11.
Mehellou, Y.; De Clercq, E. Journal of medicinal
(OD540) of the mock-infected control sample by 50%. The
concentration achieving 50% protection against the cytopathic
effect of the virus in infected cells was defined as the 50%
effective concentration (EC50).
chemistry 2010, 53, 521.
12.
Tremblay, C. L. Antimicrobial agents and chemotherapy 2012,
56, 5000.
13.
Asahchop, E. L.; Wainberg, M. A.; Sloan, R. D.;
Reverse transcriptase assay.
Croxtall, J. D. Drugs 2012, 72, 847.
14.
1495.
15.
Schafer, J. J.; Short, W. R. Antiviral therapy 2012, 17,
Recombinant wild type p66/p51 HIV-1 RT was expressed and
purified as described by 35. The RT assay is performed with the
EnzCheck Reverse Transcriptase Assay kit (Molecular Probes,
Invitrogen), as described by the manufacturer. The assay is based
on the dsDNA quantitation reagent PicoGreen. This reagent
shows a pronounced increase in fluorescence signal upon binding
to dsDNA or RNA-DNA heteroduplexes. Single-stranded nucleic
acids generate only minor fluorescence signal enhancement when
a sufficiently high dye:base pair ratio is applied. 36 This condition
is met in the assay. A poly(rA) template of approximately 350
bases long, and an oligo(dT)16 primer, are annealed in a molar
ratio of 1:1.2 (60 min. at room temperature). Fifty-two ng of the
RNA/DNA is brought into each well of a 96-well plate in a
volume of 20 µl polymerization buffer (60 mMTris-HCl, 60
mMKCl, 8 mM MgCl2, 13 mM DTT, 100 µM dTTP, pH 8.1).
Five µl of RT enzyme solution, diluted to a suitable
concentration in enzyme dilution buffer (50 mMTris-HCl, 20%
glycerol, 2 mM DTT, pH 7.6), is added. The reactions are
incubated at 25°C for 40 minutes and then stopped by the
addition of EDTA (15 mM fc). Heteroduplexes are then detected
by addition of PicoGreen. Signals are read using an excitation
wavelength of 490 nm and emission detection at 523 nm using a
spectrofluorometer (Safire 2, Tecan). To test the activity of
compounds against RT, 1 µl of compound in DMSO is added to
each well before the addition of RT enzyme solution. Control
wells without compound contain the same amount of DMSO.
Results are expressed as relative fluorescence i.e. the
fluorescence signal of the reaction mix with compound divided
by the signal of the same reaction mix without compound.
Song, Y.; Fang, Z.; Zhan, P.; Liu, X. Current medicinal
chemistry 2014, 21, 329.
16.
Monforte, A. M.; Maga, G.; Pannecouque, C.; Balzarini, J.; De
Clercq, E.; Chimirri, A.; Monforte, P. Journal of medicinal
chemistry 2005, 48, 3433.
Barreca, M. L.; Rao, A.; De Luca, L.; Zappala, M.;
17.
Barreca, M. L.; Rao, A.; De Luca, L.; Iraci, N.;
Monforte, A. M.; Maga, G.; De Clercq, E.; Pannecouque, C.;
Balzarini, J.; Chimirri, A. Bioorganic & medicinal chemistry
letters 2007, 17, 1956.
18.
Monforte, A. M.; Rao, A.; Logoteta, P.; Ferro, S.; De
Luca, L.; Barreca, M. L.; Iraci, N.; Maga, G.; De Clercq, E.;
Pannecouque, C.; Chimirri, A. Bioorganic & medicinal chemistry
2008, 16, 7429.
19.
Monforte, A. M.; Logoteta, P.; Ferro, S.; De Luca, L.;
Iraci, N.; Maga, G.; Clercq, E. D.; Pannecouque, C.; Chimirri, A.
Bioorganic & medicinal chemistry 2009, 17, 5962.
20.
Monforte, A. M.; Logoteta, P.; De Luca, L.; Iraci, N.;
Ferro, S.; Maga, G.; De Clercq, E.; Pannecouque, C.; Chimirri,
A. Bioorganic & medicinal chemistry 2010, 18, 1702.
21.
Morreale, F.; Pannecouque, C.; Balzarini, J.; Chimirri, A.
Bioorganic & medicinal chemistry 2014, 22, 1459.
22.
23.
Weaver, K. L.; Romines, K. R.; Hazen, R.; Freeman, A.; Ferris,
R. G.; Andrews, C. W.; Boone, L.; Chan, J. H.; Stammers, D. K.
Journal of medicinal chemistry 2008, 51, 5000.
Monforte, A. M.; Ferro, S.; De Luca, L.; Lo Surdo, G.;
The PyMOL Molecular Graphics System, V. S., LLC. .
Ren, J.; Chamberlain, P. P.; Stamp, A.; Short, S. A.;
24.
Ferro, S.; Buemi, M. R.; De Luca, L.; Agharbaoui, F.
Acknowledgments
E.; Pannecouque, C.; Monforte, A. M. Bioorganic & medicinal
chemistry 2017.
25.
chemistry 2010, 31, 455.
26.
The technical assistance of Mr. Kris Uyttersprot, Mrs. Kristien
Erven, and Mrs. Cindy Heens for the HIV experiments is
gratefully acknowledged.
Trott, O.; Olson, A. J. Journal of computational
Frey, K. M.; Puleo, D. E.; Spasov, K. A.; Bollini, M.;
Jorgensen, W. L.; Anderson, K. S. Journal of medicinal
chemistry 2015, 58, 2737.
References
27.
Chong, P.; Sebahar, P.; Youngman, M.; Garrido, D.;
1.
of medicinal chemistry 2015, 89, 421.
2. Xu, Z.; Guo, J.; Yang, Y.; Zhang, M.; Ba, M.; Li, Z.;
Cao, Y.; He, R.; Yu, M.; Zhou, H.; Li, X.; Huang, X.; Guo, Y.;
Guo, C. European journal of medicinal chemistry 2016, 123,
309.
Zhang, M. Z.; Chen, Q.; Yang, G. F. European journal
Zhang, H.; Stewart, E. L.; Nolte, R. T.; Wang, L.; Ferris, R. G.;
Edelstein, M.; Weaver, K.; Mathis, A.; Peat, A. Journal of
medicinal chemistry 2012, 55, 10601.
28.
Ren, J.; Nichols, C. E.; Stamp, A.; Chamberlain, P. P.;
Ferris, R.; Weaver, K. L.; Short, S. A.; Stammers, D. K. The
FEBS journal 2006, 273, 3850.
3.
4.
Yeni, P. Journal of hepatology 2006, 44, S100.
Martins, S.; Ramos, M. J.; Fernandes, P. A. Current
29.
Gowda, B. T.; Usha, K. M.; Jayalakshmi, K. L. Z
Naturforsch A 2003, 58, 801.
30.
biology & drug design 2011, 78, 816.
31.
32.
47, 6321.
33.
medicinal chemistry 2008, 15, 1083.
Meng, J.; Yu, S.; Wan, S.; Ren, S.; Jiang, T. Chemical
5.
Das, K.; Martinez, S. E.; Bauman, J. D.; Arnold, E.
Nature structural & molecular biology 2012, 19, 253.
Behbehani, H.; Ibrahim, H. M. Chem Cent J 2013, 7.
Harte, A. J.; Gunnlaugsson, T. Tetrahedron Lett 2006,
6. Rittinger, K.; Divita, G.; Goody, R. S. Proceedings of
the National Academy of Sciences of the United States of
America 1995, 92, 8046.
Husain, A. Asian J Chem 2005, 17, 2055.
Balzarini, J.; Karlsson, A.; Perez-Perez, M. J.; Vrang,
7.
Spence, R. A.; Kati, W. M.; Anderson, K. S.; Johnson,
34.
K. A. Science 1995, 267, 988.
L.; Walbers, J.; Zhang, H.; Oberg, B.; Vandamme, A. M.;
Camarasa, M. J.; De Clercq, E. Virology 1993, 192, 246.
8.
Rawal, R. K.; Murugesan, V.; Katti, S. B. Current
medicinal chemistry 2012, 19, 5364.
35.
Auwerx, J.; North, T. W.; Preston, B. D.; Klarmann, G.
9.
Prajapati, D. G.; Ramajayam, R.; Yadav, M. R.;
J.; De Clercq, E.; Balzarini, J. Molecular pharmacology 2002,
61, 400.
Giridhar, R. Bioorganic & medicinal chemistry 2009, 17, 5744.
10.
Das, K.; Arnold, E. Current opinion in virology 2013, 3,
111.