Kengo Sakaguchi

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.

Novel anti-inflammatory compounds from Myrsine seguinii, terpeno-benzoic acids, are inhibitors of mammalian DNA polymerases.

Novel anti-inflammatory compounds, terpeno-benzoic acids, were found from the plant, Myrsine seguinii. The strongest of these anti-inflammatory agents, 3-geranyl-4-hydroxy-5-(3-methyl-2-butenyl) benzoic acid (compound 1), showed an inhibitory effect against enzymes involved in replication, such as calf DNA polymerase alpha (pol. alpha), rat DNA polymerase beta (pol. beta) and one of the beta family polymerases, calf thymus terminal deoxynucleotidyl transferase (TdT). The minimum inhibitory concentration (MIC) and IC50 values were 82 and 22 microM for pol. alpha, 86 and 11 microM for pol. beta, 140 and 46 microM for TdT, respectively. However, compound 1 did not influence the activities of plant DNA polymerases, human immunodeficiency virus type-1 reverse transcriptase, any of the prokaryotic DNA polymerases or DNA and RNA metabolic enzymes tested. Dose-dependent relationships were observed between the anti-inflammatory activities and the DNA polymerase-inhibitory activities of the four derivatives. The carboxylic acid moiety in the benzoic acid of the compounds appeared to be related to the inhibitory effects. The mode of action of the terpeno-benzoic acids against the polymerases and their relationships to the anti-inflammatory activity are discussed.

Mode analysis of binding of fatty acids to mammalian DNA polymerases.

We previously reported that unsaturated long-chain fatty acids were potent DNA polymerase inhibitors (Y. Mizushina et al., J. Biol. Chem. 274 (1999) 25599-25607). In those experiments, the question remained of whether metastable oil droplets (liposomal vesicles) of the unsaturated long-chain fatty acids can non-specifically inhibit the polymerase activity. We report here that only the soluble fatty acid monomers of linoleic acid or nervonic acid could affect the activities of mammalian DNA polymerases, and the metastable oil droplets could not. When we consider the facts that nuclear membranes are a kind of liposomal vesicles, that free fatty acids occur only at the moment the lipids are digested, and that the DNA polymerization possibly occurs on the nuclear membranes, the data shown here are suggestive regarding the mechanism of regulation of DNA polymerization in vivo.