Screening of phosphatidylinositol kinase inhibitors from Streptomyces.

Phosphatidylinositol turnover is important in intracellular signal transduction in response to various growth factors, hormones and neurotransmitters1}. Transformation by oncogenes such as ras2^, 5rc3), erbB^, fmsb) and fes^ also enhances cellular phosphatidylinositol turnover. Phosphatidylinositol kinase is an enzyme involved in the pathway of phosphatidylinositol turnover. Especially, its activity is known to be enhanced by transformation with srcz>e\ erbB4^, fms6) and sis*0 and by platelet-derived growth factor6). Therefore, we have screened for inhibitors of phosphatidylinositol kinase from microbial secondary metabolites and found that 2,3-dihydroxybenzoic acid and orobol strongly inhibit the enzyme. The phosphatidylinositol kinase from A431 cells has been purified to near homogeneity7). The enzyme has a subunit weight of 55,000 and acts as a monomer. It phosphorylates the inositol moiety of phosphatidylinositol on the 4-position and is therefore phosphatidylinositol 4-kinase (Type II)8). With the A431 cell membrane, we have developed a rapid assay method for phosphatidylinositol kinase using a small packed silica gel column for separating phosphorylated lipid and unreacted [^-32P]ATP. The reaction mixture of phosphatidylinositol (60 ^g) and membrane fraction of A431cells with or without inhibitor was incubated in 90 jul of 20 mM4-(2-hydroxyethyl)1 -piperazineethanesulfonic acid (HEPES) buffer (pH 7.2) at 20°C for 10 minutes, then [r-32P]ATP was added to give about 2 ^m final concentration and incubation was continued at 20°C for 20 minutes. The reaction was stopped by addition of 700 iA of a mixture of CHC13, MeOHand 1 n HC1 (4:1 :2). The mixture was centrifuged (3,000rpmx10 minutes) to separate the two phases. Then, 200 p\ of the lower phase was