Anti-cancer gallotannin penta-O-galloyl-beta-D-glucose is a nanomolar inhibitor of select mammalian DNA polymerases

Abstract

Penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG) has been shown by us and others to inhibit the in vivo growth of human prostate cancer (PCa) xenografts in athymic nude mice and mouse lung cancer allograft in syngenic mice without evident adverse effect on their body weight. We observed a rapid inhibition of DNA synthesis in S-phase cells in PGG-exposed cancer cells and in PGG-treated isolated nuclei. The purpose of the present study was to test the hypothesis that PGG inhibits DNA replicative synthesis through a direct inhibition of one or more DNA polymerases (pols). Using purified pols, we show that PGG exhibited a selective inhibition against the activities of B-family replicative pols (alpha, delta and epsilon) and Y-family (eta, iota and kappa) of bypass synthesis pols, and the inhibitory effect of PGG on pol alpha was the strongest with IC(50) value of 13 nM. PGG also inhibited pol beta, but the potency was an order of magnitude less than against pol alpha. PGG inhibition of pol alpha and kappa activity was non-competitive with respect to the DNA template-primer and the dNTP substrate; whereas it inhibited pol beta competitively. Docking simulation on pol beta, which is the only mammalian pol with solved crystal structure, suggests several favorable interactions with the catalytic pocket/binding site for the incoming dNTP. These results support PGG as a novel inhibitor of select families of mammalian pols by distinct mechanisms, and suggest that the potent pol inhibition may contribute to its anti-cancer efficacy.

Fig. 1

(A) Chemical structure of PGG. (B) Scheme for DNA polymerase assays. (C) Differential inhibitory effects of PGG on the activities of various mammalian pols. 100 nM (gray bars) and 1,000 nM (black bars) of PGG were incubated with each enzyme (0.05 unit). Enzymatic activity was measured as described previously [17, 18]. Enzyme activity in the absence of the compounds was taken as 100%. Data are shown as the means ± SEM of four independent experiments. (D) Dose-response inhibition curves of PGG against mammalian pol α, κ and β. PGG was incubated with calf pol α (square) as a representative B family pols, mouse pol κ (triangle) as a representative Y family pols and rat pol β (circle) as a representative X family of pols (0.05 unit of each). (E) More refined dose–response curve for calf pol α to define IC₅₀ for PGG. Pol activity in the absence of the compounds was taken as 100 %. For D and E, data are shown as the mean ± SEM of three independent experiments.

Fig. 2

Kinetic analysis of the inhibition of calf thymus pol α by PGG. (A and B) Lineweaver–Burk double-reciprocal plots obtained by varying DNA template-primer (i.e., poly(dA)/oligo(dT)₁₈) concentrations (A), and dNTP substrate (i.e., dTTP) concentrations (B). (C and D) The inhibition constant (K_i) were determined as 2.5 and 2.2 nM from a Dixon plot made on the basis of the same data for A and B, respectively. The amount of pol α in the assay mixture was 0.05 unit.

Fig. 3

Kinetic analysis of the inhibition of rat pol β by PGG. (A and B) Lineweaver–Burk double-reciprocal plots obtained by varying DNA template-primer (i.e., poly(dA)/oligo(dT)₁₈) concentrations (A), and dNTP substrate (i.e., dTTP) concentrations (B). (C and D) The inhibition constant (K_i) were determined as 20.1 and 23.8 nM from a Dixon plot made on the basis of the same data for A and B, respectively. The amount of pol β in the assay mixture was 0.05 unit.

Fig. 4

(A) Schematic representation of pol β, pol λ and TdT of the X family of pols. The NLS (nuclear localization signal), BRCT (BRCA1 C-terminus) domain, proline-rich region, HhH (helix-hairpin-helix) and pol X motif are indicated. The pol β-like region includes two HhHs and a pol X motif. The inhibitory activity of PGG against these enzymes is indicated, “++” is an IC₅₀ value of < 200 nM, and “+” is an IC₅₀ value of 200 to 400 nM, based on data from C. (B) Inhibition dose-response curves of PGG against mammalian X pol family and deletion mutants. PGG was incubated with rat pol β (closed circle, ●), full length of human pol λ (closed square, ■), del-1 of human pol λ (open reverse-triangle, △), del-2 of human pol λ (open diamond, ◇), and calf TdT (closed triangle, ▲) (0.05 unit of each). Pol activity in the absence of PGG was taken as 100%. Data are shown as the means ± SEM of three independent experiments.

Fig. 5

Docking of PGG into human DNA pol β using a IBM Bluegene Supercomputer at the Hormel Institute.