The Human Kinome and Kinase Inhibition

Krisna C. Duong‐Ly1, Jeffrey R. Peterson1

1 Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.9
DOI:  10.1002/0471141755.ph0209s60
Online Posting Date:  March, 2013
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Protein and lipid kinases play key regulatory roles in a number of biological processes. Unsurprisingly, activating mutations in kinases have been linked to a number of disorders and diseases, most notably cancers. Thus, kinases have emerged as promising clinical targets. There are more than 500 human protein kinases and about 20 lipid kinases. Most protein kinases share a highly conserved domain, the eukaryotic protein kinase (ePK) domain, which contains the ATP and substrate‐binding sites. Many inhibitors in clinical use bind to the highly conserved ATP binding site. For this reason, many kinase inhibitors are not exclusively selective for their intended targets. Furthermore, despite the current interest in kinase inhibitors, very few kinases implicated in disease have validated inhibitors. This unit describes the human kinome, ePK structure, and types of kinase inhibitors, focusing on methods to identify potent and selective kinase inhibitors. Curr. Protoc. Pharmacol. 60:2.9.1‐2.9.14. © 2013 by John Wiley & Sons, Inc.

Keywords: kinase; small‐molecule inhibition; inhibitor selectivity; inhibitor screening; kinase domain; kinase assay

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Table of Contents

  • Introduction
  • The Kinome
  • The Architecture of Kinases
  • Types of Inhibition
  • Considerations in the Development of Kinase Inhibitors
  • Assay Formats for Measuring Kinase‐Inhibitor Interactions
  • Discussion
  • Acknowledgements
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Adrian, F.J., Ding, Q., Sim, T., Velentza, A., Sloan, C., Liu, Y., Zhang, G., Hur, W., Ding, S., Manley, P., Mestan, J., Fabbro, D., and Gray, N.S. 2006. Allosteric inhibitors of Bcr‐abl‐dependent cell proliferation. Nat. Chem. Biol. 2:95‐102.
   Anastassiadis, T., Deacon, S.W., Devarajan, K., Ma, H., and Peterson, J.R. 2011. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat. Biotechnol. 29:1039‐1045.
   Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R., and Cohen, P. 2007. The selectivity of protein kinase inhibitors: A further update. Biochem. J. 408:297‐315.
   Barf, T. and Kaptein, A. 2012. Irreversible protein kinase inhibitors: Balancing the benefits and risks. J. Med. Chem. 55:6243‐6262.
   Ben‐Neriah, Y., Daley, G.Q., Mes‐Masson, A.M., Witte, O.N., and Baltimore, D. 1986. The chronic myelogenous leukemia‐specific P210 protein is the product of the bcr/abl hybrid gene. Science 233:212‐214.
   Chissoe, S.L., Bodenteich, A., Wang, Y.F., Wang, Y.P., Burian, D., Clifton, S.W., Crabtree, J., Freeman, A., Iyer, K., Jian, L., Ma, Y., McLaury, H.J., Pan, H.Q., Sarhan, O.H., Toth, S., Wang, Z., Zhang, G., Heisterkamp, N., Groffen, J., and Roe, B.A. 1995. Sequence and analysis of the human ABL gene, the BCR gene, and regions involved in the Philadelphia chromosomal translocation. Genomics 27:67‐82.
   Davis, M.I., Hunt, J.P., Herrgard, S., Ciceri, P., Wodicka, L.M., Pallares, G., Hocker, M., Treiber, D.K., and Zarrinkar, P.P. 2011. Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 29:1046‐1051.
   Deacon, S.W., Beeser, A., Fukui, J.A., Rennefahrt, U.E., Myers, C., Chernoff, J., and Peterson, J.R. 2008. An isoform‐selective, small‐molecule inhibitor targets the autoregulatory mechanism of p21‐activated kinase. Chem. Biol. 15:322‐331.
   Denley, A., Cosgrove, L.J., Booker, G.W., Wallace, J.C., and Forbes, B.E. 2005. Molecular interactions of the IGF system. Cytokine Growth Factor Rev. 16:421‐439.
   Fabbro, D., Cowan‐Jacob, S.W., Mobitz, H., and Martiny‐Baron, G. 2012. Targeting cancer with small‐molecular‐weight kinase inhibitors. Methods Mol. Biol. 795:1‐34.
   Fabian, M.A., Biggs, W.H., 3rd, Treiber, D.K., Atteridge, C.E., Azimioara, M.D., Benedetti, M.G., Carter, T.A., Ciceri, P., Edeen, P.T., Floyd, M., Ford, J.M., Galvin, M., Gerlach, J.L., Grotzfeld, R.M., Herrgard, S., Insko, D.E., Insko, M.A., Lai, A.G., Lelias, J.M., Mehta, S.A., Milanov, Z.V., Velasco, A.M., Wodicka, L.M., Patel, H.K., Zarrinkar, P.P., and Lockhart, D.J. 2005. A small molecule‐kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23:329‐336.
   Fedorov, O., Marsden, B., Pogacic, V., Rellos, P., Muller, S., Bullock, A.N., Schwaller, J., Sundstrom, M., and Knapp, S. 2007. A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. Proc. Natl. Acad. Sci. U.S.A. 104:20523‐20528.
   Fedorov, O., Muller, S., and Knapp, S. 2010. The (un)targeted cancer kinome. Nat. Chem. Biol. 6:166‐169.
   Foster, K.G., Acosta‐Jaquez, H.A., Romeo, Y., Ekim, B., Soliman, G.A., Carriere, A., Roux, P.P., Ballif, B.A., and Fingar, D.C. 2010. Regulation of mTOR complex 1 (mTORC1) by raptor Ser863 and multisite phosphorylation. J. Biol. Chem. 285:80‐94.
   Garuti, L., Roberti, M., and Bottegoni, G. 2010. Non‐ATP competitive protein kinase inhibitors. Curr. Med. Chem. 17:2804‐2821.
   Geda, P., Patury, S., Ma, J., Bharucha, N., Dobry, C.J., Lawson, S.K., Gestwicki, J.E., and Kumar, A. 2008. A small molecule‐directed approach to control protein localization and function. Yeast 25:577‐594.
   Hanada, M., Feng, J., and Hemmings, B.A. 2004. Structure, regulation and function of PKB/AKT—a major therapeutic target. Biochim. Biophys. Acta 1697:3‐16.
   Hantschel, O., Nagar, B., Guettler, S., Kretzschmar, J., Dorey, K., Kuriyan, J., and Superti‐Furga, G. 2003. A myristoyl/phosphotyrosine switch regulates c‐Abl. Cell 112:845‐857.
   Heath, C.M., Stahl, P.D., and Barbieri, M.A. 2003. Lipid kinases play crucial and multiple roles in membrane trafficking and signaling. Histol. Histopathol. 18:989‐998.
   Huss, K.L., Blonigen, P.E., and Campbell, R.M. 2007. Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter‐binding assay format. J. Biomol. Screen. 12:578‐584.
   Jones, J.I. and Clemmons, D.R. 1995. Insulin‐like growth factors and their binding proteins: Biological actions. Endocr. Rev. 16:3‐34.
   Karaman, M.W., Herrgard, S., Treiber, D.K., Gallant, P., Atteridge, C.E., Campbell, B.T., Chan, K.W., Ciceri, P., Davis, M.I., Edeen, P.T., Faraoni, R., Floyd, M., Hunt, J.P., Lockhart, D.J., Milanov, Z.V., Morrison, M.J., Pallares, G., Patel, H.K., Pritchard, S., Wodicka, L.M., and Zarrinkar, P.P. 2008. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26:127‐132.
   Kazlauskas, A. and Cooper, J.A. 1989. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell 58:1121‐1133.
   Knight, Z.A. and Shokat, K.M. 2005. Features of selective kinase inhibitors. Chem. Biol. 12:621‐637.
   Kobayashi, S., Boggon, T.J., Dayaram, T., Janne, P.A., Kocher, O., Meyerson, M., Johnson, B.E., Eck, M.J., Tenen, D.G., and Halmos, B. 2005. EGFR mutation and resistance of non‐small‐cell lung cancer to gefitinib. N. Engl. J. Med. 352:786‐792.
   Kumar, R., Gururaj, A.E., and Barnes, C.J. 2006. p21‐activated kinases in cancer. Nat. Rev. Cancer 6:459‐471.
   Lahiry, P., Torkamani, A., Schork, N.J., and Hegele, R.A. 2010. Kinase mutations in human disease: Interpreting genotype‐phenotype relationships. Nat. Rev. Genet. 11:60‐74.
   Liu, Y. and Gray, N.S. 2006. Rational design of inhibitors that bind to inactive kinase conformations. Nat. Chem. Biol. 2:358‐364.
   Lochhead, P.A., Kinstrie, R., Sibbet, G., Rawjee, T., Morrice, N., and Cleghon, V. 2006. A chaperone‐dependent GSK3beta transitional intermediate mediates activation‐loop autophosphorylation. Mol. Cell. 24:627‐633.
   Lougheed, J.C., Chen, R.H., Mak, P., and Stout, T.J. 2004. Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42‐associated tyrosine kinase ACK1. J. Biol. Chem. 279:44039‐44045.
   Manning, G., Whyte, D.B., Martinez, R., Hunter, T., and Sudarsanam, S. 2002. The protein kinase complement of the human genome. Science 298:1912‐1934.
   Matsumoto, T., Bohman, S., Dixelius, J., Berge, T., Dimberg, A., Magnusson, P., Wang, L., Wikner, C., Qi, J.H., Wernstedt, C., Wu, J., Bruheim, S., Mugishima, H., Mukhopadhyay, D., Spurkland, A., and Claesson‐Welsh, L. 2005. VEGF receptor‐2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis. EMBO J. 24:2342‐2353.
   Matthews, D.J. and Gerritsen, M.E. 2010. Targeting Protein Kinases for Cancer Therapy. John Wiley & Sons, Hoboken, N.J.
   Meggio, F. and Pinna, L.A. 2003. One‐thousand‐and‐one substrates of protein kinase CK2? FASEB J. 17:349‐368.
   Miick, S.M., Jalali, S., Dwyer, B.P., Havens, J., Thomas, D., Jimenez, M.A., Simpson, M.T., Zile, B., Huss, K.L., and Campbell, R.M. 2005. Development of a microplate‐based, electrophoretic fluorescent protein kinase A assay: Comparison with filter‐binding and fluorescence polarization assay formats. J. Biomol. Screen. 10:329‐338.
   Moshinsky, D.J., Ruslim, L., Blake, R.A., and Tang, F. 2003. A widely applicable, high‐throughput TR‐FRET assay for the measurement of kinase autophosphorylation: VEGFR‐2 as a prototype. J. Biomol. Screen. 8:447‐452.
   Niefind, K., Putter, M., Guerra, B., Issinger, O.G., and Schomburg, D. 1999. GTP plus water mimic ATP in the active site of protein kinase CK2. Nat. Struct. Biol. 6:1100‐1103.
   Oliver, A.W., Knapp, S., and Pearl, L.H. 2007. Activation segment exchange: a common mechanism of kinase autophosphorylation? Trends Biochem. Sci. 32:351‐356.
   Pao, W., Miller, V.A., Politi, K.A., Riely, G.J., Somwar, R., Zakowski, M.F., Kris, M.G., and Varmus, H. 2005. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2:e73.
   Pearce, L.R., Komander, D., and Alessi, D.R. 2010. The nuts and bolts of AGC protein kinases. Nat. Rev Mol. Cell. Biol. 11:9‐22.
   Raimondi, C. and Falasca, M. 2011. Targeting PDK1 in cancer. Curr. Med. Chem. 18:2763‐2769.
   Reedijk, M., Liu, X., van der Geer, P., Letwin, K., Waterfield, M.D., Hunter, T., and Pawson, T. 1992. Tyr721 regulates specific binding of the CSF‐1 receptor kinase insert to PI 3'‐kinase SH2 domains: A model for SH2‐mediated receptor‐target interactions. EMBO J. 11:1365‐1372.
   Rennefahrt, U.E., Deacon, S.W., Parker, S.A., Devarajan, K., Beeser, A., Chernoff, J., Knapp, S., Turk, B.E., and Peterson, J.R. 2007. Specificity profiling of Pak kinases allows identification of novel phosphorylation sites. J. Biol. Chem. 282:15667‐15678.
   Schindler, T., Bornmann, W., Pellicena, P., Miller, W.T., Clarkson, B., and Kuriyan, J. 2000. Structural mechanism for STI‐571 inhibition of abelson tyrosine kinase. Science 289:1938‐1942.
   Shah, N.P., Nicoll, J.M., Nagar, B., Gorre, M.E., Paquette, R.L., Kuriyan, J., and Sawyers, C.L. 2002. Multiple BCR‐ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2:117‐125.
   Shtivelman, E., Lifshitz, B., Gale, R.P., and Canaani, E. 1985. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315:550‐554.
   Singh, J., Petter, R.D., and Kluge, A.F. 2010. Targeted covalent drugs of the kinase family. Curr. Opin. Chem. Biol. 14:475‐480.
   Tanramluk, D., Schreyer, A., Pitt, W.R., and Blundell, T.L. 2009. On the origins of enzyme inhibitor selectivity and promiscuity: A case study of protein kinase binding to staurosporine. Chem. Biol. Drug Des. 74:16‐24.
   Thommes, K., Lennartsson, J., Carlberg, M., and Ronnstrand, L. 1999. Identification of Tyr‐703 and Tyr‐936 as the primary association sites for Grb2 and Grb7 in the c‐Kit/stem cell factor receptor, Biochem. J. 341:211‐216.
   Uitdehaag, J.C., Verkaar, F., Alwan, H., de Man, J., Buijsman, R.C., and Zaman, G.J. 2012. A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets. Br. J. Pharmacol. 166:858‐876.
   Vaasa, A., Viil, I., Enkvist, E., Viht, K., Raidaru, G., Lavogina, D., and Uri, A. 2009. High‐affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK. Anal. Biochem. 385:85‐93.
   Vedadi, M., Niesen, F.H., Allali‐Hassani, A., Fedorov, O.Y., Finerty, P.J. Jr., Wasney, G.A., Yeung, R., Arrowsmith, C., Ball, L.J., Berglund, H., Hui, R., Marsden, B.D., Nordlund, P., Sundstrom, M., Weigelt, J., and Edwards, A.M. 2006. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. U.S.A. 103:15835‐15840.
   Viaud, J. and Peterson, J.R. 2009. An allosteric kinase inhibitor binds the p21‐activated kinase autoregulatory domain covalently. Mol. Cancer Ther. 8:2559‐2565.
   White, M.F., Shoelson, S.E., Keutmann, H., and Kahn, C.R. 1988. A cascade of tyrosine autophosphorylation in the beta‐subunit activates the phosphotransferase of the insulin receptor. J. Biol. Chem. 263:2969‐2980.
   Wood, L.D., Parsons, D.W., Jones, S., Lin, J., Sjoblom, T., Leary, R.J., Shen, D., Boca, S.M., Barber, T., Ptak, J., Silliman, N., Szabo, S., Dezso, Z., Ustyanksky, V., Nikolskaya, T., Nikolsky, Y., Karchin, R., Wilson, P.A., Kaminker, J.S., Zhang, Z., Croshaw, R., Willis, J., Dawson, D., Shipitsin, M., Willson, J.K., Sukumar, S., Polyak, K., Park, B.H., Pethiyagoda, C.L., Pant, P.V., Ballinger, D.G., Sparks, A.B., Hartigan, J., Smith, D.R., Suh, E., Papadopoulos, N., Buckhaults, P., Markowitz, S.D., Parmigiani, G., Kinzler, K.W., Velculescu, V.E., and Vogelstein, B. 2007. The genomic landscapes of human breast and colorectal cancers. Science 318:1108‐1113.
   Wu, J., Li, W., Craddock, B.P., Foreman, K.W., Mulvihill, M.J., Ji, Q.S., Miller, W.T., and Hubbard, S.R. 2008. Small‐molecule inhibition and activation‐loop trans‐phosphorylation of the IGF1 receptor. EMBO J. 27:1985‐1994.
   Xu, W., Doshi, A., Lei, M., Eck, M.J., and Harrison, S.C. 1999. Crystal structures of c‐Src reveal features of its autoinhibitory mechanism. Mol. Cell. 3:629‐638.
   Young, M.A., Gonfloni, S., Superti‐Furga, G., Roux, B., and Kuriyan, J. 2001. Dynamic coupling between the SH2 and SH3 domains of c‐Src and Hck underlies their inactivation by C‐terminal tyrosine phosphorylation. Cell 105:115‐126.
   Zegzouti, H., Zdanovskaia, M., Hsiao, K., and Goueli, S.A. 2009. ADP‐Glo: A bioluminescent and homogeneous ADP monitoring assay for kinases. Assay Drug Dev. Technol. 7:560‐572.
   Zhang, B., Tavare, J.M., Ellis, L., and Roth, R.A. 1991. The regulatory role of known tyrosine autophosphorylation sites of the insulin receptor kinase domain. An assessment by replacement with neutral and negatively charged amino acids. J. Biol. Chem. 266:990‐996.
   Zhang, J., Yang, P.L., and Gray, N.S. 2009. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 9:28‐39.
   Zhang, J., Adrian, F.J., Jahnke, W., Cowan‐Jacob, S.W., Li, A.G., Iacob, R.E., Sim, T., Powers, J., Dierks, C., Sun, F., Guo, G.R., Ding, Q., Okram, B., Choi, Y., Wojciechowski, A., Deng, X., Liu, G., Fendrich, G., Strauss, A., Vajpai, N., Grzesiek, S., Tuntland, T., Liu, Y., Bursulaya, B., Azam, M., Manley, P.W., Engen, J.R., Daley, G.Q., Warmuth, M., and Gray, N.S. 2010. Targeting Bcr‐Abl by combining allosteric with ATP‐binding‐site inhibitors. Nature 463:501‐506.
   Zhou, H. and Huang, S. 2010. The complexes of mammalian target of rapamycin. Curr. Protein Pept. Sci. 11:409‐424.
   Zuccotto, F., Ardini, E., Casale, E., and Angiolini, M. 2010. Through the “Gatekeeper Door”: Exploiting the active kinase conformation. J. Med. Chem. 53:2681‐2694.
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