Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.
Abstract Ever since their discovery in 1980's, phosphoinositide 3-kinases (PI3Ks) have played a vital role in many intracellular signaling pathways and PI3K inhibitors are prominent targets in drug discovery, especially in cancer. PI3Ks catalyse the phosphorylation of phosphatidylinositol at the 3′-position of the inositol ring and the resulting secondary by-products are implicated in various cellular activities such as cell survival, growth and proliferation. Although class I PI3K has been studied extensively, not much is known about members of class II PI3K. The class II PI3K has gained attention recently as they are believed to be up-regulated in certain types of cancers. The class II PI3Ks consists of three mammalian isoforms: PI3KC2α, PI3KC2β and PI3KC2γ however, as of yet there are no potent, selective inhibitors of class II PI3Ks. We have generated a homology model of PI3KC2β and carried out molecular docking studies of known class I PI3K inhibitors that also show activity against PI3KC2β. Our docking results have revealed that while the overall binding poses are common for the two classes, there are non-conserved residues involved in interactions with these compounds that could be targeted for selectivity. To pursue a screening program based on these concepts, we have expressed and purified active PI3KC2β. Compounds from within our 400 compound PI3K inhibitor library are being screened as our first step to develop novel PI3KC2β-selective inhibitors. In conclusion, identified differences in the catalytic pocket of PI3KC2β, compared to other PI3Ks which may aid discovery of the first novel potent inhibitors of a class II PI3K. Citation Format: Krithika Sundaram, Ian Jennings, Alexandre Arcaro, Philip Thompson. Probing the inhibitor selectivity site of class II PI3KC2β. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4246. doi:10.1158/1538-7445.AM2014-4246
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