELA CELL; HUMAN; HUMAN CELL; IC 50; PROTEIN LOCALIZATION; STRUCTURE ACTIVITY RELATION;

ACTIVE TRANSPORT, CELL NUCLEUS; ADJUVANTS, IMMUNOLOGIC; ANTI-HIV AGENTS; COUMARINS; HELA CELLS; HIV-1; HUMANS; PLANT EXTRACTS; PLANTS, MEDICINAL; REV GENE PRODUCTS, HUMAN IMMUNODEFICIENCY VIRUS; STRUCTURE-ACTIVITY RELATIONSHIP;

CNIDIUM MONNIERI; SCHIZOSACCHAROMYCETACEAE;

EID: 77954212463     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.04.081     Document Type: Article

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12. 2 for 24 h. Transfection of pCG-HA-Rev (plasmid encoding HA-tagged Rev protein) and pCRRE/ΔRev (plasmid encoding Gag protein) plasmids into HeLa cells were performed using PolyFect transfection reagent kit (QIAGEN) for 16 h according to the manufacturer's instructions. After the cells were washed, each solution of tested sample at an appropriate concentration in the medium containing 1% DMSO was inoculated and the whole was incubated at 37 °C for further 12 h. Cells were rinsed with cold D-PBS (-) twice and fixed with 4% formaldehyde/ D-PBS (-) for 20 min. Then the cells were defatted with MeOH under shaking for 10 min and washed with cold D-PBS (-) thrice. After treatment with 10% FBS in Dulbecco's MEM medium for 30 min, the samples were incubated with anti-HA antibody (Roche) for 45 min followed by incubation with FITC-labeled anti-mouse IgG antibody (Vector) for 45 min. Localization of the HA-tagged Rev protein in the cells was examined with a fluorescence microscope, then image analysis was conducted by Scion image software (Scion) to determine Rev-export inhibitory activity.
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14. 2 for 24 h. After the whole was washed, the cells were treated with 1 μM concentration of biotinylated LMB probe 3 in 1 mL of the medium containing 1% DMSO for 3 h. For competitive experiments, osthol (1), 7-demethoxyanalog 4, and LMB (Cosmo Bio) were injected 1 h prior to addition of 3, respectively. The cells were harvested, then 0.2 mL of TBS lysis buffer (pH 7.5, 20 mM Tris-HCl, 0.1% NonidetP40, 0.15 mM NaCl, 2 M 2-mercaptoethanol, 1% protease inhibitor cocktail-DMSO) was added and the mixture was sonicated for 10 min at 0 °C. After centrifugation at 15,000 rpm for 30 min, the supernatant was treated with 50 μL of 50% (v/v) beads immobilized with streptavidin in TBS lysis buffer under rotation at 4 °C overnight. The beads were rinsed thrice by the lysis buffer, then the bound proteins were eluted by SDS-PAGE sample buffer (50 μL) under boiling at 95 °C for 5 min. Each eluate was separated by 5-20% SDS-PAGE, then the proteins were transferred to PVDF membrane and the blot was blocked with 5% milk in TBS-T at 4 °C overnight. The membrane was incubated with primary antibody to CRM1 (Santa Cruz Biotech) at room temperature for 1 h. The bound antibodies were detected by treatment with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech) at room temperature for 1 h, then the blots were visualized using enhanced chemiluminescence.
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17. 2+H; 215.1072, found: 215.1098.
18. 3+H; 247.1334, found: 247.1332.