Table 1. Activity data of compounds against enzymes.
IC50 a (M)
16. Maes, D.; Debenedetti, S.; De Kimpe, N. Biochem. Syst. Ecol.
2006, 34, 165.
17. Alarcóna, R.; Pacciaroni, A.; Peñalozac, L.; Uriburuc, M.;
Boemoc, A.; Sosa, V. Biochem. Syst. Ecol. 2010, 38, 1059.
18. Experimental: PCR Assays. The assayed compounds were all
dissolved in DMSO. The PCR master mixture consisted of 40 mM
Trisacetate pH 8.3, 15 mM MgCl2, 2.5 U of Taq DNA polymerase
(Sigma-Aldrich), 20 mM each oligonucleotide primer, and 2.5
mM each desoxynucleotide triphosphate (dNTP). Inhibition
studies were carried out with varying compound concentrations.
All PCRs were done in 20 L reaction volumes. The sequence of
the sense primer was 5´-TAG AGC GTG AGG TCG ACA C-3´,
and the antisense primer, 5´ TCA AGT TAG ACG TGG CCG TC
3´. Thermocycling conditions consisted of 35 cycles of
denaturation at 95 °C for 1 min followed by primer annealing at
56 °C and primer extension at 72 °C for 90 seg.
Compound
Taq DNA pol
48.08 ± 9.26
57.88 ± 1.26
No activity
RT-MMLV
b
13
14
20
28
b
38.62 ± 3.25
No activity
50.98 ± 1.79
a Concentration that inhibits enzyme activity by 50%.
b It cannot be determinate
In summary, we have isolated four natural coumarins from P.
virgatum and P. alopecuroides and prepare twenty semi-synthetic
analogues besides commercial drugs. Their inhibitory activity
against Taq DNA polymerase and MMLV-RT was evaluated and
two molecules (compound 13 and 14) with similar structures
showed inhibitory activity against Taq DNA polymerase. Also,
coumarin (20) and compound 28 were able to inhibit the reverse
transcriptase with interesting values of activity. Thus, novel leads
from these coumarins can be further developed into potential
chemotherapeutic agents in antiviral treatment.
RT-PCR Assays. The inverse transcription process consisted of
1g RNA dissolved in 4L of commercial buffer and 2.0 U of
MMLV-RT (Sigma-Aldrich), 20 Random Primers, and 1.0
mM each desoxynucleotide triphosphate (dNTP). Inhibition
studies were carried out with varying compound concentrations
and adding compounds in this stage. All RT- PCRs were done in
6.8 L reaction volumes. Reaction conditions consisted of heating
at 70 °C for 10 min and incubating 1h at 56 °C.
Analysis of PCR Products. Relative intensities of ethidium
bromide stained PCR products were analyzed by using the optical
scanner and the image program. The image of stained agarose gel
was captured using a photography camera Kodak 2320 and then
was scanned (Hewlett-Packard 3200 C). The digitized band
images were processed using the Image processing program
(Scion Image, public domain program); IC50 values were
determined by the GraphPad Prism program.
Acknowledgements
This research was supported by CONICET (PIP 00628),
UNSL (Project 7301 and 2-1412) and ANPCyT (PICT-10714,
9759). H.A.G. thank CONICET for post-doctoral fellowship. We
wish to thank to Lic. Ferrari, Lic. Mascotti, Dr. Ardanáz and Dr.
Rossomando for their help. C.R.P., G.M.C and C.E.T. belong to
CIC-CONICET. This work is a part of the doctoral thesis of
H.A.G.
19. Kumar, V.; Mahajana, A.; Chibale, K. Bioorg. Med. Chem. 2009,
17, 2236.
20. Romanenko, V.; Kukhar, V. Tetrahedron. 2008, 64, 6153.
21. Spectroscopy data for natural compounds 1-4 and active tested
compounds 13, 14, 20, 28.
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