Fumio Sugawara

Synthesis of sulfoquinovosylacylglycerols, inhibitors of Eukaryotic DNA Polymerase α and β

Abstract Sulfoquinovosyldiacylglycerols (SQDGs) and sulfoquinovosylmonoacylglycerols (SQMGs), bearing diverse fatty acids, were synthesized from d -glucose, and were examined for enzymatic inhibitions of DNA polymerase α and β. These results indicated that the carbon numbers of the fatty acids were highly related to the activities, at least in vitro, of eukaryotic DNA polymerase inhibition.

Structure‐Activity Relationship of a Novel Group of Mammalian DNA Polymerase Inhibitors, Synthetic Sulfoquinovosylacylglycerols

We reported previously that sulfolipids in the sulfoquinovosylacylglycerol class from a fern and an alga are potent inhibitors of DNA polymerase α and β and potent anti‐neoplastic agents. In developing a procedure for chemical synthesis of sulfolipids, we synthesized many derivatives and stereoisomers of sulfoquinovosylmonoacylglycerol (SQMG)/sulfoquinovosyldiacylglycerol (SQDG). Some of these molecules were stronger inhibitors than the SQMG/SQDG originally reported as natural compounds. In this study, we examined the structure‐inhibitory function relationship of synthetic SQMG/SQDG and its relationship to cytotoxic activity. The inhibitory effect is probably mainly dependent on the fatty acid effect, which we reported previously, although each of the SQMG/SQDG was a much stronger inhibitor than the fatty acid alone that was present in the SQMG/SQDG. The inhibitory effect could be influenced by the chain size of fatty acids in the SQMG/SQDG. The sulfate moiety in the quinovose was also important for the inhibition. Lineweaver‐Burk plots of SQMG/SQDG indicated that DNA polymerase α was non‐competitively inhibited, but the SQMG/SQDG were effective as antagonists of both template‐primer DNA‐binding and nucleotide substrate‐binding of DNA polymerase β. The SQMG had an cytotoxic effect, but the SQDG tested did not. The SQDG might not be able to penetrate into cells. Based on these results, we discuss the molecular action of SQMG/SQDG and propose drug design strategies for developing new anti‐neoplastic agents.

Dehydroaltenusin, a Mammalian DNA Polymerase α Inhibitor

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

Structural determination of sulfoquinovosyldiacylglycerol by chiral syntheses

Abstract Chiral sulfoquinovosyldiacylglycerols (SQDGs) have been synthesized to determine the absolute stereochemistry and the biological activities. The 1 H NMR spectrum of a natural SQDG is comparable to that of synthetic (2 S )-SQDG rather than that of the (2 R ) analogue. The biological activity of the respective isomers for DNA polymerase α and β inhibition was not distinguishable in the enzymatic assay.

Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases

Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase. We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon). Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report. Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively. On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities. RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM. The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases. As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM. The cells were halted at G1 phase in the cell cycle by RAL.

Selective inhibition of the activities of both eukaryotic DNA polymerases and DNA topoisomerases by elenic acid.

(R)-(-)-Elenic acid (R-2,4-dimethyl-22-(p-hydroxyphenyl)-docos-3(E)-enoic acid) (EA) is a DNA topoisomerase II inhibitor found in an Indonesian sponge, Plakinastrella sp. We found and report here that it is a potent inhibitor of calf DNA polymerase alpha (IC(50)=7.7 microM) and rat DNA polymerase beta (IC(50)=12.9 microM). EA did not bind to DNA directly. EA did not influence the activities of DNA polymerases such as plant DNA polymerases I and II and prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I, or other DNA metabolic enzymes such as human immunodeficiency virus (HIV) reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. Interestingly, EA was also an inhibitor of DNA topoisomerases I and II, although the enzymatic characteristics including modes of action, amino acid sequences and three-dimensional structures were markedly different from those of DNA polymerases. EA could prevent the growth of NUGC-3 cancer cells, and the LD(50) value was 22.5 microM. The cells were halted at G1 and G2/M phase in the cell cycle. From these results, the action mode of EA is discussed.