S. Tamura et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2555–2557
2557
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 lL) under boiling at 95 °C
Taking the reactants of 1 with N-acetyl-cysteine methyl ester and
synthesis of several analogs into consideration, 10-S configuration
and the two acetyl functions as well as 10-acetoxyl-20-ene moiety
proved crucial for Rev-export inhibitory activity of 1. In addition,
ACA (1) was shown to inhibit export of the genuine Rev protein
in HeLa cells expressing HA-tagged Rev by indirect fluorescent
antibody technique. Exploration for synthetic leads possessing
more potent activity than 1 is in progress in our laboratory.
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.
11. Corey, E. J.; Bakshi, R. K.; Shibata, S.; Chen, C. P.; Singh, V. K. J. Am. Chem. Soc.
1987, 109, 7925.
Acknowledgments
12. Compound 5: colorless oil. ½a D24
ꢃ
+49.5 (c 1.0, EtOH). IR (KBr): 3019, 1765, 1741,
1606 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.37 (2H, d, J = 8.5 Hz, 2-H), 7.07 (2H,
.
This work was supported in part by Grants-in-Aid for Scientific
Research (Grant No. 19590100) from the Ministry of Education,
Science, Culture and Sports. The authors are grateful to the Naito
Foundation and the Takeda Science Foundation for financial
support.
d, J = 8.5 Hz, 3-H), 6.26 (1H, d, J = 6.1 Hz, 10-H), 5.98 (1H, ddd, J = 6.1, 11.0,
17.1 Hz, 20-H), 5.30 (1H, dd, J = 1.2, 17.1 Hz, 30-Ha), 5.25 (1H, dd, J = 1.2,
11.0 Hz, 30-Hb), 2.30 (3H, s, 4-OAc), 2.11 (3H, s, 10-OAc). FAB-MS m/z: 235
[M+H+]. FAB-HRMS m/z: Calcd for C13H15O4: 235.0970, Found: 235.0966.
Compound 9a: colorless oil. ½a D24
ꢁ33.5 (c 1.0, EtOH). IR (KBr): 3350, 3025,
ꢃ
1608 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.24 (2H, d, J = 8.3 Hz, 2-H), 6.80 (2H,
.
d, J = 8.3 Hz, 3-H), 6.04 (1H, ddd, J = 16.5, 10.5, 6.0 Hz, 20-H), 5.33 (1H, d,
J = 16.5 Hz, 30-Ha), 5.17 (1H, d, J = 10.5 Hz, 30-Hb), 5.16 (1H, d, J = 6.0 Hz, 10-H).
FAB-MS m/z: 151 [M+H]+. FAB-HRMS m/z: Calcd for C9H11O2: 151.0759, Found:
151.0762.
References and notes
Compound 10: colorless oil. ½a D24
ꢁ36.3 (c 1.0, EtOH). IR (KBr): 3265, 3022,
ꢃ
1. Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C. Science 1984, 224, 497.
2. del Rio, C. Arch. Med. Res. 2005, 36, 682.
3. Daly, T. J.; Cook, K. S.; Gray, G. S.; Maione, T. E.; Rusche, J. R. Nature 1989, 342,
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4. Kudo, N.; Wolff, B.; Sekimoto, T.; Schreiner, E. P.; Yoneda, Y.; Yanagida, M.;
Horinouchi, S.; Yoshida, M. Exp. Cell Res. 1998, 242, 540.
5. Kjems, J.; Askjaer, P. Adv. Pharmacol. 2000, 48, 251.
6. Murakami, N.; Ye, Y.; Kawanishi, M.; Aoki, S.; Kudo, N.; Yoshida, M.; Nakayama,
E. E.; Shioda, T.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2002, 12, 2807.
7. Noro, T.; Sekiya, T.; Katoh, M.; Oda, Y.; Miyase, T.; Kuroyanagi, M.; Ueno, A.;
Fukushima, S. Chem. Pharm. Bull. 1988, 36, 244.
1767, 1606 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.38 (2H, d, J = 8.5 Hz, 2-H), 7.07
.
(2H, d, J = 8.5 Hz, 3-H), 6.03 (1H, ddd, J = 16.8, 10.0, 5.6 Hz, 20-H), 5.35 (1H, d,
J = 16.8 Hz, 30-Ha), 5.25 (1H, d, J = 10.0 Hz, 30-Hb), 5.21 (1H, d, J = 5.6 Hz, 10-H),
2.30 (3H, s, OAc). FAB-MS m/z: 193 [M+H]+. FAB-HRMS m/z: Calcd for C11H13O3:
193.0864, Found: 193.0866.
13. Kimura, T.; Hashimoto, I.; Yamamoto, T.; Nishikawa, M.; Fujisawa, J.-I. Genes
Cells 2000, 5, 289.
14. HeLa cells (1.0 ꢂ 105 cells) were maintained on coverslips in 24-well
microplate with 1 mL of Dulbecco’s MEM medium supplemented with 10%
FBS at 37 °C in 5% CO2 for 24 h. Transfection of pCG-HA-Rev (plasmid encoding
8. After picking of an aliquot of colony of S. pombe on the agar, the yeasts were
transferred and cultured in thiamine-free MM-medium with inducing the
fusion protein for 24 h at 37 °C. Then the cells were seeded in 96-well plates
along with test samples in the medium containing 1% DMSO and incubated at
37 °C for further 3 h. The distribution of the GST-NLS-GFP-RevNES-fused
protein was monitored by fluorescence microscope to determine MIC values.
9. Kudo, N.; Matsumori, N.; Taoka, H.; Fujiwara, D.; Schreiner, E. P.; Wolff, B.;
Yoshida, M.; Horinouchi, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9112.
10. In culture dishes (3 cm i.d.), HeLa cells (6.0 ꢂ 105 cells) were cultured in 3 mL
of Dulbecco’s MEM medium containing with 10% fetal bovine serum at 37 °C in
HA-tagged Rev protein) and pCRRE/DRev (plasmid encoding Gag protein)
plasmids into HeLa cells were performed using PolyFectÒ 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.
5% CO2 for 24 h. After the whole was washed, the cells were treated with 3 lM
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 lL of 50% (v/v) beads