Xác định và Đặc trưng hóa một Inhibitor Mới và Đặc hiệu của Kinase ATM Đột biến Ataxia-Telangiectasia

American Association for Cancer Research (AACR) - Tập 64 Số 24 - Trang 9152-9159 - 2004
Ian Hickson1, Yan Zhao2, Caroline J. Richardson1, Sharon J. Green1, Niall M.B. Martin1, Alisdair I. Orr1, Philip M. Reaper3, Stephen P. Jackson3, Nicola J. Curtin2, Graeme C.M. Smith1
11KuDOS Pharmaceuticals Ltd., Cambridge Science Park, Milton Road, Cambridge;
22Northern Institute for Cancer Research, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne; and
33Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Zoology, Cambridge University, Cambridge, United Kingdom

Tóm tắt

Tóm tắt Kinase protein serine/threonine ATM phát tín hiệu đến các thành phần chu kỳ tế bào và sửa chữa DNA bằng cách phosphoryl hóa các mục tiêu hạ lưu như p53, CHK2, NBS1 và BRCA1. Đột biến của ATM xảy ra trong rối loạn tự gen lặn ở người là ataxia-telangiectasia, được đặc trưng bởi độ nhạy cảm cao với bức xạ ion hóa và sự thất bại của tế bào trong việc dừng chu kỳ tế bào sau khi xảy ra đứt gãy DNA hai chuỗi. Do đó, đã được đề xuất rằng việc ức chế ATM sẽ gây ra sự nhạy cảm của tế bào đối với bức xạ và hóa trị liệu. Thông qua việc sàng lọc một thư viện hợp chất phân tử nhỏ được phát triển cho họ kinase giống phosphatidylinositol 3′-kinase, chúng tôi đã xác định một chất ức chế cạnh tranh ATP, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), có khả năng ức chế ATM với IC50 là 13 nmol/L và Ki là 2.2 nmol/L. KU-55933 cho thấy tính đặc hiệu trong việc ức chế các kinase giống phosphatidylinositol 3′-kinase khác. Việc ức chế ATM ở tế bào bởi KU-55933 được chứng minh bằng việc loại bỏ sự phosphoryl hóa phụ thuộc vào bức xạ ion hóa của một loạt các mục tiêu ATM, bao gồm p53, γH2AX, NBS1 và SMC1. KU-55933 không cho thấy sự ức chế của các sự kiện phosphoryl hóa tế bào gây ra bởi tổn thương DNA do ánh sáng UV. Sự tiếp xúc của tế bào với KU-55933 dẫn đến sự nhạy cảm đáng kể với các tác động độc tế bào của bức xạ ion hóa và các tác nhân hóa trị liệu gây đứt gãy DNA hai chuỗi, như etoposide, doxorubicin và camptothecin. Việc ức chế ATM bởi KU-55933 cũng dẫn đến việc mất khả năng ngừng chu kỳ tế bào do bức xạ ion hóa. Ngược lại, KU-55933 không tăng cường tác động độc tế bào của bức xạ ion hóa trên các tế bào ataxia-telangiectasia, cũng như không ảnh hưởng đến hồ sơ chu kỳ tế bào của chúng sau tổn thương DNA. Chúng tôi kết luận rằng KU-55933 là một chất ức chế kinase ATM mới, đặc hiệu và mạnh mẽ.

Từ khóa


Tài liệu tham khảo

Rouse J, Jackson SP Interfaces between the detection, signaling and repair of DNA damage. Science (Wash. DC) 2002; 297: 547-51.

Zakian VA ATM-related genes: what do they tell us about functions of the human gene?. Cell 1995; 82: 685-7.

Abraham RT Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001; 15: 2177-96.

Shiloh Y ATM and related protein kinases: safeguarding genome integrity. Nat Cancer Rev 2003; 3: 155-68.

Lavin MF, Shiloh Y The genetic defect in ataxia-telangiectasia. Annu Rev Immunol 1997; 15: 177-202.

Rotman G, Shiloh Y ATM. from gene to function. Hum Mol Genet 1988; 7: 1555-63.

Kastan MB, Lim DS The many substrates and functions of ATM. Nat Rev Mol Cell Biol 2000; 1: 179-86.

Shiloh Y ATM and ATR: networking cellular responses to DNA damage. Curr Opin Genet Dev 2001; 11: 71-7.

Xu B, Kim ST, Lim DS, Kastan MB Two molecularly distinct G2-M checkpoints are induced by ionizing irradiation. Mol Cell Biol 2002; 22: 1049-59.

Canman CE, Lim DS, Cimprich KA, et al Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science (Wash. DC) 1988; 281: 1677-9.

Banin S, Moyal L, Shieh S, et al Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science (Wash. DC) 1998; 281: 1674-7.

Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc Natl Acad Sci 2000; 97: 10389-94.

Maya R, Balass M, Kim ST, et al ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev 1999; 15: 1067-77.

Zhao S, Weng YC, Yuan SS, et al Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products. Nature (Lond.) 2000; 405: 473-7.

Bao S, Tibbetts RS, Brumbaugh KM, et al ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature (Lond.) 2001; 411: 969-74.

Xu B, Kim ST, Kastan MB Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation. Mol Cell Biol 2001; 21: 3445-50.

Fornace AL, Jr, Little JB Normal repair of DNA single-strand breaks in patients with ataxia-telangiectasia. Biochem Biophys Acta 1980; 607: 432-7.

Lavin MF, Davidson M Repair of strand breaks in superhelical DNA of ataxia-telangiectasia lymphoblastoid cells. J Cell Sci 1981; 48: 383-91.

Cornforth MN, Bedford JS On the nature of a defect in cells from individuals with ataxia-telangiectasia. Science (Wash. DC) 1985; 227: 1589-91.

Coquerelle TM, Weibezahn KF, Lucke-Huhle C Rejoining of double strand breaks in normal human and ataxia-telangiectasia fibroblasts after exposure to 60Co gamma-rays, 241Am alpha-particles or bleomycin. Int J Radiat Biol Relat Stud Phys Chem Med 1987; 51: 209-18.

Foray N, Priestley A, Alsbeih G, et al Hypersensitivity of ataxia-telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks. Int J Radiat Biol 1997; 72: 271-83.

Sarkaria JN, Eshleman JS ATM as a target for novel radiosensitizers. Semin Radiat Oncol 2001; 11: 316-27.

Sarkaria JN, Tibbetts RS, Busby EC, Kennedy AP, Hill DE, Abraham RT Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 1998; 58: 4375-82.

Izzard RA, Jackson SP, Smith GCM Competitive and non-competitive inhibition of the DNA dependent protein kinase. Cancer Res 1999; 59: 2581-6.

Ui M, Okada T, Hazeki K, Hazeki O Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem Sci 1995; 20: 303-7.

Zhou B-B S, Chaturvedi P, Spring K, et al Caffeine abolishes the mammalian G2-M DNA damage checkpoint by inhibiting ataxia-telangiectasia–mutated kinase activity. J Biol Chem 2000; 5: 10342-8.

Block WD, Merkle D, Meek K, Lees-Miller SP Selective inhibition of the DNA-dependent protein kinase (DNA-PK) by the radiosensitizing agent caffeine. Nucleic Acid Res 2004; 32: 1967-72.

Vlahos CJ, Matter WF, Hui KY, Brown RF A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994; 269: 5241-8.

Blunt T, Finnie NJ, Taccioli GE, et al Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995; 80: 813-23.

Tibbetts RS, Brumbaugh KM, Williams JM, et al A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 1999; 13: 152-7.

Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC, Abraham RT Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J 1996; 15: 5256-67.

Veuger SJ, Curtin NJ, Richardson CJ, Smith GCM, Durkacz BW Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly (ADP-ribose) polymerase-1. Cancer Res 2003; 63: 6008-15.

Zhao X-H, Bondeva T, Balla T Characterization of recombinant phosphatidylinositol 4-kinase beta reveals auto- and heterophosphorylation of the enzyme. J Biol Chem 2000; 275: 14642-8.

Allan LA, Fried M p53-dependent apoptosis or growth arrest induced by different forms of radiation in U2OS cells: p21WAF1/CIP1 repression in UV induced apoptosis. Oncogene 1999; 18: 5403-12.

Ormerod MG Analysis of DNA-general methods Ormerod MG eds. . Flow cytometry 200083-97. Oxford University Press Oxford, UK

Walker EH, Pacold ME, Perisic O, et al Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 2000; 6: 909-19.

Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001; 276: 42462-7.

Lim DS, Kim ST, Xu B, et al ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature (Lond.) 2000; 404: 613-7.

Liu Q, Guntuku S, Cui XS, et al Chk1 is an essential kinase that is regulated by Atr and required for the G2-M DNA damage checkpoint. Genes Dev 2000; 14: 1448-59.

Kim ST, Xu B, Kastan MB Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev 2002; 16: 560-70.

Caporossi D, Porfirio B, Nicoletti B, et al Hypersensitivity of lymphoblastoid lines derived from ataxia-telangiectasia patients to the induction of chromosomal aberrations by etoposide (VP-16). Mutat Res 1993; 290: 265-72.

Henner WD, Blazka ME Hypersensitivity of cultured ataxia-telangiectasia cells to etoposide. J Natl Cancer Inst (Bethesda) 1986; 76: 1007-11.

Fedier A, Schlamminger M, Schwarz VA, Haller U, Howell SB, Fink D Loss of atm sensitises p53-deficient cells to topoisomerase poisons and antimetabolites. Ann Oncol 2003; 14: 938-45.

Canman CE, Wolff AC, Chen C-Y, Fornace AJ, Jr, Kastan MB The p53-dependent G1 cell cycle checkpoint pathway and ataxia-telangiectasia. Cancer Res 1994; 54: 5054-8.

Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. Proc Natl Acad Sci USA 1999; 96: 13777-82.

Stiff T, O’Driscoll M, Rief N, Iwabuchi K, Lobrich M, Jeggo PA ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res 2004; 64: 2390-6.

Bakkenist CJ, Kastan MB DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature (Lond.) 2003; 421: 499-506.

Gosink EC, Chong MJ, McKinnon PJ Ataxia-telangiectasia mutated deficiency affects astrocyte growth but not radiosensitivity. Cancer Res 1999; 59: 5294-8.

Takimoto CH, Arbuck SG Topoisomerase I targeting agents: the camptothecins Chabner BA Longo DL eds. . Cancer chemotherapy and biotherapy: principles and practice 2001579-646. Lippincott, Williams and Wilkins Philadelphia

Smith PJ, Makinson TA, Watson JV Enhanced sensitivity to camptothecin in ataxia-telangiectasia cells and its relationship with the expression of DNA topoisomerase I. Int J Radiat Biol 1989; 55: 217-31.

Pommier YG, Goldwasser F, Strumberg D Topoisomerase II inhibitors: epipodophyllotoxins, acridines, ellipticines, and bisdioxopiperazines Chabner BA Longo DL eds. . Cancer chemotherapy and biotherapy: principles and practice 2001538-78. Lippincott, Williams & Wilkins Philadelphia

Shiloh Y, Becker Y Kinetics of O6-methylguanine repair in human normal and ataxia-telangiectasia cell lines and correlation of repair capacity with cellular sensitivity to methylating agents. Cancer Res 1981; 41: 5114-20.

Jaspers NG, de Wit J, Regulski MR, Bootsma D Abnormal regulation of DNA replication and increased lethality in ataxia-telangiectasia cells exposed to carcinogenic agents. Cancer Res 1982; 42: 335-41.

Zhang N, Chen P, Gatei M, Scott S, Khanna KK, Lavin MF An anti-sense construct of full-length ATM cDNA imposes a radiosensitive phenotype on normal cells. Oncogene 1998; 17: 811-8.

Uhrhammer N, Fritz E, Boyden L, Meyn MS Human fibroblasts transfected with an ATM antisense vector respond abnormally to ionizing radiation. Int J Mol Med 1999; 4: 43-7.

Guha C, Guha U, Tribius S, et al Antisense ATM gene therapy: a strategy to increase the radiosensitivity of human tumors. Gene Ther 2000; 7: 852-8.

Collis SJ, Swartz MJ, Nelson WG, DeWeese TL Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 2003; 63: 1550-4.