5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase lambda and terminal deoxynucleotidyltransferase

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee (Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as alpha, delta, and epsilon, nor even the activity of pol beta which is from the same family and thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol lambda and TdT. The inhibitory effect of HMF on intact pol lambda (i.e., residues 1-575), a truncated pol lambda lacking the N-terminal BRCA1 C-terminus domain (133-575, del-1 pol lambda) and another truncated pol lambda lacking the N-terminal proline-rich region (245-575, del-2 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 microM, respectively. The IC(50) value of HMF for TdT was the same as that for del-2 pol lambda (5.5 microM). The HMF-induced inhibition of both pol lambda and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol beta-like region of pol lambda and TdT.     

Inhibitory effect of tocotrienol on eukaryotic DNA polymerase lambda and angiogenesis

Tocotrienols, vitamin E compounds that have an unsaturated side chain with three double bonds, selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. These compounds did not influence the activities of replicative pols such as alpha, delta, and epsilon, or even the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since delta-tocotrienol had the strongest inhibitory effect among the four (alpha- to delta-) tocotrienols, the isomer's structure might be an important factor in the inhibition of pol lambda. The inhibitory effect of delta-tocotrienol on both intact pol lambda (residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (residues 133-575, del-1 pol lambda) was dose-dependent, with 50% inhibition observed at a concentration of 18.4 and 90.1microM, respectively. However, del-2 pol lambda (residues 245-575) containing the C-terminal pol beta-like region was unaffected. Tocotrienols also inhibited the proliferation of and formation of tubes by bovine aortic endothelial cells, with delta-tocotrienol having the greatest effect. These results indicated that tocotrienols targeted both pol lambda and angiogenesis as anti-cancer agents. The relationship between the inhibition of pol lambda and anti-angiogenesis by delta-tocotrienol was discussed.     

Hymenoic acid, a novel specific inhibitor of human DNA polymerase lambda from a fungus of Hymenochaetaceae sp

Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1′E,5′S)-5′-carboxy-1′-methyl-1′-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase λ (pol λ) in vitro, and 50% inhibition was observed at a concentration of 91.7 μM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols , γ, δ, ε, η, ι, and κ), but also showed no effect even on the activity of pol β, which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol λ. This compound had no inhibitory activities against two N-terminal truncated pol λ, del-1 pol λ (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133–575]), and del-2 pol λ (lacking NLS, BRCT, domain and proline-rich region [residues 245–575]). The compound 1-induced inhibition of intact pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.     

Petasiphenol: a DNA polymerase lambda inhibitor

Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicus, selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta, and epsilon but also showed no effect even on the pol beta activity, the three-dimensional structure of which is thought to be highly similar to pol lambda. The inhibitory effect of petasiphenol on intact pol lambda including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8 microM. The petasiphenol-induced inhibition of the pol lambda activity was noncompetitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol lambda including the pol beta-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol lambda but did not bind to the C-terminal region. On the basis of these results, the pol lambda inhibitory mechanism of petasiphenol is discussed.     

Cholesterol hemisuccinate: a selective inhibitor of family X DNA polymerases

Cholesterol hemisuccinate (compound 5), which consists of succinic acid esterified to the beta-hydroxyl group of cholesterol, selectively and strongly inhibited the activities of mammalian DNA polymerases (pols) such as pol beta, pol lambda, and terminal deoxynucleotidyltransferase (TdT), which are family X pols, in vitro, and the IC50 values were 2.9, 6.3, and 6.5 microM, respectively. The compound moderately suppressed the activities of other mammalian pols such as pol A (i.e., pol gamma), pol B (i.e., pols alpha, delta, and epsilon), and pol Y (i.e., pols iota, eta, and kappa) with 50% inhibition observed at concentrations of 131, 89.2-98.0, and 120-125 microM, respectively. The compound had no influence on the activities of plant pols alpha and beta, prokaryotic pols and other DNA metabolic enzymes tested. Since other cholesterol-related compounds such as cholesterol, cholesteryl chloride, cholesteryl bromide, cholesteryl acetate, and cholesteryl-5alpha, 6alpha-epoxide (compounds 1-4 and 6, respectively) did not influence the activities of any enzymes tested, the hemisuccinate group of compound 5 could be important for inhibition of the pol X family. Surface plasmon resonance analysis demonstrated that compound 5 bound selectively to the C-terminal 31 kDa domain of pol beta and pol lambda containing a pol beta-like region. On the basis of these results, the inhibitory mechanism of compound 5 on the pol X family was discussed.     

Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors

The inhibitory activities against DNA polymerases (pols) of catechin derivatives (i.e., flavan-3-ols) such as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate (EGCg) were investigated. Among the eight catechins, some catechins inhibited mammalian pols, with EGCg being the strongest inhibitor of pol alpha and lambda with IC(50) values of 5.1 and 3.8 microM, respectively. EGCg did not influence the activities of plant (cauliflower) pol alpha and beta or prokaryotic pols, and further had no effect on the activities of DNA metabolic enzymes such as calf terminal deoxynucleotidyl transferase, T7 RNA polymerase, and bovine deoxyribonuclease I. EGCg-induced inhibition of pol alpha and lambda was competitive with respect to the DNA template-primer and non-competitive with respect to the dNTP (2'-deoxyribonucleotide 5'-triphosphate) substrate. Tea catechins also suppressed TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity was the same as that of anti-inflammation. EGCg at 250 microg was the strongest suppressor of inflammation (65.6% inhibition) among the compounds tested. The relationship between the structure of tea catechins and the inhibition of mammalian pols and inflammation was discussed.     

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.     

Inhibitory effect of novel 5-O-acyl juglones on mammalian DNA polymerase activity, cancer cell growth and inflammatory response

We previously found that vitamin K(3) (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on juglone (5-hydroxy-1,4-naphthoquinone), which is a 1,4-naphthoquinone derivative, and chemically synthesized novel juglones conjugated with C2:0 to C22:6 fatty acid (5-O-acyl juglones). The chemically modified juglones enhanced mammalian pol inhibition and their cytotoxic and anti-inflammatory activities. The juglone conjugated with oleic acid (C18:1-acyl juglone) showed the strongest inhibition of DNA replicative pol α activity and human colon carcinoma (HCT116) cell growth in 10 synthesized 5-O-acyl juglones. C12:0-Acyl juglone was the strongest inhibitor of DNA repair-related pol λ, as well as the strongest suppression of the production of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS) in the compounds tested. Moreover, this compound caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ears. C12:0- and C18:1-Acyl juglones selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. These data indicate that the novel 5-O-acyl juglones target anti-cancer and/or anti-inflammatory agents based on mammalian pol inhibition. Moreover, the results suggest that acylation of juglone is an effective chemical modification to improve the anti-cancer and anti-inflammation of vitamin K(3) derivatives, such as juglone.     

Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol alpha) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol alpha. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as delta and epsilon; it also showed no effect even on the pol alpha activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 microm. Dehydroaltenusin-induced inhibition of mammalian pol alpha activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol alpha, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol alpha. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol alpha, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.     

Inhibitory effect of mispyric acid on mammalian DNA polymerases

Mispyric acid is a novel natural triterpene dicarboxylic acid which has inhibitory activity against DNA polymerase beta (pol beta) isolated from the plant, Mischocarpus pyriformis. In this report, we examine the selectivity of the inhibitory activity against mammalian pols and the mode of inhibition in vitro. Natural mispyric acid (compound 1) inhibited the activities of all the mammalian pols tested (pol alpha, beta, gamma, delta and epsilon) with an IC50 value in the range of 3.6-44.5 microM. The inhibition was strongest for pol gamma among these five pols. The enantiomer of mispyric acid (compound 2, ent-mispyric acid) had similar effects to those of the natural compound. However, derivatives of compounds 1 and 2 with hydroxyl groups instead of carboxyl groups (i.e., compounds 3 and 4, respectively) exhibited no inhibitory effect on mammalian pols. The moiety of two carboxylic acids in mispyric acid was important for the inhibition of pols, and the stereoisomers of mispyric acid had no inhibitory effect.     

Formosusin A,a novel specific inhibitor of mammalian DNA polymerase β from the fungus Paecilomyces formosus

Variotin (1) and three novel compounds, formosusin A (2), B (3), and C (4), were isolated from the cultures of the fungus Paecilomyces formosus, and their structures were determined by spectroscopic analyses. Compound 2 is (6Z,8E,10E)-variotin, a new cis-olefin analog of compound 1. Compound 2 selectively inhibited the activity of mammalian DNA polymerase β (pol β) in vitro, with an IC₅₀ of 35.6 μM. By contrast, compounds 1, 3, and 4 did not influence the activity of pol β. These four compounds showed no effect on the activities of other 10 mammalian pols (i.e., pols α, γ, δ, ε, η, ι, κ, λ, and μ, and terminal deoxynucleotidyl transferase). These compounds also did not inhibit the activities of fish, insect, plant, and prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 2 could be a selective inhibitor of mammalian pol β. The compound 2-induced inhibition of rat pol β activity was competitive and non-competitive with respect to the DNA template–primer substrate and the dNTP substrate, respectively. On the basis of these results, the relationship between the three-dimensional structure and pol β inhibitory mechanism of compound 2 is discussed.     

DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.