ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3 kinase isoforms.
Class I phosphatidylinositol 3 kinases (PI3K) phosphorylate phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate. These molecules play an important role in fundamental cellular responses. Four isoforms of class I PI3K are known to have different functions, and abnormalities in their activities have been related to various diseases such as cancer and inflammation. We previously identified a novel PI3K inhibitor, ZSTK474, which showed potent antitumor activity in vivo against a human cancer xenograft without observable toxicity. However, the mode of its molecular action was not investigated in detail. Our previous study only suggested that ZSTK474 possibly competes with ATP for the ATP-binding pocket of PI3Kgamma. In the present study, we have used an in vitro homogenous time-resolved fluorescence kinase assay to examine whether ZSTK474 is indeed an ATP-competing inhibitor of PI3K, and also to determine whether the inhibitory activity of ZSTK474 was isoform-specific. Lineweaver-Burk plot analysis revealed that ZSTK474 inhibits all four PI3K isoforms in an ATP-competitive manner. Among all of the PI3K isoforms, PI3Kdelta was inhibited most potently by ZSTK474 with a K(i) of 1.8 nM, and the other isoforms were inhibited at higher doses. We have also used a kinase activity ELISA to determine whether ZSTK474 inhibits mammalian target of rapamycin, a key kinase acting downstream of PI3K to promote protein synthesis and cell proliferation. Even at a concentration of 100 microM, ZSTK474 inhibited mammalian target of rapamycin activity rather weakly. These results indicate that ZSTK474 is an ATP-competitive pan-class I PI3K inhibitor.
Kong
Dexin
D
Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan.
Yamori
Takao
T
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001667:Binding, Competitive; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015200:Fluoroimmunoassay; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014227:Triazines / Q000494:pharmacology*
D000255:Adenosine Triphosphate; D002867:Chromones; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D014227:Triazines; C510150:ZSTK474; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C546842:MTOR protein, human
Science. 2002 Dec 6;298(5600):1912-34
12471243
Biochem J. 1998 Aug 1;333 ( Pt 3):471-90
9677303
Curr Opin Immunol. 1996 Jun;8(3):412-8
8793996
Mol Cancer Ther. 2005 Sep;4(9):1349-57
16170026
Cell. 2000 Jan 7;100(1):113-27
10647936
Science. 2000 Feb 11;287(5455):1040-6
10669416
Biochem Biophys Res Commun. 2006 Feb 10;340(2):469-75
16376301
Cancer Cell. 2006 May;9(5):341-9
16697955
Oncogene. 2006 Oct 26;25(50):6648-59
16702948
Anal Biochem. 2003 Feb 15;313(2):234-45
12605860
Nat Med. 2005 Sep;11(9):933-5
16127435
Science. 2000 Feb 11;287(5455):1046-9
10669417
J Biol Chem. 1994 Feb 18;269(7):5241-8
8106507
Clin Cancer Res. 2000 Mar;6(3):880-6
10741711
J Immunol. 2003 Mar 1;170(5):2647-54
12594293
Nat Rev Drug Discov. 2005 Dec;4(12):988-1004
16341064
Nat Med. 2005 Sep;11(9):936-43
16127437
Blood. 2005 Aug 1;106(3):1063-6
15840695
EMBO J. 1996 Oct 1;15(19):5256-67
8895571
Clin Cancer Res. 2005 Apr 15;11(8):2875-8
15837735
J Biol Chem. 1994 Feb 4;269(5):3563-7
8106399
Trends Biochem Sci. 2005 Apr;30(4):194-204
15817396
Cell. 2006 May 19;125(4):733-47
16647110
Curr Opin Cell Biol. 2005 Dec;17(6):596-603
16226444
Science. 2004 Apr 23;304(5670):554
15016963
Anal Biochem. 2006 Sep 15;356(2):273-81
16814737
J Natl Cancer Inst. 2006 Apr 19;98(8):545-56
16622124
Mol Cancer Ther. 2004 Jul;3(7):763-72
15252137
Biochem J. 2000 Oct 1;351(Pt 1):95-105
10998351
Cell. 2002 May 3;109(3):275-82
12015977
Science. 2000 Feb 11;287(5455):1049-53
10669418
Nature. 1988 Apr 14;332(6165):644-6
2833705
Cell. 1997 Feb 21;88(4):435-7
9038334
Nat Med. 2005 May;11(5):507-14
15834429