New Rev-export inhibitor from Alpinia galanga and structure-activity relationship

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 9, 2009, Pages 2555-2557

  Tamura, Satoru ^a   Shiomi, Atsushi ^a   Kaneko, Masafumi ^b   Ye, Ying ^a   Yoshida, Minoru ^c   Yoshikawa, Masayuki ^d   Kimura, Tominori ^e,f   Kobayashi, Motomasa ^a   Murakami, Nobutoshi ^a,b  

^a OSAKA UNIVERSITY   (Japan)

^b JAPAN SCIENCE AND TECHNOLOGY AGENCY   (Japan)

^c RIKEN   (Japan)

^d KYOTO PHARMACEUTICAL UNIVERSITY   (Japan)

^e RITSUMEIKAN UNIVERSITY   (Japan)

^f KANSAI MEDICAL UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

1' ACETOXYCHAVICOL ACETATE; ALPINIA GALANGA EXTRACT; LEPTOMYCIN B; REGULATOR PROTEIN; REV PROTEIN; UNCLASSIFIED DRUG; VIRUS PROTEIN;

ALPINIA; ARTICLE; BIOASSAY; BIOLOGICAL ACTIVITY; BIOTINYLATION; CONTROLLED STUDY; DRUG MECHANISM; DRUG STRUCTURE; HELA CELL; HUMAN; HUMAN CELL; HUMAN IMMUNODEFICIENCY VIRUS 1; MINIMUM INHIBITORY CONCENTRATION; MOLECULAR PROBE; NUCLEAR EXPORT SIGNAL; PLANT ROOT; SEPARATION TECHNIQUE; STRUCTURE ACTIVITY RELATION; YEAST;

ACQUIRED IMMUNODEFICIENCY SYNDROME; ALPINIA; ANTI-HIV AGENTS; BENZYL ALCOHOLS; BIOLOGICAL ASSAY; BIOTINYLATION; CHEMISTRY, PHARMACEUTICAL; DRUG DESIGN; HELA CELLS; HIV-1; HUMANS; PLANT EXTRACTS; PLANT ROOTS; REV GENE PRODUCTS, HUMAN IMMUNODEFICIENCY VIRUS; STRUCTURE-ACTIVITY RELATIONSHIP;

ALPINIA GALANGA; HUMAN IMMUNODEFICIENCY VIRUS 1; SCHIZOSACCHAROMYCETACEAE;

EID: 64249097969     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2009.03.047     Document Type: Article

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10. 2 for 24 h. After the whole was washed, the cells were treated with 3 μM concentration of biotinylated LMB probe 2 in 1 mL of the medium containing 1% DMSO for 3 h. For competitive experiments, ACA (1) and LMB were injected 1 h prior to addition of 2, 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 15000 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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14. ® 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 under a fluorescence microscope, then image analysis was conducted by Scion image software (Scion) to determine Rev-export inhibitory activity. In the depicted pictures, several cells free from transfection displayed disperse weak fluorescence due to nonspecific binding of the antibodies.