2934
M. Sechi et al. / Bioorg. Med. Chem. 17 (2009) 2925–2935
3.3.3. Integrase assays
To determine the extent of strand transfer, wild-type IN was
preincubated at a final concentration of 200 nM with the inhibitor
Sardegna and to Università di Sassari for their partial financial sup-
port. The work in NN’s laboratory was supported by funds from
the Campbell Foundation. The authors thank the ‘Laboratorio di
Strutturistica Mario Nardelli’ of the University of Parma for facilities.
in reaction buffer (50 mM NaCl, 1 mM HEPES, pH 7.5, 50
lM EDTA,
50 M dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL
l
bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl
sulfoxide, and 25 mM MOPS, pH 7.2) at 30 °C for 30 min. Then,
20 nM of the 50-end 32P-labeled linear oligonucleotide substrate
was added, and incubation was continued for an additional 1 h.
Reactions were quenched by the addition of an equal volume
References and notes
1. Barbaro, G.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Curr. Pharm. Des.
2005, 11, 1843.
2. Cohen, J. Science 2002, 296, 2320.
3. De Clercq, E. Nat. Rev. Drug Discov. 2002, 1, 13.
4. Neamati, N.; Marchand, C.; Pommier, Y. Adv. Pharmacol. 2000, 49, 147.
5. d’Angelo, J.; Mouscadet, J. F.; Desmaele, D.; Zouhiri, F.; Leh, H. Pathol. Biol. 2001,
49, 237.
6. Neamati, N. Exp. Opin. Invest. Drugs 2001, 10, 281.
7. Anthony, N. J. Curr. Top. Med. Chem. 2004, 4, 979.
8. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev. Drug Discov. 2005, 4, 236.
9. Brown, P. O. Integration. In Retroviruses; Coffin, J. M., Hughes, S. H., Varmus, H.
E., Eds.; Cold Spring Harbor Laboratory Press: NY, 1997; pp 161–203.
10. Pais, G. C. G.; Burke, T. R. Drugs Future 2002, 27, 1101.
11. Kiyama, R.; Kawasuji, T. PCT Int. Appl. 2001, WO-01/95905.
12. Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock, K.; Grobler, J. A.;
Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau, C.; Miller, M. D. Science 2000, 287,
646.
(16
0.025% xylene cyanol and 0.025% bromophenol blue). An aliquot
(5 L) was electrophoresed on a denaturing 20% polyacrylamide
lL) of loading dye (98% deionized formamide, 10 mM EDTA,
l
gel (0.09 M tris–borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8M
urea).
Gels were dried, exposed in a PhosphorImager cassette, and
analyzed using a Typhoon 8610 Variable Mode Imager (Amersham
Biosciences) and quantitated using ImageQuant 5.2. Percent inhibi-
tion (% I) was calculated using the following equation:
%I ¼ 100 ꢂ ½1 ꢀ ðD ꢀ CÞ=ðN ꢀ CÞꢃ
13. Goldgur, Y.; Dyda, F.; Hickman, A. B.; Jenkins, T. M.; Craigie, R.; Davies, D. R.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9150.
where C, N, and D are the fractions of 21-mer substrate converted to
strand transfer products for DNA alone, DNA plus IN, and IN plus
drug, respectively. The IC50 values were determined by plotting
the logarithm of drug concentration versus percent inhibition to ob-
tain concentration that produced 50% inhibition.
14. Marchand, C.; Zhang, X.; Pais, G. C. G.; Cowansage, K.; Neamati, N.; Burke, T. R.,
Jr.; Pommier, Y. J. Biol. Chem. 2002, 277, 12596.
15. Pais, G. C. G.; Zhang, X.; Marchand, C.; Neamati, N.; Cowansage, K.; Svarovskaia,
E. S.; Pathak, V. K.; Tang, Y.; Nicklaus, M.; Pommier, Y.; Burke, T. R., Jr. J. Med.
Chem. 2002, 45, 3184.
16. Pluymers, W.; Pais, G.; Van Maele, B.; Pannecouque, C.; Fikkert, V.; Burke, T. R.,
Jr.; De Clercq, E.; Witvrouw, M.; Neamati, N.; Debyser, Z. Antimicrob. Agents
Chemother. 2002, 46, 3292.
17. Sechi, M.; Derudas, M.; Dallocchio, R.; Dessi, A.; Bacchi, A.; Sannia, L.; Carta, F.;
Palomba, M.; Ragab, O.; Chan, C.; Shoemaker, R.; Sei, S.; Dayam, R.; Neamati, N.
J. Med. Chem. 2004, 47, 5298.
3.3.4. Cell culture
The HCT116 P53+/+ and HCT116 P53ꢀ/ꢀ cells were kindly pro-
vided by Dr. Bert Vogelstein (Johns Hopkins Medical Institutions,
Baltimore, MD). Cells were maintained as monolayer cultures in
RPMI 1640 media supplemented with 10% fetal bovine serum
18. Billich, A. Curr. Opin. Invest. Drugs 2003, 4, 206.
19. Johnson, A. A.; Marchand, C.; Pommier, Y. Curr. Top. Med. Chem. 2004, 4, 671.
20. Wang, Y.; Serradell, N.; Bolos, J.; Rosa, E. Drugs Future 2007, 32, 118.
21. Sorbera, L. A.; Serradell, N. Drugs Future 2006, 31, 310.
22. Nicklaus, M. C.; Neamati, N.; Hong, H.; Mazumder, A.; Sunder, S.; Chen, J.;
Milne, G. W.; Pommier, Y. J. Med. Chem. 1997, 40, 920.
23. Neamati, N.; Hong, H.; Mazumder, A.; Wang, S.; Sunder, S.; Nicklaus, M. C.;
Milne, G. W.; Proksa, B.; Pommier, Y. J. Med. Chem. 1997, 40, 942.
24. Chen, I.-J.; Neamati, N.; MacKerrel, A. D., Jr. Curr. Drug Target Infect. Dis. 2002, 2,
217.
25. Gupta, S. P.; Nagappa, A. N. Curr. Med. Chem. 2003, 10, 1779.
26. Maurin, C.; Bailly, F.; Cotelle, P. Curr. Med. Chem. 2003, 10, 1795.
27. Long, Y. Q.; Jiang, X. H.; Dayam, R.; Sanchez, T.; Shoemaker, R.; Sei, S.; Neamati,
N. J. Med. Chem. 2004, 47, 2561.
28. Barreca, M. L.; Rao, A.; De Luca, L.; Zappala, M.; Gurnari, C.; Monforte, P.; De
Clercq, E.; Van Maele, B.; Debyser, Z.; Witvrouw, M.; Briggs, J. M.; Chimirri, A. J.
Chem. Inf. Comput. Sci. 2004, 44, 1450.
(Gemini-Bioproducts, Woodland, CA) and 2 mmol/L L-glutamine
at 37 °C in a humidified atmosphere of 5% CO2. To remove the
adherent cells from the flask for passaging and counting, cells were
washed with PBS without calcium or magnesium, incubated with a
small volume of 0.25% trypsin–EDTA solution (Sigma–Aldrich, St.
Louis, MO) for 5–10 min, and washed with culture medium and
centrifuged. All experiments were performed using cells at expo-
nential growth stage. Cells were routinely checked for mycoplasma
contamination using a PCR-based assay (Stratagene, Cambridge,
UK).
29. Dayam, R.; Sanchez, T.; Clement, O.; Shoemaker, R.; Sei, S.; Neamati, N. J. Med.
Chem. 2005, 48, 111.
30. Sechi, M.; Sannia, L.; Carta, F.; Palomba, M.; Dallocchio, R.; Dessì, A.; Derudas,
M.; Zawahir, Z.; Neamati, N. Antiviral Chem. Chemother. 2005, 16, 41.
31. Deng, J.; Lee, K. W.; Sanchez, T.; Cui, M.; Neamati, N.; Briggs, J. M. J. Med. Chem.
2005, 48, 1496.
32. Dayam, R.; Sanchez, T.; Neamati, N. J. Med. Chem. 2005, 48, 8009.
33. Dayam, R.; Sanchez, T.; Neamati, N. ChemMedChem 2006, 1, 238.
34. Dayam, R.; Al-Mawsawi, L. Q.; Zawahir, Z.; Witvrouw, M.; Debyser, Z.; Neamati,
N. J. Med. Chem. 2008, 51, 1136.
3.3.5. Drugs
Stock solutions (10 mM) of compounds were prepared in DMSO
and stored at 20 °C. Further dilutions were made fresh in PBS or
cell-culture media.
3.3.6. Cytotoxicity assays
Cytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay as previously de-
scribed.51 Briefly, cells were seeded in 96-well microtiter plates
and allowed to attach. Cells were subsequently treated with con-
tinuous exposure to the corresponding drug for 72 h. An MTT solu-
tion (at a final concentration of 0.5 mg/mL) was added to each well,
and cells were incubated for 4 h at 37 °C. After removal of the med-
ium, DMSO was added and the absorbance was read at 570 nm. All
assays were done in triplicate. The IC50 was then determined for
each drug from a plot of log (drug concentration) versus percent-
age of cells killed.
35. Maurin, C.; Bailly, F.; Buisine, E.; Vezin, H.; Mbemba, G.; Mouscadet, J. F.;
Cotelle, P. J. Med. Chem. 2004, 47, 5583.
36. Sechi, M.; Bacchi, A.; Carcelli, M.; Compari, C.; Duce, E.; Fisicaro, E.; Rogolino,
D.; Gates, P.; Derudas, M.; Al-Mawsawi, L. Q.; Neamati, N. J. Med. Chem. 2006,
49, 4248.
37. Sotriffer, C. A.; Ni, H.; McCammon, A. J. J. Am. Chem. Soc. 2000, 122, 6136.
38. Barreca, M. L.; Lee, K. W.; Chimirri, A.; Briggs, J. M. Biophys. J. 2003, 84, 1450.
39. Dayam, R.; Neamati, N. Bioorg. Med. Chem. 2004, 12, 6371.
40. Espeseth, A. S.; Felock, P.; Wolfe, A.; Witmer, M.; Grobler, J.; Anthony, N.;
Egbertson, M.; Melamed, J. Y.; Young, S.; Hamill, T.; Cole, J. L.; Hazuda, D. J. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 11244.
41. Grobler, J. A.; Stillmock, K.; Binghua, H.; Witmer, M.; Felock, P.; Espeseth, A. S.;
Wolfe, A.; Egbertson, M.; Bourgeois, M.; Melamed, J.; Wai, J. S.; Young, S.;
Vacca, J.; Hazuda, D. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6661.
42. Kawasuji, T.; Fuji, M.; Yoshinaga, T.; Sato, A.; Fujiwara, T.; Kiyama, R. Bioorg.
Med. Chem. 2006, 14, 8420.
Acknowledgments
43. Katritzky, A. R.; Lang, H.; Lan, X. Tetrahedron 1993, 49, 7445.
44. Gavezzotti, A. Cryst. Eng. Commun. 2008, 10, 389.
We thank Dr. Maria Orecchioni and Mr. Paolo Fiori for assistance
with NMR spectroscopy. M.S. is grateful to Fondazione Banco di
45. Marchand, C.; Neamati, N.; Pommier, Y. Methods Enzymol. 2001, 340,
624.