3-O-methylfunicone, a selective inhibitor of mammalian Y-family DNA polymerases from an Australian sea salt fungal strain

Abstract

We isolated a pol inhibitor from the cultured mycelia extract of a fungal strain isolated from natural salt from a sea salt pan in Australia, which was identified as 3-O-methylfunicone by spectroscopic analyses. This compound selectively inhibited the activities of mammalian Y-family DNA polymerases (pols) (i.e., pols eta, iota and kappa). Among these pols, human pol kappa activity was most strongly inhibited, with an IC(50) value of 12.5 microM. On the other hand, the compound barely influenced the activities of the other families of mammalian pols, such as A-family (i.e., pol gamma), B-family (i.e., pols alpha, delta and epsilon) or X-family (i.e., pols beta, lambda and terminal deoxynucleotidyl transferase), and showed no effect on the activities of fish pol delta, plant pols, prokaryotic pols and other DNA metabolic enzymes, such as calf primase of pol alpha, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I. This compound also suppressed the growth of two cultured human cancer cell lines, HCT116 (colon carcinoma cells) and HeLa (cervix carcinoma cells), and UV-treated HeLa cells exhibited lower clonogenic survival in the presence of inhibitor.

Keywords: 3-O-methylfunicone; Australian sea salt; DNA polymerase κ; Y-family DNA polymerase; anti-cancer drug; enzyme inhibitor; marine fungal strains.

Figure 1.

Structure of compound 1 (3-O-methylfunicone).

Figure 2.

Effect of 3-O-methylfunicone (1) on the activities of various DNA polymerases and other DNA metabolic enzymes. (A) Mammalian pols, (B) fish, insect, plant and prokaryotic pols, and (C) other DNA metabolic enzymes. Compound 1 (100 μM) was incubated with each enzyme (0.05 units). % of relative activity. Enzymatic activities were measured as described in the Experimental section. Activities in the absence of the compounds were taken as 100%. Data are shown as the means ± SEM of four independent experiments.

Figure 3.

Mammalian Y-family DNA polymerase inhibition dose-response curves of 3-O-methylfunicone (1). Compound 1 was incubated with human pol η (square), mouse pol ι (triangle), and human pol κ (circle) (0.05 units of each). Pol activity was measured as described in the Experimental section. Activity in the absence of the compounds was taken as 100%. Data are shown as the means ± SEM of three independent experiments.

Figure 4.

Kinetic analysis of the inhibition of human pol κ by compound 1. (A) Human pol κ activity was measured in the absence (closed square) or presence of 4 μM (closed circle), 8 μM (closed triangle) or 12 μM (closed diamond) compound 1 using the indicated concentrations of the DNA template-primer. (B) Human pol κ activity was assayed with the indicated concentrations of the substrate dTTP in the presence of 4 μM (open circle), 8 μM (open triangle) or 12 μM (open diamond) or in the absence (open square) of compound 1. (C) and (D) The inhibition constants (K_i) were determined as 1.3 and 4.4 μM from a Dixon plot made on the basis of the same data for (A) and (B), respectively. Amount of human pol κ in the assay mixture was 0.05 units.

Figure 5.

(A) Human cancer cell growth inhibition dose-response curves of 3-O-methyl-funicone (1). Various concentrations (0 to 150 μM) of compound 1 were incubated with human colon carcinoma (HCT116) cells (circle) and human cervix carcinoma (HeLa) cells (square) for 24 h. Rate of cell growth was determined by MTT assay [44] as described in the Experimental section. Data are shown as the means ± SEM of five independent experiments. (B) Clonogenic survival of UV-treated HeLa cells by compound 1. Various concentrations (0 to 100 μM) of compound 1 were incubated with 10 J/m² UV-treated (closed circle) or untreated (open circle) HeLa cells. After 7 days, the number of colonies in each treatment group was counted. Values are the means ± SEM of two independent experiments.

Figure 6.

(Left) Isolation methods of marine fungal strains from Australian sea salt. (Right) Purification procedure of a compound with pol inhibitory activity from mycelia of “AF1-2” fungal strain.

Figure 7.

Assay method of pol inhibitor.

Figure 8.

Marine fungal strain AF1-2.