Autodisplay of human PIP5K1α lipid kinase on Escherichia coli and inhibitor testing.

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) plays a major role in the PI3K/AKT/mTOR signaling pathway. As it has been shown before that hPIP5K1α is involved in the development of different types of cancer in particular prostate cancer, inhibitors of the enzyme might be a new option for the treatment of this disease. Here we report on the expression of hPIP5K1α on the surface of E. coli using Autodisplay. Autodisplay is defined as the surface display of a recombinant protein on a gramnegative bacterium by the autotransporter secretion pathway. After verification of surface expression, enzyme activity of whole cells displaying hPIP5K1α was determined by a capillary electrophoresis based assay. When using cells at an OD of 2.5, the artificial substrate phosphatidylinositol4-phosphate (PI(4)P) fluorescein was converted by a rate of 10.7 ± 0.2 fmol/min. Using this substrate inhibition of three pyranobenzoquinone type compounds was tested. The most active compound was 4-(2-amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-chromen-4-yl) benzoic acid with an IC value of 8.6 μM. Because until now, all attempts to purify hPIP5K1α failed, we suggest the use of whole cells of E. coli displaying the enzyme as a convenient tool for inhibitor identification.