Takayuki Yamazaki

Mammalian mitotic centromere-associated kinesin (MCAK): a new molecular target of sulfoquinovosylacylglycerols novel antitumor and immunosuppressive agents.

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 KDu2003=u20033.1u2003×u200310−4 to 6.2u2003×u200310−5m. 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.

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(α-d-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.

Downregulation of Tie2 gene by a novel antitumor sulfolipid, 3′-sulfoquinovosyl-1′-monoacylglycerol, targeting angiogenesis

We previously reported that 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG) was effective in suppressing the growth of solid tumors due to hemorrhagic necrosis in vivo. In the present study, we investigated the antiangiogenic effect of SQMG. In vivo assessment of antitumor assays showed that some tumor cell lines, but not others, were sensitive to SQMG. Microscopic study suggested that in SQMG‐sensitive tumors, but not SQMG‐resistant tumors, angiogenesis was reduced. We next investigated gene expression relating to angiogenesis in tumor tissues by quantitative real‐time polymerase chain reaction. Consequently, although vascular endothelial growth factor gene expression was not detected with significant differences among the cases, significant downregulation of Tie2 gene expression was observed in all SQMG‐sensitive tumors as compared with controls, but not in SQMG‐resistant tumors. These data suggested that the antitumor effects of SQMG could be attributed to antiangiogenic effects, possibly via the downregulation of Tie2 gene expression in SQMG‐sensitive tumors. (Cancer Sci 2008; 99: 1063–1070)

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 β (pol β) 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 β. SQMG competed with both the substrate and the template‐primer for binding to pol β. 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 β, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol β 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.