Midkine inhibitors: application of a simple assay procedure to screening of inhibitory compounds

BACKGROUND Midkine is a heparin-binding cytokine and is involved in etiology of various diseases. Thus, midkine inhibitors are expected to be helpful in treatment of many diseases. METHODS We developed a simple assay for midkine activity based on midkine-dependent migration of osteblastic cells. Midkine inhibitors were searched as materials that inhibit this midkine activity. To develop peptides that inhibit midkine activity, we constructed models in which C-terminal half of midkine interacted with alpha4beta1-integrin. Low molecular weight compounds which are expected to bind to midkine with high affinity were searched by in silico screening with the aid of Presto-X2 program. RESULTS Among peptides in putative binding sites of midkine and the integrin, a peptide derived from beta1-integrin and that derived from the first beta sheet of the C-terminal half of midkine significantly inhibited midkine activity. Two low molecular weight compounds found by in silico screening exhibited no toxicity to target cells, but inhibited midkine activity. They are trifluoro compounds: one (PubChem 4603792) is 2-(2,6-dimethylpiperidin-1-yl)-4-thiophen-2-yl-6-(trifluoromethy)pyrimidine, and the other has a related structure. CONCLUSIONS The assay procedure is helpful in screening midkine inhibitors. All reagents described here might become mother material to develop clinically effective midkine inhibitors.