pmc.ncbi.nlm.nih.gov

Specificity and mechanism of action of some commonly used protein kinase inhibitors

Abstract

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Full Text

The Full Text of this article is available as a PDF (149.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995 Nov 17;270(46):27489–27494. doi: 10.1074/jbc.270.46.27489. [DOI] [PubMed] [Google Scholar]
  2. Alessi D. R. The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase-1beta (Rsk-2) and p70 S6 kinase. FEBS Lett. 1997 Feb 3;402(2-3):121–123. doi: 10.1016/s0014-5793(96)01510-4. [DOI] [PubMed] [Google Scholar]
  3. Amano M., Chihara K., Nakamura N., Kaneko T., Matsuura Y., Kaibuchi K. The COOH terminus of Rho-kinase negatively regulates rho-kinase activity. J Biol Chem. 1999 Nov 5;274(45):32418–32424. doi: 10.1074/jbc.274.45.32418. [DOI] [PubMed] [Google Scholar]
  4. Badger A. M., Bradbeer J. N., Votta B., Lee J. C., Adams J. L., Griswold D. E. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther. 1996 Dec;279(3):1453–1461. [PubMed] [Google Scholar]
  5. Brown E. J., Beal P. A., Keith C. T., Chen J., Shin T. B., Schreiber S. L. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature. 1995 Oct 5;377(6548):441–446. doi: 10.1038/377441a0. [DOI] [PubMed] [Google Scholar]
  6. Börsch-Haubold A. G., Pasquet S., Watson S. P. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase. J Biol Chem. 1998 Oct 30;273(44):28766–28772. doi: 10.1074/jbc.273.44.28766. [DOI] [PubMed] [Google Scholar]
  7. Caivano M., Cohen P. Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1 beta in RAW264 macrophages. J Immunol. 2000 Mar 15;164(6):3018–3025. doi: 10.4049/jimmunol.164.6.3018. [DOI] [PubMed] [Google Scholar]
  8. Carroll M., Ohno-Jones S., Tamura S., Buchdunger E., Zimmermann J., Lydon N. B., Gilliland D. G., Druker B. J. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood. 1997 Dec 15;90(12):4947–4952. [PubMed] [Google Scholar]
  9. Cheng K., Creacy S., Larner J. 'Insulin-like' effects of lithium ion on isolated rat adipocytes. II. Specific activation of glycogen synthase. Mol Cell Biochem. 1983;56(2):183–189. doi: 10.1007/BF00227219. [DOI] [PubMed] [Google Scholar]
  10. Cohen P. The development and therapeutic potential of protein kinase inhibitors. Curr Opin Chem Biol. 1999 Aug;3(4):459–465. doi: 10.1016/S1367-5931(99)80067-2. [DOI] [PubMed] [Google Scholar]
  11. Cohen P. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol. 1997 Sep;7(9):353–361. doi: 10.1016/S0962-8924(97)01105-7. [DOI] [PubMed] [Google Scholar]
  12. Cuenda A., Rouse J., Doza Y. N., Meier R., Cohen P., Gallagher T. F., Young P. R., Lee J. C. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 1995 May 8;364(2):229–233. doi: 10.1016/0014-5793(95)00357-f. [DOI] [PubMed] [Google Scholar]
  13. Davis P. D., Hill C. H., Keech E., Lawton G., Nixon J. S., Sedgwick A. D., Wadsworth J., Westmacott D., Wilkinson S. E. Potent selective inhibitors of protein kinase C. FEBS Lett. 1989 Dec 18;259(1):61–63. doi: 10.1016/0014-5793(89)81494-2. [DOI] [PubMed] [Google Scholar]
  14. DeSilva D. R., Jones E. A., Favata M. F., Jaffee B. D., Magolda R. L., Trzaskos J. M., Scherle P. A. Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy. J Immunol. 1998 May 1;160(9):4175–4181. [PubMed] [Google Scholar]
  15. Deak M., Clifton A. D., Lucocq L. M., Alessi D. R. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 1998 Aug 3;17(15):4426–4441. doi: 10.1093/emboj/17.15.4426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dudley D. T., Pang L., Decker S. J., Bridges A. J., Saltiel A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7686–7689. doi: 10.1073/pnas.92.17.7686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eyers P. A., Craxton M., Morrice N., Cohen P., Goedert M. Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution. Chem Biol. 1998 Jun;5(6):321–328. doi: 10.1016/s1074-5521(98)90170-3. [DOI] [PubMed] [Google Scholar]
  18. Eyers P. A., van den IJssel P., Quinlan R. A., Goedert M., Cohen P. Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580. FEBS Lett. 1999 May 21;451(2):191–196. doi: 10.1016/s0014-5793(99)00552-9. [DOI] [PubMed] [Google Scholar]
  19. Favata M. F., Horiuchi K. Y., Manos E. J., Daulerio A. J., Stradley D. A., Feeser W. S., Van Dyk D. E., Pitts W. J., Earl R. A., Hobbs F. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998 Jul 17;273(29):18623–18632. doi: 10.1074/jbc.273.29.18623. [DOI] [PubMed] [Google Scholar]
  20. Fong T. A., Shawver L. K., Sun L., Tang C., App H., Powell T. J., Kim Y. H., Schreck R., Wang X., Risau W. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 1999 Jan 1;59(1):99–106. [PubMed] [Google Scholar]
  21. Frantz B., Klatt T., Pang M., Parsons J., Rolando A., Williams H., Tocci M. J., O'Keefe S. J., O'Neill E. A. The activation state of p38 mitogen-activated protein kinase determines the efficiency of ATP competition for pyridinylimidazole inhibitor binding. Biochemistry. 1998 Sep 29;37(39):13846–13853. doi: 10.1021/bi980832y. [DOI] [PubMed] [Google Scholar]
  22. Graves P. R., Yu L., Schwarz J. K., Gales J., Sausville E. A., O'Connor P. M., Piwnica-Worms H. The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem. 2000 Feb 25;275(8):5600–5605. doi: 10.1074/jbc.275.8.5600. [DOI] [PubMed] [Google Scholar]
  23. Gschwendt M., Müller H. J., Kielbassa K., Zang R., Kittstein W., Rincke G., Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun. 1994 Feb 28;199(1):93–98. doi: 10.1006/bbrc.1994.1199. [DOI] [PubMed] [Google Scholar]
  24. Gum R. J., McLaughlin M. M., Kumar S., Wang Z., Bower M. J., Lee J. C., Adams J. L., Livi G. P., Goldsmith E. J., Young P. R. Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket. J Biol Chem. 1998 Jun 19;273(25):15605–15610. doi: 10.1074/jbc.273.25.15605. [DOI] [PubMed] [Google Scholar]
  25. Hall-Jackson C. A., Eyers P. A., Cohen P., Goedert M., Boyle F. T., Hewitt N., Plant H., Hedge P. Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol. 1999 Aug;6(8):559–568. doi: 10.1016/s1074-5521(99)80088-x. [DOI] [PubMed] [Google Scholar]
  26. Hall-Jackson C. A., Goedert M., Hedge P., Cohen P. Effect of SB 203580 on the activity of c-Raf in vitro and in vivo. Oncogene. 1999 Mar 25;18(12):2047–2054. doi: 10.1038/sj.onc.1202603. [DOI] [PubMed] [Google Scholar]
  27. Hers I., Tavaré J. M., Denton R. M. The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of glycogen synthase kinase-3 activity. FEBS Lett. 1999 Nov 5;460(3):433–436. doi: 10.1016/s0014-5793(99)01389-7. [DOI] [PubMed] [Google Scholar]
  28. Hidaka H., Yokokura H. Molecular and cellular pharmacology of a calcium/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, and proposal of CaM kinase phosphorylation cascades. Adv Pharmacol. 1996;36:193–219. doi: 10.1016/s1054-3589(08)60583-9. [DOI] [PubMed] [Google Scholar]
  29. Itoh K., Yoshioka K., Akedo H., Uehata M., Ishizaki T., Narumiya S. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med. 1999 Feb;5(2):221–225. doi: 10.1038/5587. [DOI] [PubMed] [Google Scholar]
  30. Kamakura S., Moriguchi T., Nishida E. Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases. Identification and characterization of a signaling pathway to the nucleus. J Biol Chem. 1999 Sep 10;274(37):26563–26571. doi: 10.1074/jbc.274.37.26563. [DOI] [PubMed] [Google Scholar]
  31. Kimura K., Ito M., Amano M., Chihara K., Fukata Y., Nakafuku M., Yamamori B., Feng J., Nakano T., Okawa K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase) Science. 1996 Jul 12;273(5272):245–248. doi: 10.1126/science.273.5272.245. [DOI] [PubMed] [Google Scholar]
  32. Lali F. V., Hunt A. E., Turner S. J., Foxwell B. M. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem. 2000 Mar 10;275(10):7395–7402. doi: 10.1074/jbc.275.10.7395. [DOI] [PubMed] [Google Scholar]
  33. Leopoldt D., Hanck T., Exner T., Maier U., Wetzker R., Nürnberg B. Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit. J Biol Chem. 1998 Mar 20;273(12):7024–7029. doi: 10.1074/jbc.273.12.7024. [DOI] [PubMed] [Google Scholar]
  34. Lingameneni R., Vysotskaya T. N., Duch D. S., Hemmings H. C., Jr Inhibition of voltage-dependent sodium channels by Ro 31-8220, a 'specific' protein kinase C inhibitor. FEBS Lett. 2000 May 12;473(2):265–268. doi: 10.1016/s0014-5793(00)01532-5. [DOI] [PubMed] [Google Scholar]
  35. Mohammadi M., Froum S., Hamby J. M., Schroeder M. C., Panek R. L., Lu G. H., Eliseenkova A. V., Green D., Schlessinger J., Hubbard S. R. Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J. 1998 Oct 15;17(20):5896–5904. doi: 10.1093/emboj/17.20.5896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moyer J. D., Barbacci E. G., Iwata K. K., Arnold L., Boman B., Cunningham A., DiOrio C., Doty J., Morin M. J., Moyer M. P. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res. 1997 Nov 1;57(21):4838–4848. [PubMed] [Google Scholar]
  37. Nakanishi S., Kakita S., Takahashi I., Kawahara K., Tsukuda E., Sano T., Yamada K., Yoshida M., Kase H., Matsuda Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem. 1992 Feb 5;267(4):2157–2163. [PubMed] [Google Scholar]
  38. Niggli V. Rho-kinase in human neutrophils: a role in signalling for myosin light chain phosphorylation and cell migration. FEBS Lett. 1999 Feb 19;445(1):69–72. doi: 10.1016/s0014-5793(99)00098-8. [DOI] [PubMed] [Google Scholar]
  39. Picton C., Woodgett J., Hemmings B., Cohen P. Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3. FEBS Lett. 1982 Dec 13;150(1):191–196. doi: 10.1016/0014-5793(82)81332-x. [DOI] [PubMed] [Google Scholar]
  40. Sahai E., Ishizaki T., Narumiya S., Treisman R. Transformation mediated by RhoA requires activity of ROCK kinases. Curr Biol. 1999 Feb 11;9(3):136–145. doi: 10.1016/s0960-9822(99)80067-0. [DOI] [PubMed] [Google Scholar]
  41. Sassone-Corsi P., Mizzen C. A., Cheung P., Crosio C., Monaco L., Jacquot S., Hanauer A., Allis C. D. Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3. Science. 1999 Aug 6;285(5429):886–891. doi: 10.1126/science.285.5429.886. [DOI] [PubMed] [Google Scholar]
  42. Sebolt-Leopold J. S., Dudley D. T., Herrera R., Van Becelaere K., Wiland A., Gowan R. C., Tecle H., Barrett S. D., Bridges A., Przybranowski S. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat Med. 1999 Jul;5(7):810–816. doi: 10.1038/10533. [DOI] [PubMed] [Google Scholar]
  43. Shaw M., Cohen P., Alessi D. R. The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Biochem J. 1998 Nov 15;336(Pt 1):241–246. doi: 10.1042/bj3360241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shawver L. K., Schwartz D. P., Mann E., Chen H., Tsai J., Chu L., Taylorson L., Longhi M., Meredith S., Germain L. Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide. Clin Cancer Res. 1997 Jul;3(7):1167–1177. [PubMed] [Google Scholar]
  45. Stambolic V., Ruel L., Woodgett J. R. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol. 1996 Dec 1;6(12):1664–1668. doi: 10.1016/s0960-9822(02)70790-2. [DOI] [PubMed] [Google Scholar]
  46. Sutherland C., Leighton I. A., Cohen P. Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem J. 1993 Nov 15;296(Pt 1):15–19. doi: 10.1042/bj2960015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tachibana E., Harada T., Shibuya M., Saito K., Takayasu M., Suzuki Y., Yoshida J. Intra-arterial infusion of fasudil hydrochloride for treating vasospasm following subarachnoid haemorrhage. Acta Neurochir (Wien) 1999;141(1):13–19. doi: 10.1007/s007010050260. [DOI] [PubMed] [Google Scholar]
  48. Thomas G., Hall M. N. TOR signalling and control of cell growth. Curr Opin Cell Biol. 1997 Dec;9(6):782–787. doi: 10.1016/s0955-0674(97)80078-6. [DOI] [PubMed] [Google Scholar]
  49. Thomson S., Clayton A. L., Hazzalin C. A., Rose S., Barratt M. J., Mahadevan L. C. The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J. 1999 Sep 1;18(17):4779–4793. doi: 10.1093/emboj/18.17.4779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tokumitsu H., Chijiwa T., Hagiwara M., Mizutani A., Terasawa M., Hidaka H. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 1990 Mar 15;265(8):4315–4320. [PubMed] [Google Scholar]
  51. Tong L., Pav S., White D. M., Rogers S., Crane K. M., Cywin C. L., Brown M. L., Pargellis C. A. A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. Nat Struct Biol. 1997 Apr;4(4):311–316. doi: 10.1038/nsb0497-311. [DOI] [PubMed] [Google Scholar]
  52. Uehata M., Ishizaki T., Satoh H., Ono T., Kawahara T., Morishita T., Tamakawa H., Yamagami K., Inui J., Maekawa M. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997 Oct 30;389(6654):990–994. doi: 10.1038/40187. [DOI] [PubMed] [Google Scholar]
  53. Wilson K. P., McCaffrey P. G., Hsiao K., Pazhanisamy S., Galullo V., Bemis G. W., Fitzgibbon M. J., Caron P. R., Murcko M. A., Su M. S. The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase. Chem Biol. 1997 Jun;4(6):423–431. doi: 10.1016/s1074-5521(97)90194-0. [DOI] [PubMed] [Google Scholar]
  54. Yu L., Orlandi L., Wang P., Orr M. S., Senderowicz A. M., Sausville E. A., Silvestrini R., Watanabe N., Piwnica-Worms H., O'Connor P. M. UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase. J Biol Chem. 1998 Dec 11;273(50):33455–33464. doi: 10.1074/jbc.273.50.33455. [DOI] [PubMed] [Google Scholar]