Wortmannin, kinase inhibition

Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, Wymann MP. 2000. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 6 909-919. 

The first example of an irreversible inhibitor is the natural product Wortmannin, which was isolated from Penicillium wortmannii [30], Wortmannin effectively inhibits PI3K at low nanomolar concentration and was shown to be specific across a large panel of kinases [31]. Covalent attachment to PI3K occurs after attack by Lys-883, which is essential for phosphate transfer (see Section 7.2), at the furan ring of Wortmannin [32], Attack at this ring specifically occurs within the catalytic site of the PI3K kinase and is unaffected by nucleophiles in aqueous solution. 

Wortmannin is a fungus-derived inhibitor of PI 3-kinase. The agent binds and inhibits the enzyme covalently and irreversibly. It is very potent and considered to be highly specific (IC5o in most cells in the low nanomolar range). 

LY294002 is a synthetic drug which reversibly inhibits PI 3-kinases. It is less toxic and also less potent than wortmannin. The IC50 in most cells is in the micromolar range. 

Family of enzymes phosphorylating phosphatidylinositol (Ptdlns), PtdIns(4)phosphate, and PtdIns(4,5)phosphate in the 3-position. The Ptdlns(3 phospholipids are second messengers in processes like cell growth, cytoskeletal rearrangement, and vesicular transport. PI 3-kinases are heterodimers composed of a catalytic and a regulatory subunit. The enzymes are activated by insulin, many growth factors, and by a variety of cytokines. Their activity can be inhibited by wortmannin and LY294002. 

Wortmannin PI 3-kinase (Arcaro and Wymann, 1993) May also inhibit mTOR (Brunn et al., 1996)... 

Brunn, G.J., Williams, J., Sabers, C., Weiderrecht, G., Lawrence, J. C., and Abraham, R. T. (1996). Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBOJ. 15, 5256-5267. 

LY294002 and wortmannin inhibit the enzyme PI-3 kinase required for the closure of pseudopodia to form intracellular vesicles (61,73-75,78,122). Compared to wortmannin, which is relatively unstable in aqueous media, the inhibitory effects of LY294002 are more specific, reversible (recovery after 10 minutes), and not light dependent. Therefore, LY294002 can be used for time-lapse experiments. Several studies have indicated that both substances have little or no effect on the other pathways described (75). However, both substances might block the uptake of Tfn as well (76). 

The following inhibiting agents are often used NEM, the PI-3 kinase inhibitors wortmannin and LY294002, filipin, nocodazole, colchicine, cyto-chalasin D, and the tyrosine kinase inhibitors herbimycin and genistein (all described above). 

Members of the phosphoinositide (PI)-3 kinase family appear to be involved in the phosphorylation of H2A.X. The SQ motif matches a common target site for these kinases and the formation of y-H2A.X in response to double stranded breaks is inhibited by wortmannin, an inhibitor of PI-3 kinases [63]. Examination of cell lines deficient in the PI-3 kinase ATM indicated that it has a major role in phosphorylating H2A.X in response to double strand breaks [64]. ATM can phosphorylate H2A.X in vitro suggesting that it may directly phosphorylate H2A.X in vivo [64]. Another PI-3 kinase ATR appears to be involved in phosphorylating H2A.X in response to replicational stress induced by treatment of dividing cells with hydroxyurea or by irradiating them with ultraviolet light [65]. It has been hypothesized that PI-3 kinases such as ATM are recruited to, or activated at, the site of the double stranded break and then phosphorylate H2A.X molecules around the break point [40,64,66]. 

Okada, T. Sakuma, L. Fukui, Y Hazeki, O. Ui, M. Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J. Biol. Chem., 269, 3563-3567 (1994)... 

PI3 kinase Wortmannin, LY 294002 Inhibit kinase Multiple growth factors and cytokines Inhibit neutrophil (T4) and eosinophil function (B16)... 

T4. Thelen, M., Wymann, M. P., and Langen, H., Wortmannin binds specifically to 1-pho-sphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes. Proc. Natl. Acad. Sci. USA 91, 4960-4964 (1994). 

Yano H, Nakanishi S, Kimura K et al (1993) Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem... 

Liu Y, Shreder KR, Gai W, Corral S, Ferris DK, Rosenblum JS (2005) Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian Polo-like kinase. Chem Biol 12 99-107... 

Blonunaart, E.F., Krause, U., Schellens, J.P., Vreehug-Siudelarova, H., aud Meijer, A.J. (1997). The phosphatidyhuositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243 240-246. 

Phosphatidyl-inositol-3-OH kinase (PI(3)kinase) plays an important role in the fusion of endosomes. Phosphatidyl-inositol-3-phosphate (PI(3)P), a product of this enzyme, is enriched in early endosomes, and blocking PI(3)kinase activity with the small molecule wortmannin prevents endosome fusion. This fungal natural product has been shown to inhibit the endocytosis of transferrin, horseradish peroxidase, and albumin (44, 45). 

In contrast to this polyspecific ABPP probe suitable for profiling a large fraction of the protein kinase family, several other approaches have been introduced, which focus on specific subfamilies. Wortmannin, for example, is a potent natural product specific for the inhibition of phosphoinositide 3- and polo-like kinases in vitro and in vivo. Its role in ABPP probe design will be discussed in the natural product-derived probe section (Section 9.17.3.3)97 98... 

Figure 26 Wortmannin- and microcystin-based probes and their mechanistical action via embedded Michael acceptors, (a) The Michael acceptor system of wortmannin and its derived probes irreversibly inhibits its kinase target by covalent modification of the conserved active site lysine, (b) The cyclic peptide microcystine and its derived probes react covalently with the active site cysteine of phosphatases.

T. Rahn, M. Ridderstrale, H. Tornqvist, V. Manganiello, G. Fredrikson, P. Belfrage, and E. Degerman, Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin, FEBS Lett., 1994, 350, 314-317. 

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