New Test System for Serine/Threonine Protein Kinase Inhibitors Screening: E. coli APHVIII/Pk25 design

An efficient test system for serine/threonine protein kinase inhibitors screening has been developed based on theE. coliprotein system APHVIII/Pk25. Phosphorylation of aminoglycoside phosphotransferase VIII (APHVIII) by protein kinases enhances resistance of the bacterial cell to aminoglycoside antibiotics, e.g. kanamycin. Addition of protein kinase inhibitors prevents phosphorylation and increases cell sensitivity to kanamycin. We have obtained modifications of APHVIII in which phosphorylatable Ser146 was encompassed into the canonical autophosphorylation sequence ofStreptomyces coelicolorPk25 protein kinase. Mutant and wild-typeaphVIII were cloned intoE. coliwith the catalytic domain ofpk25. As a result of the expression of these genes, accumulation of corresponding proteins was clearly observed. Extracted from bacterial lysates, Pk25 demonstrated its ability to autophosphorylate. It was shown that variants ofE. colicontaining bothaphVIIIand рк25were more resistant to kanamycin than those carrying onlyaphVIII. Protein kinase inhibitors of the indolylmaleimide class actively inhibited Pk25 and reduced cell resistance to kanamycin. Modeling of APHVIII and Pk25 3D structures showed that pSer146 is an analog of phosphoserine in the ribose pocket of protein kinase A. Pk25 conformation was similar to that of РknB ofMycobacterium tuberculosis. Potential indolylmaleimide inhibitors were docked into the ATP-binding pocket of Pk25. The designed test system can be used for the primary selection of ATP-competitive small molecule protein kinase inhibitors.