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 compounds, and found that a
fatty acid,
linoleic acid (C18:2), from a basidiomycete, inhibited Cdt1-geminin interaction in vitro. Of the commercially purchased linear-chain
fatty acids tested, the inhibitory effect of
oleic acid (C18:1) was the strongest, with 50% inhibition observed at concentrations of 9.6 microM. Since trans-configuration, the
ester form, and the addition of the
hydroxyl group of
oleic acid had no influence on C18:1
fatty acid derivatives, both parts of a
carboxylic acid and an alkyl chain containing cis-type double bonds of
fatty acid might be essential for inhibition. Surface plasmon resonance analysis demonstrated that
oleic acid was able to bind selectively to Cdt1, but did not interact with
geminin. Using a three-dimensional computer modeling analysis,
oleic acid was conjectured to interact with the
geminin interaction interface on Cdt1, and the carboxyl group of
oleic acid was assumed to form hydrogen bonds with the residue of Arg342 of Cdt1. These results suggested that, at least in vitro,
oleic acid-containing cell membranes of the
lipid bilayer inhibit Cdt1-geminin complex formation by binding to Cdt1 and thereby liberating Cdt1 from inhibition by
geminin.