Inhibitors Of Mammalian DNA Polymerases Research Articles

Synthesis and absolute configuration of formosusin A, a specific inhibitor of mammalian DNA polymerase β

Formosusin A, a specific inhibitor of an X-family DNA polymerase (rat DNA polymerase β), was synthesized using a Sharpless asymmetric dihydroxylation as the key step for the preparation of a chiral building block. This Letter describes the first example of the total synthesis of formosusin A. The absolute configuration of the natural formosusin A was determined to be (R) from the specific rotation value. Racemization of formosusin A was observed under acidic conditions, suggesting that the reported natural product might have low optical purity.

Screening of Mammalian DNA Polymerase Inhibitors from Rosemary Leaves and Analysis of the Anti-inflammatory and Antiallergic Effects of the Isolated Compounds

In this study, on screening mammalian DNA polymerase (pol) inhibitors from the extracts of 20 edible plants, we found that leaves of rosemary (Rosmarinus officinalis L.) showed the strongest pol inhibition; furthermore, we isolated four components, carnosic acid, carnosol, ursolic acid, and rosmarinic acid, from an extract of the leaves. Carnosic acid inhibited the activities of all 11 mammalian pols tested. In descending order of their inhibitory effect on pol λ, which is a DNA repair/recombination pol, the four compounds ranked as follows: carnosic acid > ursolic acid > carnosol > rosmarinic acid. The inhibition of pol λ by these compounds was significantly correlated with both in vivo anti-inflammatory and antiallergic effects, including on 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, and immunoglobulin E-induced passive cutaneous anaphylactic reaction in mice. These results indicate that rosemary and its constituent, carnosic acid, are potential therapeutic food candidates for inflammatory and allergic diseases.

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 35.6μM. 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.

Dehydroaltenusin is a specific inhibitor of mammalian DNA polymerase α

Introduction:We carried out a screen for small molecule selective inhibitors of eukaryotic DNA polymerases (pols). Dehydroaltenusin, isolated from a fungus (Alternaria tenuis), was found to be a specific inhibitor of pol α.Areas covered:We succeeded in chemically synthesizing dehydroaltenusin along with five analogs. Of these compounds, dehydroaltenusin was the strongest and most specific inhibitor of mammalian pol α, with an IC50 value of 0.68 μM. The inhibitory mode of action of dehydroaltenusin against mammalian pol α activity was competitive with respect to the DNA template primer and non-competitive with respect to the 2′-deoxyribonucleoside 5′-triphosphate substrate. Dehydroaltenusin inhibited the cell proliferation of a human cervical cancer cell line, HeLa, by arresting the cells at the S-phase, and preventing the incorporation of thymidine into the cells. These observations indicate that dehydroaltenusin blocks in vivo DNA replication by inhibiting pol α.Expert opinion:Dehydroaltenusin was effective in suppressing the growth of solid tumors and, therefore, is of interest as a candidate drug for anti-cancer treatment.

Syntheses of Dehydroaltenusin, a Selective Inhibitor of Mammalian DNA Polymerase α

Dehydroaltenusin has been isolated from Alternaria tenuis and other fungal species. It exhibits various biological activities such as growth inhibition of wood-damaging fungi, inhibition of calmodulin-dependent myosin light chain kinase, anti-HIV activity, and inhibition of mammalian DNA polymerase α (pol α). These significant biological activities, as well as the unique structural properties of dehydroaltenusin, have stimulated considerable interest in its synthesis. In this review, we summarize the synthesis and structural analysis of dehydroaltenusin and its derivatives. We also focus on the structure-activity relationships of dehydroaltenusin as a mammalian pol α inhibitor. Dehydroaltenusin specifically inhibits mammalian pol α, an enzyme that plays an important role in DNA replication and cell division. The human genome encodes 14 pols that control cellular DNA synthesis, and synthetic dehydroaltenusin derivatives, which are specific inhibitors of mammalian pol α, are useful tools and molecular probes for distinguishing pols and for clarifying their biological and in vivo functions. Moreover, these compounds might be potentially useful as anti-cancer chemotherapeutic drugs for in vivo studies.

Structure and activity relationship of monogalactosyl diacylglycerols, which selectively inhibited in vitro mammalian replicative DNA polymerase activity and human cancer cell growth

The glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), containing two α-linolenic acids (C18:3), was isolated from bitter melon as a potent and selective inhibitor of mammalian DNA polymerase (pol) species such as pols α, γ, δ, and ε with IC 50 values of 17.6–37.2 μM. This MGDG also suppressed the growth of six human cancer cell lines, although normal human cell lines were not affected. This compound (i.e., MGDG-C18:3-C18:3) was a stronger inhibitor than both MGDG-C18:1-C18:0 and MGDG-C18:0-C18:0. In this study, we discussed the structure–function relationship in the selective inhibition of mammalian replicative pols and human cancer cell proliferation by MGDGs.

Effect of Dehydroaltenusin-C12 Derivative, a Selective DNA Polymerase α Inhibitor, on DNA Replication in Cultured Cells

Dehydroaltenusin is a selective inhibitor of mammalian DNA polymerase α (pol α) from a fungus (Alternaria tennuis). We have designed, synthesized, and characterized a derivative of dehydroaltenusin conjugated with a C12-alkyl side chain (dehydroaltenusin-C12 [C12]). C12 was the strongest pol α inhibitor in vitro. We introduced C12 into NIH3T3 cells with the help of a hypotonic shift, that is, a transient exposure of cultured cells in hypotonic buffer with small molecules which can not penetrate cells. The cells that took in C12 by hypotonic shift showed cell growth inhibition. At a low concentration (5 μM), DNA replication was inhibited and several large replication protein A (RPA) foci, which is different from dUTP foci. Furthermore, when C12 was incubated with aphidicolin, RPA foci were not observed in cells. Finally, these findings suggest that C12 inhibited DNA replication through pol α inhibition, and generated single-stranded DNA, resulted in uncoupling of the leading strand and lagging strand synthesis. These findings suggest that C12 could be more interesting as a molecule probe or anticancer agent than aphidicolin. C12 might provide novel markers for the development of antiproliferative drugs.

New Cancer Chemotherapy Agents: Inhibitors of DNA Polymerase

The purpose of this review is 1) to introduce our studies of newly-discovered mammalian DNA polymerase inhibitors; 2) to elucidate their precise molecular action based on three-dimensional structural models and comparisons with the spatial positioning on the smallest DNA polymerase, β, of specific amino acids binding to them; and 3) to document their promising clinical anti-tumor activity. Multiple DNA polymerases have been identified in eukaryotes, and recent investigations have revealed at least sixteen types. An effective approach to study the in vivo roles of each of these is to use selective DNA polymerase inhibitors to distinguish between them. Using a broad natural product screening approach, we have identified many different types of novel DNA polymerase inhibitor which we have exploited to analyze the structure and function of the DNA polymerases. The aim was not only to understand the function of each inhibitor in vitro, but also to develop a drug design strategy for cancer chemotherapy agents. We have found a class of such DNA polymerase inhibitors, the sulfoquinovosylacylglycerols (SQAG), which could represent clinically-promising anti-tumor agents. We discussed about the mechanism of the anti-tumor action, and suggested new concept about cancer chemotherapy.

Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol α from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol α with IC50 value of 0.5μM, and did not influence the activities of other replicative pols such as pols δ and ε, but also showed no effect on pol α activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD50 values of 38.0–44.4μM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol α-specific inhibitor, but also as a candidate drug for anti-cancer treatment.

The In Vitro and In Vivo Inhibitory Effect of Dehydroaltenusin: A Specific Inhibitor of Mammalian DNA Polymerase α

In the screening of the selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol from a fungus (Alternaria tennuis). This compound inhibited only mammalian polα, and did not influence the activities of other replicative pols such as polsαs and Ƹ , but also showed no effect even on polαactivity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence DNA metabolic enzymes tested. The inhibitory effect of dehydroaltenusin on mammalian polαwas dose-dependent with IC50 value of 0.5 ?M. This effect was 10-fold stronger than that of aphidicolin, a well-known potent eukaryotic polαinhibitor. The inhibitory mode of dehydroaltenusin for mammalian polαactivity was competitive with the DNA template-primer and noncompetitive with the dNTP substrate. We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited the cell proliferation of human gastric cancer cell lines by arresting the cells at the S-phase, and preventing the incorporation of thymidine into the cells, indicating that it blocks in vivo DNA replication by inhibiting polα. This compound also induced cell apoptosis. Furthermore, we investigated in vivo anti-tumor effects on nude mice bearing solid tumors of the human cervical cancer cell line HeLa. Dehydroaltenusin was significantly effective in suppressing the growth of solid tumors, therefore, it was of interest as a candidate drug for anti-cancer treatment. These results suggested that dehydroaltenusin is a mammalian polα-specific inhibitor useful in both in vivo and in vitro experiments.

Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase β

We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data.

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 α and λ with IC 50 values of 5.1 and 3.8 μM, respectively. EGCg did not influence the activities of plant (cauliflower) pol α and β 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 α and λ 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 μg 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.

Mammalian mitotic centromere‐associated kinesin (MCAK)

Sulfoquinovosylacylglycerols (SQAGs), in particular compounds with C18 fatty acid(s) on the glycerol moiety, may be clinically promising antitumor and/or immunosuppressive agents. They were found originally as inhibitors of mammalian DNA polymerases. However, SQAGs can arrest cultured mammalian cells not only at S phase but also at M phase, suggesting they have several molecular targets. A screen for candidate target molecules using a T7 phage display method identified an amino acid sequence. An homology search showed this to be a mammalian mitotic centromere-associated kinesin (MCAK), rather than a DNA polymerase. Analyses showed SQAGs bound to recombinant MCAK with a K(D)=3.1x10(-4) to 6.2x10-5 M. An in vivo microtubule depolymerization assay, using EGFP-full length MCAK fusion constructs, indicated inhibition of the microtubule depolymerization activity of MCAK. From these results, we conclude that clinically promising SQAGs have at least two different molecular targets, DNA polymerases and MCAK. It should be stressed that inhibitors of MCAK have never been reported previously so that there is a major potential for clinical utility.

Stereoselective synthesis of procyanidin B3-3- O-gallate and 3,3″-di- O-gallate, and their abilities as antioxidant and DNA polymerase inhibitor

A simple method for the synthesis of procyanidin B3 substituted with a galloyl group at the 3 and 3″ position is described. Condensation of a benzylated catechin-3- O-gallate electrophile with a nucleophile, catechin and catechin-3- O-gallate, proceeded smoothly and stereoselectively to afford the corresponding dimer gallates, procyanidin B3-3- O-gallate and procyanidin B3-3,3″-di- O-gallate, in good yields. Further, their antioxidant activities on UV-induced lipid peroxide formation, DPPH radical scavenging activity and inhibitory activity of DNA polymerase were also investigated. Among three procyanidin B3 congeners (procyanidin B3, 3- O-gallate and 3,3″-di- O-gallate), the 3,3″-di- O-gallate derivative showed the strongest antioxidant and radical scavenging activity. Interestingly, the 3- O-gallate derivative was the strongest inhibitor of mammalian DNA polymerase α with IC 50 value of 0.26 μM, although it showed the weakest antioxidant and radical scavenging activity. It became apparent that the presence of a galloyl group at the C-3 position in the proanthocyanidin oligomer was very important for biological activity, however, the antioxidant activity of these compounds was not parallel to the DNA polymerase inhibitory activity.

Precise structural elucidation of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

The X-ray crystal structure of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α, has previously been reported. We show that dehydroaltenusin exists in an equilibrium mixture of two tautomers possessing γ-lactone or δ-lactone in polar solvents by NMR experiments. Acetylation of dehydroaltenusin afforded two types of diacetates and two types of monoacetate, possessing γ-lactone or δ-lactone, respectively. The inhibitory activities of these acetate derivatives against DNA polymerase α were all much weaker than that of dehydroaltenusin.

Taxol derivatives are selective inhibitors of DNA polymerase α

During screening for mammalian DNA polymerase inhibitors, we found and succeeded in isolating a potent inhibitor from a higher plant, Taxus cuspidate. The compound was unexpectedly determined to be taxinine, an intermediate of paclitaxel (taxol) metabolism. Taxinine was found to selectively inhibit DNA polymerase α (pol.α) and β (pol.β). We therefore, tested taxol and other derivatives and found that taxol itself had no such inhibitory effect, and only taxinine could inhibit both pol.α and β. The other compounds used, one derivative, cephalomannine, and five intermediates synthesized chemically inhibited only the pol.α activity in vitro. None of the compounds, including taxinine, influenced the activities of the other DNA polymerases, which are reportedly targeted by many pol.β inhibitors. With both pol.α and β, all of the compounds tested noncompetitively inhibited with respect to both the DNA template-primer and the dNTP substrate.

Mechanism of cell cycle arrest by sulfoquinovosyl monoacylglycerol with a C18-saturated fatty acid (C18-SQMG)

We have screened the inhibitors of mammalian DNA polymerases from natural products, and in the process found that either sulfoglycolipids or sulfoquinovosyl monoacylglycerol with a C18-saturated fatty acid (C18-SQMG), potently and selectively inhibited the activity of mammalian DNA polymerase (pol) ε and moderately the pol α [Biochem. J. 370 (2003) 299]. C18-SQMG was a cancer cell growth suppressor and a promissive anti-tumor agent [Jpn. J. Cancer Res. 93 (2002) 85]. The purpose of this study was to elucidate the cell growth inhibition mechanism of C18-SQMG using HeLa cells. Analyses of the cell cycle and cyclin expression suggested that C18-SQMG arrested the cell cycle at intra-S phase, and the inhibition manner of DNA replication by C18-SQMG was similar to that by hydroxyurea. However, the DNA replication block by C18-SQMG did not induce degradation of Cdc25A protein, which was required for the replication block by hydroxyurea. C18-SQMG somewhat delayed mitosis because it induced phosphorylation of protein kinases, such as checkpoint kinases 1 and 2. These results suggest that C18-SQMG at first blocked DNA replication at the S phase by inhibiting replicative DNA polymerases, such as α, and then as the result of the inhibition, the other checkpoint signals associated with the pol ε might have responded.

Inhibitory action of emulsified sulfoquinovosyl acylglycerol on mammalian DNA polymerases.

We reported previously that sulfoquinovosyl diacylglycerol and sulfoquinovosyl monoacylglycerol (SQDG/SQMG) are potent inhibitors of mammalian DNA polymerases and DNA topoisomerase II, and can be potent immunosuppressive agents and anticancer chemotherapy agents [Matsumoto, Y., Sahara, H., Fujita T., Shimozawa, K., Takenouchi, M., Torigoe, T., Hanashima, S., Yamazaki, T., Takahashi, S., Sugawara, F., et al., An Immunosuppressive Effect by Synthetic Sulfonolipids Deduced from Sulfonoquinovosyl Diacylglycerols of Sea Urchin, Transplantation 74, 261-267 (2002); Sahara, H., Hanashima, S., Yamazaki, T., Takahashi, S., Sugawara, F., Ohtani, S., Ishikawa, M., Mizushina, Y., Ohta, K., Shimozawa, K., et al., Anti-tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols, Jpn. J. Cancer Res. 93, 85-92 (2002)]. In those experiments, the in vivo effectiveness greatly depended on the degree of water solubility of SQDG/SQMG. In the present work, we studied the emulsification of SQDG/SQMG in terms of their use in in vivo experiments. Lipid emulsions containing SQDG/SQMG (oil-in-water emulsions) in which the particle size was smaller than 100 nm were designed and synthesized, and then the biochemical modes of emulsified SQDG/SQMG were studied in comparison with those of SQDG/SQMG solubilized by DMSO. Emulsified SQDG/SQMG are also selective mammalian DNA polymerase inhibitors and potent antineoplastic agents but do not inhibit the DNA topoisomerase II activity. The growth inhibition effect of emulsified SQMG to NUGC-3 cancer cells was twofold stronger than DMSO-soluble SQMG (69 and 151 microM, respectively). From these results, the properties of lipid emulsions containing SQDG/SQMG and their possible use in in vivo experiments including clinical use are discussed.

Some anti-chronic inflammatory compounds are DNA polymerase λ-specific inhibitors

We previously reported that a phenolic compound, petasiphenol, was a selective inhibitor of DNA polymerase λ (pol λ) in vitro [Biochemistry 41 (2002) 14463]. We found here that another phenolic compound, curcumin (diferuloylmethane), which is known as an anti-chronic inflammatory agent and is structurally quite similar to petasiphenol, was also a potent pol λ inhibitor. The ic 50 values of petasiphenol and curcumin were 7.8 and 7.0 μM, respectively. Curcumin, as well as petasiphenol, did not influence the activities of replicative DNA polymerases, such as α, γ, δ, and ε, but also showed no effect even on the pol β activity belonging to the X family. Curcumin could prevent the growth of human NUGC-3 cancer cells with ld 50 values of 13 μM, and halted them at the G2/M phase in the cell cycle, whereas petasiphenol suppressed the cell growth at 66 μM and arrested the cells at the G1 phase. These data showed that curcumin and petasiphenol were slightly different functionally. We also previously reported that novel anti-inflammatory terpeno benzoic acids and triterpenoids were inhibitors of mammalian DNA polymerases [Biochem. Biophys. Acta 1475 (2000) 1; Biochem. Biophys. Acta 1596 (2002) 193]. They could also efficiently inhibit the pol λ activity, although they influenced the other polymerase species to the same extent, suggesting that there may be a physiological relationship between pol λ inhibition and anti-12- O-tetradecanoylphorbol-13-acetate-induced inflammation. Expectedly, petasiphenol also showed an anti-12- O-tetradecanoylphorbol-13-acetate-induced inflammatory effect in mice. This finding may provide clues to investigating the molecular mechanism of inflammation.

Structure–function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian DNA polymerase inhibitors

We reported previously that sulfo-glycolipids such as sulfoquinovosyl-diacylglycerol (SQDG) and sulfoquinovosyl-monoacylglycerol (SQMG) are potent inhibitors of DNA polymerase α and β and antineoplastic agents. Then, we succeeded in synthesizing SQDG and SQMG chemically, including their stereoisomers, glucopyranosyl-diacylglycerol (GDG) and glucopyranosyl-monoacylglycerol (GMG). In this study, we demonstrated the structure–function relationship of the synthetic sulfo-glycolipids to DNA polymerase α and β and their relationship to the cytotoxic activity. Both SQDG and SQMG inhibited the activity of mammalian DNA polymerase α with IC 50 values of 3–5 μM, but GMG only moderately inhibited it. GDG, diacylglycerol (DG), and monoacylglycerol (MG) did not influence any of the DNA polymerase activities. The sulfate moiety in the quinovose was important in inhibiting the enzyme activity. The one-fatty-acid-sulfo-glycolipids, SQMG, GMG, and MG, prevented the growth of NUGC-3 human gastric cancer cells and induced apoptotic cell death, but the two-fatty-acid-sulfo-glycolipids, SQDG, GDG, and DG, did not. SQMG and GMG could halt the cell cycle at the G1 phase, but the cell cycle was not changed by MG. The relationship between the DNA polymerase inhibition and the cell growth effect by these compounds are discussed.