113
4 DNA Dependent DNA Polymerases as Targets for Low-Weight …
[110]. Some of these compounds demonstrated broad-spectrum inhibition of the
herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases. DHTPs, in contrast to the kinetics determined
for the 4-oxo-DHQs, proved to be competitive inhibitors of dTTP incorporation into
primer template by HCMV DNA polymerase [108].
However, as we mentioned above, the majority of non-nucleotide inhibitors
influence the activity of eukaryotic DNA-dependent pols. Despite the significant
number of known ones, search and development of new selective NNI is continuing up to now. For instance, inhibitors of several DNA polymerases were found
among flavonoide derivates. Shiomi et al. [88] investigated the inhibitory activities of 16 major bioflavonoids against mammalian DNA polymerases. Myricetin
(3,3′,4′,5,5′,7-hexahydroxyflavone) was the most potent inhibitor of pols among
the compounds tested, with IC 50 values of 21.3–40.9 μM. This compound did not
affect the activities of plant (cauliflower) pol α or prokaryotic pols. Myricetin also
inhibited human DNA topoisomerase II (topo II) activity with an IC 50 value of
27.5 μM, but did not inhibit the activities of other DNA metabolic enzymes tested
[88]. Myricetin also did not influence the direct binding to double stranded DNA as
determined by the thermal transition analysis. It was found to prevent the proliferation of human colon HCT116 carcinoma cells with an LD 50 of 28.2 μM, halt the cell
cycle in G2/M phase, and induce apoptosis. These results suggest that the decrease
of proliferation may be a result of the inhibition of cellular topoisomerase (topo) II
rather than pols [88].
Significant inhibitory effectiveness against representatives of B and Y pol families is demonstrated for natural plant gallotannin penta-O-galloyl-beta-d-glucose
(PGG) that has been shown to inhibit the in vivo growth of several types of tumors
without evident adverse side effects [111–113].
PGG exhibits a selective inhibition against the activities of pol α and pol κ in
nanomolar concentrations. The inhibitory effect of PGG on pol α is the strongest
among known low-weight inhibitors, with IC 50 value of 13 nM. PGG activity
against pol κ is slightly less—IC 50 in this case is 30 nM [94]. PGG is also able to inhibit pol β, but its potency is an order of magnitude less than that against pol α—the
corresponding value of IC 50 is in the range of 108–160 nM. PGG inhibition of pol α
and κ activity is non-competitive with respect to the DNA template-primer and the
dNTP substrate; in contrast to the inhibition of pol β activity which is competitive
[94]. The structural model of ‘pol β–PGG’ interaction is also proposed (see the next
chapter for details). PGG seems to be a very promising compound for fundamental
investigations (distinct inhibitory mechanisms for different pol families) as well as
for practical application (potential anticancer drug).
4.4 Structural Analysis of Pol–Inhibitor Interactions
There is essential difference between analytical approaches suitable for the analysis
of structural mechanisms/features of DNA polymerase interactions with nucleoside
and non-nucleoside inhibitors. Being the analogs of incoming dNTP, the nucleoside
4 DNA Dependent DNA Polymerases as Targets for Low-Weight …
[110]. Some of these compounds demonstrated broad-spectrum inhibition of the
herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases. DHTPs, in contrast to the kinetics determined
for the 4-oxo-DHQs, proved to be competitive inhibitors of dTTP incorporation into
primer template by HCMV DNA polymerase [108].
However, as we mentioned above, the majority of non-nucleotide inhibitors
influence the activity of eukaryotic DNA-dependent pols. Despite the significant
number of known ones, search and development of new selective NNI is continuing up to now. For instance, inhibitors of several DNA polymerases were found
among flavonoide derivates. Shiomi et al. [88] investigated the inhibitory activities of 16 major bioflavonoids against mammalian DNA polymerases. Myricetin
(3,3′,4′,5,5′,7-hexahydroxyflavone) was the most potent inhibitor of pols among
the compounds tested, with IC 50 values of 21.3–40.9 μM. This compound did not
affect the activities of plant (cauliflower) pol α or prokaryotic pols. Myricetin also
inhibited human DNA topoisomerase II (topo II) activity with an IC 50 value of
27.5 μM, but did not inhibit the activities of other DNA metabolic enzymes tested
[88]. Myricetin also did not influence the direct binding to double stranded DNA as
determined by the thermal transition analysis. It was found to prevent the proliferation of human colon HCT116 carcinoma cells with an LD 50 of 28.2 μM, halt the cell
cycle in G2/M phase, and induce apoptosis. These results suggest that the decrease
of proliferation may be a result of the inhibition of cellular topoisomerase (topo) II
rather than pols [88].
Significant inhibitory effectiveness against representatives of B and Y pol families is demonstrated for natural plant gallotannin penta-O-galloyl-beta-d-glucose
(PGG) that has been shown to inhibit the in vivo growth of several types of tumors
without evident adverse side effects [111–113].
PGG exhibits a selective inhibition against the activities of pol α and pol κ in
nanomolar concentrations. The inhibitory effect of PGG on pol α is the strongest
among known low-weight inhibitors, with IC 50 value of 13 nM. PGG activity
against pol κ is slightly less—IC 50 in this case is 30 nM [94]. PGG is also able to inhibit pol β, but its potency is an order of magnitude less than that against pol α—the
corresponding value of IC 50 is in the range of 108–160 nM. PGG inhibition of pol α
and κ activity is non-competitive with respect to the DNA template-primer and the
dNTP substrate; in contrast to the inhibition of pol β activity which is competitive
[94]. The structural model of ‘pol β–PGG’ interaction is also proposed (see the next
chapter for details). PGG seems to be a very promising compound for fundamental
investigations (distinct inhibitory mechanisms for different pol families) as well as
for practical application (potential anticancer drug).
4.4 Structural Analysis of Pol–Inhibitor Interactions
There is essential difference between analytical approaches suitable for the analysis
of structural mechanisms/features of DNA polymerase interactions with nucleoside
and non-nucleoside inhibitors. Being the analogs of incoming dNTP, the nucleoside