A human replication initiation
protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and
geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by
geminin silencing with
small interfering RNA, induces
DNA re-replication and eventual cell death in some
cancer-derived cell lines. We established a high throughput screening system based on a modified
enzyme linked
immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a
glycolipid, sulfoquinovosyl
diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major
glycolipids, such as monogalactosyl
diacylglycerol (MGDG) and digalactosyl
diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with
geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the
geminin interaction interface on Cdt1, and the
sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions.