Developing chemical biology approaches for the activity based investigations of reversible protein phosphorylation mediating enzymes

  DEVELOPING CHEMICAL BIOLOGY APPROACHES FOR THE ACTIVITY-BASED INVESTIGATIONS OF REVERSIBLE PROTEIN PHOSPHORYLATIONMEDIATING ENZYMES KARUNAKARAN NAIR A. KALESH (M.Sc, Indian Institute of Technology, Madras, India) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010     Acknowledgement First and foremost, I express my deepest gratitude to my supervisor A/P Yao Shao Qin for being nothing less than a wonderful research advisor. Words are too few to express how much he has influenced me and how much he has inspired me in this journey. He has given me an incredibly encouraging and motivating environment to science, he taught me how to find answers to my questions, and every scientific discussion with him has fueled my passion for science. His unparallel commitment and dedication to science, professionalism and quick and intelligent approaches to problem solving have deeply influenced me and I hope they will guide me in my scientific journey in the years ahead. Thanks are due to my colleagues in the Yao lab (Chemistry and Biology) for all your help, collaborations, discussions and most importantly your friendship which turned all the inevitable difficulties in research into wonderful learning experience, which I will cherish for ever. Souvik, Mingyu, Raja, Junqi, Liu Kai, Haibin, Lay Pheng, Candy, Jingyan, Hongyan, Bahulayan, Kitty, Liquian, Pengyu, Jigang, Wu Hao, Mahesh, Joo Leng, Li Bing, Derek, Wee Liang, Grace, Farhana, Wang Jun, Li Lin, Chongjing, Xiamin, Zhenkun, Su Ying, Cathy, Catherine, Ching Tian, Su Ling, Shen Yuan – working with all of them have been great experience. From day one, I thank Raja for introducing me to the Chemistry lab, for showing me for the first time how to run a column, for all your support at every stage of my life in the lab. I thank Souvik for all his help, both professional and personal, throughout my life at Yao lab. He was there always to discuss science, to help me troubleshoot bio-experiments, with utmost sincerity, nothing less than what one could expect from the closest friend or relative. ii      Special thanks are due to my collaborators- Liu Kai for providing me all the kinases, for the PTP-pull-down experiments and for all the biological experiments with the NDA-AD cross-linker. Lay Pheng for providing me all the PTPs and for helping me in the PTP-labeling experiments, Joo Leng for helping me in the synthesis of the caged PTP-probes, Li Bing and Wee Liang for helping me in the synthesis of the NDA-AD cross-linker, Derek for helping me in the synthesis of the dialdehyde and Liquian and Hongyan for their help in the peptide synthesis - I have been extremely fortunate to have worked all of you. There are a number of people outside Yao lab who made this journey more enjoyable- Santhosh, Rajesh, Abhilash I thank you all for your true friendship. I thank all staff from the Chemistry office, in particular Suria. I appreciate the support of the laboratory staff from the NMR and MS labs for the training and technical assistance. I would never have accomplished this without the support, prayers and sacrifices of my parents. Kala-my sister, I would never have overcome difficulties in life without her support. Words are too few to express how much she has helped me to stabilize, emotionally, at all difficult times - both in personal life and in professional life. I dedicate this thesis to you Kala- my dear sister. Last but not the least I thank the NUS for financial support in the form of the research scholarship. iii      Table of Contents Contents Chapter 1: Introduction Page 1. Proteomic approaches for the global analysis of protein expression and functions. 1. 1. Methods based on liquid chromatography-tandem mass spectrometry (LC- MS/MS) 1. 1. Methods based on isotope coded affinity tagging-tandem massspectrometry (ICAT- MS/MS) 1. 1. Yeast-two-hybrid assays 1. 1. Activity-based protein profiling (ABPP) 1. 1. 4. General design considerations of ABPs 1. 1. 4. “Label-free” versions of ABPs 13 1. 1. 4. “Label-free” clickable versions of AfBPs 15 1. 1. 4. “Non-directed approaches” in ABP designs 16 1. Protein phosphorylation - An important post-translational modification (PTM) 16 1. 2. Protein kinases 18 1. 2. Protein phosphatases 20 iv      1. 2. Catalytic mechanism of Protein tyrosine phosphatases (PTPs) 21 1. Enzyme inhibitor developments - Fragment-based approaches and high-throughput chemistry 22 1. 3. “Click chemistry” in enzyme inhibitor developments 23 1. 3. “In-situ click chemistry” facilitated enzyme inhibitor developments 24 Chapter 2: Development of Peptide-Based Activity-Based Probes (ABPs) for Protein Tyrosine Phosphatases (PTPs) 27 Summary 27 2. Introduction 27 2. Synthesis of the unnatural amino acid, 2-FMPT 30 2. Solid-phase synthesis of substrate peptides and peptide-based activity-based probes 31 2. Expression and purification of PTPs 34 2. Results and discussions 36 2. 5. Labeling experiments with purified proteins 36 2. 5. Detection limits of the probes 39 2. 5. Labeling experiments with mutant PTPs 39 2. 5. Effect of H2O2 on PTP activity assessed with the probes 40 2. 5. Kinetic characterizations and substrate specificities of the probes v      and the corresponding phosphopeptides 42 2. 5. Labeling experiments in the presence of complex proteomes 47 2. 5. 6. Labeling in the presence of bacterial cell lysates 47 2. 5. 6. Labeling in the presence of mammalian proteome 48 2. Conclusions 50 2. General procedures for sample preparations and labeling experiments using proteomes 51 2. 7. Preparation of bacterial cell lysates and labeling experiments using the lysates 51 2. 7. Procedure for ‘Western Blot’ analysis 51 2. 7. Procedure for ‘Pull-down’ of biotinylated probe labeled PTP 52 2. Synthetic details and characterizations of compounds 53 Chapter 3: Caged Activity-Based Probes for Protein Tyrosine Phosphatases Summary 59 3. Introduction 59 3. Synthesis of caged 2-FMPT 65 3. Synthesis of caged peptide-based activity-based probes 67 3. Results and discussion 69 3. Conclusions 71 vi      3. Synthetic details and chemical characterizations 72 Chapter 4: High-Throughput Synthesis of Abelson Tyrosine Kinase (Abl) Inhibitors using Click Chemistry 77 Summary 77 4. Introduction 77 4. Results and discussions 79 4. 2. The first-generation kinase click inhibitors 79 4. 2. The second-generation kinase click inhibitors 82 4. 2. Kinase inhibition assays 84 4. 2. 3. Screening of the inhibitor library and generation of heat-map 84 4. 2. 3. IC50 evaluation of the click-inhibitors against Abl and Src kinases 4. 2. Cell culturing and anti-proliferative assay 4. Conclusions 4. General experimental procedures 4. 4. The click-assembly of inhibitors 87 89 99 100 100 4. 4. 1. General procedures for the click-assembly of 344-member library formed from ADP-alkyne and azides 100 vii      4. 4. 1. General procedures for the click-assembly of 90-member Imatinib analogue library formed from the two warheads (W1 & W2) and azides 100 4. 4. General procedures for Kinase inhibition assays 101 4. 4. General procedures for cell-culturing and anti-proliferation assays 102 4. Synthetic details and characterizations of compounds 103 Chapter 5: A Mechanism-Based Cross-Linker for Protein Kinase-Substrate Complexes 114 Summary 114 5. Introduction 114 5. Synthesis of the cross-linkers 117 5. 2. Synthesis of OPA-AD 117 5. 2. Synthesis of NDA-AD 118 5. Synthesis of peptide pseudosubstrates 119 5. Results and discussions 120 5. Conclusions 123 5. Synthetic details and characterizations of compounds 124 Chapter Small-Molecule Probes that Target Abl Kinase 130 Summary 130 viii      6. Introduction 130 6. Synthesis of the probes 134 6. Results and discussions 136 6. 3. Labeling experiments with the dialdehyde-7 136 6. 3. 1. Labeling experiments with pure kinases and kinase spiked in cellular lysates 136 6. 3. 1. pH-dependence of labeling reaction 138 6. 3. 1. Effect of exogenous thiols on the efficiency of labeling 139 6. 3. 1. Effect of exogenous amines on the efficiency of labeling 140 6. 3. 1. IC50 evaluation of the probe 140 6. 3. Labeling experiments with the photo cross-linkers 6. 3. 2. Comparative labeling experiments 142 142 6. 3. 2. Detection limit of pure Abl with the photo-cross-linker 6-13 146 6. 3. 2. Labeling experiments with the clickable probe (6-13) in the presence of K562 cell lysate 146 6. Conclusions 148 6. Synthetic details and characterizations of compounds 149 ix      Chapter 7: Future directions 153 Summary 153 7. Protein-based PTP probes to identify/validate the PTPs responsible for dephosphorylating a given substrate protein 153 7. Synthesis of a scaffold for the development of affinity-based probes (AfBPs) and bidentate inhibitors of protein kinases with a compact gatekeeper residue 161 Chapter 8: Concluding remarks 169 Chapter 9: References 172 Appendix 190 x      64. T. P. Geladopoulos, T. G. Sotiroudis and A. E. Evangelopoulos, Anal. Biochem., 1991, 192, 112-116. 65. L. V. Ravichandran, H. Chen, Y. Li and M. J. Quon, Mol. Endocrinol., 2001, 15, 1768-1780. 66. (a) J. M. Xie and P. G. Schultz, Nat. Rev. Mol. Cell. Biol., 2006, 7, 775-782. (b) T. W. Muir, Annu. Rev. Biochem., 2003, 72, 249-289. 67. G. Mayer and A. Heckel, Angew. Chem. Int. Ed., 2006, 45, 4900-4921. 68. J. H. Kaplan, B. Forbush III and J. F. Hoffman, Biochemistry, 1978, 17, 19291935. 69. J. Engels and E. –J. Schlaeger, J. Med. Chem., 1977, 20, 907-911. 70. (a) T. Furuta and M. Iwamura, Methods in Enzymology, 1998, 291, 50-63. (b) T. Furuta, S. S. H. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee, E. M. Callaway, W. Denk and R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 1193-1200. (c) A. Z. Suzuki, T. Watanabe, M. Kawamoto, K. Nishiyama, H. Yamashita, M. Ishii, M. Iwamura and T. Furuta, Org. Lett., 2003, 5, 4867-4870. (d) V. Hagen, S. Frings, B. Wiesner, S. Helm, U. B. Kaupp and J. Bendig, ChemBioChem, 2003, 4, 434-442. 71. A. D. Turner, S. V. Pizzo, G. Rozakis and N. A. Porter, J. Am. Chem. Soc., 1988, 110, 244-250. 72. (a) R. S. Givens and C. –H. Park, Tetrahedron Lett., 1996, 37, 6259-6262. (b) C. – H. Park and R. S. Givens, J. Am. Chem. Soc., 1997, 119, 2453-2463. (c) R. S. Givens, A. Jung, C. –H. Park, J. Weber and W. Bartlett, J. Am. Chem. Soc., 1997, 180      119, 8369-8370. (d) G. Arabaci, X. –C. Guo, K. D. Beebe, K. M. Coggeshall and D. Pei, J. Am. Chem. Soc., 1999, 121, 5085-5086. (e) R. S. Givens, J. F. W. Weber, P. G. Conrad, G. Orosz, S. L. Donahue and S. A. Thayer, J. Am. Chem. Soc., 2000, 122, 2687-2697. (f) K. Zou, W. T. Miller, R. S. Givens and H. Bayley, Angew. Chem. Int. Ed., 2001, 40, 3049-3051. 73. M. Lukeman and J. C. Scaiano, J. Am. Chem. Soc., 2005, 127, 7698-7699. 74. (a) J. W. Walker, G. P. Reid, J. A. McCray and D. R. Trentham, J. Am. Chem. Soc., 1988, 110, 7170-7177. (b) J. E. T. Corrie, A. Barth, V. R. N. Munasinghe, D. R. Trentham and M. C. Hutter, J. Am. Chem. Soc., 2003, 125, 8546-8554. 75. (a) K. Curley and D. S. Lawrence, J. Am. Chem. Soc., 1998, 120, 8573-8574. (b) Chung-yu Chang, T. Fernandez, R. Panchal and H. Bayley, J. Am. Chem. Soc., 1998, 120, 7661-7662. 76. J. S. Wood, M. Koszelak, J. Liu and D. S. Lawrence, J. Am. Chem. Soc., 1998, 120, 7145-7146. 77. W. F. Veldhuyzen, Q. Nguyen, G. McMaster and D. S. Lawrence, J. Am. Chem. Soc., 2003, 125, 13358-13359. 78. D. Humphrey, Z. Rajfur, M. E. Vazquez, D. Scheswohl, M. D. Schaller, K. Jacobson and B. Imperiali, J. Biol. Chem., 2005, 280, 22091-22101. 79. T. Kawakami, H. Cheng, S. Hashiro, Y. Nomura, S. Tsukiji, T. Furuta and T. Nagamune, ChemBioChem, 2008, 9, 1583-1586. 80. H. Li, Jung-Mi Hah and D. S. Lawrence, J. Am. Chem. Soc., 2008, 130, 1047410475. 181      81. I. A. Yudushkin, A. Schleifenbaum, A. Kinkhabwala, B. G. Neel, C. Schultz and P. I. H. Bastiaens, Science, 2007, 315, 115-119. 82. (a) D. M. Rothman, M. E. Vázquez, E. M. Vogel and B. Imperiali, Org. Lett., 2002, 4, 2865-2868. (b) D. M. Rothman, E. J. Petersson, M. E. Vázquez, G. S. Brandt, D. A. Dougherty and B. Imperiali, J. Am. Chem. Soc., 2005, 127, 846-847. 83. R. Reinhard and B. F. Schmidt, J. Org. Chem., 1998, 63, 2434-2441. 84. K. Gumireddy, S. J. Baker, S. C. Cosenza, P. John, A. D. Kang, K. A. Robell, , M. V. R. Reddy and E. P. Reddy, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 19921997. 85. F. J. Adriàn, Q. Ding, T. Sim, A. Velentza, C. Sloan, Y. Liu, G. Zhang, W. Hur, S. Ding, P. Manley, J. Mestan, D. Fabbro and N.S. Gray, Nat. Chem. Biol., 2006, 2, 95-102. 86. (a) Y. Liu and N. S. Gray, Nat. Chem. Biol., 2006, 2, 358-364. (b) C. Pargellis, L. Tong, L. Churchill, P.F. Cirillo, T. Gilmore, A. G. Graham, P. M Grob, E. R. Hickey, N. Moss, S. Pav and J. Regan, Nat. Struct. Biol., 2002, 9, 268-272. (c) J. G. Cumming, C.L. McKenzie, S.G. Bowden, D. Campbell, D.J. Masters, J. Breed, and P. J. Jewsbury, Bioorg. Med. Chem. Lett., 2004, 14, 5389-5394. (d) P. W Manley, G. Bold, J. Brüggen, G. Fendrich, P. Furet, J. Mestan, C. Schnell, B. Stolz, T. Meyer, B. Meyhack, W. Stark, A. Strauss and J. Wood, Biochim. Biophys. Acta., 2004, 1697, 17-27. (e) P. T. C. Wan, M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz, V. M. Good, C. M. Jones, C. J. Marshall, C. J. Springer, D. Barford and R. Marais, Cell, 2004, 116, 855-867. (f) A. L. Gill, M. Frederickson, A. Cleasby, S. J. Woodhead, M. G. Carr, A. J. Woodhead, M. T. 182      Walker, M. S. Congreve, L. A. Devine, D. Tisi, M. O’Reilly, L. C. A. Seavers, D. J. Davis, J. Curry, R. Anthony, A. Padova, C. W. Murray, R. A. E. Carr and H. Jhoti, J. Med. Chem., 2005, 48, 414-426. (g) A. Ricouart, J. C. Gesquiere, A. Tartar and C. Sergheraert, J. Med. Chem., 1991, 34, 73-78. (h) K. Parang, J. H. Till, A. J. Ablooglu, R. A. Kohanski, S. R. Hubbard and P.A. Cole, Nat. Struct. Biol., 2001, 8, 37-41. (i) J. H. Lee, S. Kumar and D.S. Lawrence, ChemBioChem, 2008, 9, 507-517. (j) S. C. Meyer, C. D. Shomin, T. Gaj and I. Ghosh, J. Am. Chem. Soc., 2007, 129, 13812-13813. (k) E. Enkvist, D. Lavogina, G. Raidaru, A. Vaasa, I. Viil, M. Lust, K. Viht and A. Uri, J. Med. Chem., 2006, 49, 7150-7159. 87. M. W. N. Deininger, J. M. Goldman and J.V. Melo, Blood, 2000, 96, 3343-3356. 88. B. Nagar, O. Hantschel, M. A. Young, K. Scheffzek, D. Veach, W. Bornmann, B. Clarkson, G. Superti-Furga and J. Kuriyan, Cell, 2003, 112, 859-871. 89. T. Schindler, W. Bornmann, P. Pellicena, W. T. Miller, B. Clarkson and J. Kuriyan, Science, 2000, 289, 1938-1942. 90. (a) P. W. Manley, S. W. Cowan-Jacob and J. Mestan, Biochim. et Biophy. Acta, 2005, 1754, 3-13. (b) M. E. Gorre, M, Mohammed, K. Ellwood, N. Hsu, R. Paquette, P. N. Rao and C. L. Sawyers, Science, 2001, 293, 876-880. (c) N. von Bubnoff, F. Schneller, C. Peschel and J. Duyster, Lancet, 2002, 359, 487-491. 91. (a) T. O’Hare, D. K. Walters, M. W. N. Deininger and B. J. Druker, Cancer Cell, 2005, 7, 117-119. (b) P. La Rosee, A. S. Corbin, E. P. Stoffregen, M. W. Deininger and B. J. Druker, Cancer Res., 2002, 62, 7149-7153. (c) J. S. Tokarski, J. A. Newitt, C. Y. J. Chang, J. D. Cheng, M. Wittekind, S. E. Kiefer, K. Kish, F. Y. F. Lee, R. Borzillerri, L. J. Lombardo, D. Xie, Y. Zhang and H. E. Klei, 183      Cancer Res., 2006, 66, 5790-5797. (d) P. W. Manley, W. Breitenstein, J. Brüggen, S.W. Cowan-Jacob, P. Furet, J. Mestan and T. Meyer, Bioorg. Med. Chem. Lett., 2004, 14, 5793-5797. (e) E. Weisberg, P. W. Manley, S. W. Cowan-Jacob, A. Hochhaus and J. D. Griffin, Nat. Rev. Cancer, 2007, 7, 345-356. 92. For reviews, see: (a) K. A. Kalesh, P. –Y. Yang, R. Srinivasan and S. Q. Yao, QSAR Comb. Sci., 2007, 26, 1135-1144. (b) A. Birk, C. -Y. Wu and C. -H. Wong, Org. Biomol. Chem., 2006, 4, 1446-1457. 93. L.V. Lee, M.L. Mitchell, S-J. Huang, V.V. Fokin, K.B. Sharpless and C-H. Wong, J. Am. Chem. Soc., 2003, 125, 9588-9589. 94. Y. –F. Liu, C. –L. Wang, Y. –J. Bai, N. Han, J. –P. Jiao and X. –L. Qi,. Org. Process Res. Dev., 2008, 12, 490-495. 95. (a) D. A. Walsh and D. B. Glass, Methods in Enzymology, 1991, 201, 304-316. (b) H. Hidaka, M. Watanabe and K. Kobayashi, Methods in Enzymology, 1991, 201, 328-339. (c) T. Tamaoki, Methods in Enzymology, 1991, 201, 340-347. 96. N. W. Roehm, G. H. Rodgers, S. M. Hatfield and A. L. Glasebrook, J. Immunol. Methods, 1991, 142, 257-265. 97. M. A. Seeliger, M. Young, M. N. Henderson, P. Pellicena, D. S King, A. M. Falick and J. Kuriyan, Protein Sci., 2005, 14, 3135-3139. 98. J. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc., 1961, 83, 649-658. 99. Z. S. Szakács, S. Béni, Z. Varga, L. Örfi, G. Kéri and B. Noszál, J. Med. Chem., 2005, 48, 249-255. 184      100. G. B. Bennett, R. B. Mason, L.J. Alden and J. B. Roach, Jr, J. Med. Chem., 1978, 21, 623-628. 101. M. Lautens, J-F. Paquin, S. Piguel and M. Dahlmann, J. Org. Chem., 2001, 66, 8127-8134. 102. J. Villén, S. A. Beausoleil, S. A. Gerber and S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 1488-1493. 103. (a) D. T. McLachlin and B. T. Chait, Curr. Opin. Chem. Biol., 2001, 5, 591-602. (b) Z. A. Knight, B. Schilling, R. H. Row, D. M. Kenski, B. W. Gibson and K. M. Shokat, Nat. Biotechnol., 2003, 21, 1047-1054. 104. (a) J. Ptacek and M. Snyder, Trends Genet., 2006, 22, 545-554. (b) Z. Songyang, S. Blechner, N. Hoagland, M. F. Hoekstra, H. Piwnica-Worms and L. C. Cantley, Curr. Biol., 1994, 4, 973-982. (c) J. Ptacek, Nature, 2005, 438, 679-684. (d) J. J. Allen, M. Li, C. S. Brinkworth, J. L. Paulson, D. Wang, A. Hübner, W. H. Chou, R. J. Davis, A. L. Burlingame, R. O. Messing, C. D. Katayama, S. M. Hedrick and K. M. Shokat, Nat. Methods, 2007, 4, 511-516. (e) J. D. Blethrow, J. S. Glavy, D. O. Morgan and K. M. Shokat, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 14421447. 105. D. J. Maly, J. A. Allen, K. M. Shokat, J. Am. Chem. Soc., 2004, 126, 9160-9161. 106. P. Zuman, Chem. Rev., 2004, 104, 3217-3238. 107. S. P. Haris, Y. Zhang, B. L. Bourdonnec, C. R. McCurdy and P. S. Portoghese, J. Med. Chem., 2007, 50, 3392-3396. 185      108. K. Liu, K. A. Kalesh, L. B. Ong and S. Q, Yao, ChemBioChem, 2008, 9, 18831888. 109. (a) M. S. Cohen, H. Hadjivassiliou and J. Taunton, Nat. Chem. Biol., 2007, 3, 156-160. (b) J. A. Blair, D. Rauh, C. Kung, C. –H. Yun, Q. -W. Fan, H. Rode, C. Zhang, M. J. Eck, W. A. Weiss and K. M. Shokat, Nat. Chem. Biol., 2007, 3, 229238. 110. (a) M. –C. Yee, S. C. Fas, M. M. Stohlmeyer, T. J. Wandless and K. A. Cimprich, J. Biol. Chem., 2005, 280, 29053-29059. (b) Y. Liu, K. R. Shreder, W. Gai, S. Corral, D. K. Ferris and J. S. Rosenblum, Chem. Biol., 2005, 12, 99-107. 111. S. J. Ratcliffe, T. Yi and S. S. Khandekar, J. Biomol. Screen., 2007, 12, 126-132. 112. M. P. Patricelli, A. K. Szardenings, M. Liyanage, T. K. Nomanbhoy, M. Wu, H. Weissig, A. Aban, D. Chun, S. Tanner and J. W. Kozarich, Biochemistry, 2007, 46, 350-358. 113. (a) E. D. Lowe, I. Tews, K. Y. Cheng, N. R. Brown, S. Gul, M. E. Noble, S. J. Gamblin and L. N. Johnson, Biochemistry, 2002, 41, 15625-15634. (b) W. F. Waas and K. N. Dalby, J. Biol. Chem., 2002, 277, 12532-12540. (c) B. E. Aubol, B. Nolen, D. Vu, G. Ghosh and J. A. Adams, Biochemistry, 2002, 41, 1000210009. (d) D. A. Critton, A. Tortajada, G. Stetson, W. Peti and R. Page, Biochemistry, 2008, 47, 13336-13345. 114. C. Blanchetot, M. Chagnon, N. Dubé, M. Hallé and M. L. Tremblay, Methods, 2005, 35, 44-53. 115. (a) M. T. Brown and J. A. Cooper, Biochem. Biophys. Acta, 1996, 1287, 121-149. (b) S. M. Thomas and J. S. Brugge, Annu. Rev. Cell Dev. Biol., 1997, 13, 513-609. 186      116. (a) S. Nada, M. Okada, A. MacAuley, J. A. Cooper and H. Nakagawa, Nature, 1991, 351, 69-72. (b) R. R. Roussel, S. R. Brodeur, D. Shalloway and A. P. Laudano, , Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 10696-10700. (c) K. B. Bibbins, H. Boeuf and H. E. Varmus, Mol. Cell. Biol., 1993, 13, 7278-7287. (d) X. Liu, S. R. Brodeur, G. Gish, Z. Songyang, L. C. Cantley, A. P. Laudano and T. Pawson, Oncogene, 1993, 8, 1119-1126. (e) M. Ruzzene, Z. Songyang, O. Marin, A. Donella-Deana, A. M. Brunati, B. Guerra, P. Agostinis, L. C. Cantley and L. A. Pinna, Eur. J. Biochem., 1997, 246, 433-439. (f) D. Sondhi, W. Xu, Z. Songyang, M. J. Eck and P. A. Cole, Biochemistry, 1998, 37, 165-172. (g) P. A. Cole, K. Shen, Y. Qiao and D. Wang, Curr. Opin. Chem. Biol., 2003, 7, 580-585. 117. (a) C. Jacobs and H. Rubsamen, Cancer Res., 1983, 43, 1696-1702. (b) C. A. Cartwright, A. I. Meisler and W. Eckhart, Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 558-562. (c) D. K. Luttrell, A. Lee, T. J. Lansing, R. M. Crosby, K. D. Jung, D. Willard, M. Luther, M. Rodriguez, J. Berman and T. M. Gilmer, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 83-87. (d) M. S. Talamonti, M. S. Roh, S. A. Curley and G. E. Gallick, J. Clin. Invest., 1993, 91, 53-60. (e) N. Resen, J. B. Bolen, A. M. Schwartz, P. Cohen, V. Deseau and M. A. Israel, J. Biol. Chem., 1986, 261, 13754-13759. (f) A. E. Ottenhoff-Kalff, G. Rijksen, E. A. van Beurden, A. Hennipman, A. A. Michels and G. E. Staal, Cancer Res., 1992, 52, 4773-4778. (g) C. Egan, A. Pang, D. Durda, H. C. Cheng, J. H. Wang and D. J. Fujita, Oncogene, 1999, 18, 1227-1237. 118. (a) S. Bagrodia, I. Chackalaparampil, T. E. Kmiecik and D. Shalloway, Nature, 1991, 349, 172-175. (b) I. Chackalaparampil and D. Shalloway, Cell, 1988, 52, 801-810. (c) R. M. Kypta, Y. Goldberg, E. T. Ulug and S. A. Courtneidge, Cell, 1990, 62, 481-492. 187      119. J. D. Bjorge, A. Pang and D. J. Fujita, J. Biol. Chem., 2002, 275, 41439-41446. 120. (a) T. W. Muir, D. Sondhi and P. A. Cole, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 6705-6710. (b) D. Wang and P. A. Cole, J. Am. Chem. Soc., 2001, 123, 88838886. (c) K. Shen and P. A. Cole, J. Am. Chem. Soc., 2003, 125, 16172-16173. 121. (a) M. –Q. Xu and F. B. Perler, EMBO J., 1996, 15, 5146-5153. (b) S. Chong, F. B. Mersha, D. G. Comb, M. E. Scott, D. Landry, L. M. Vence, F. B. Perler, J. Benner, R. B. Kucera, C. A. Hirvonen, J. J. Pelletier, H. Paulus and Ming-Qun Xu, Gene, 1997, 192, 271-281. 122. P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science, 1994, 266, 776-779. 123. (a) S. Mitra and A. M. Barrios, Bioorg. Med. Chem. Lett., 2005, 15, 5142-5145. (b) S. Mitra and A. M. Barrios, Anal. Biochem., 2007, 370, 249-251. 124. (a) K. Shah, Y. Liu, C. Deirmengian and K. M. Shokat, Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 3565-3570. (b) Y. Liu, K. Shah, F. Yang, L. Witucki and K. M. Shokat, Chem. Biol., 1998, 5, 91-101. (c) T. Fox, J. Coll, X. Xie, P. Ford, U. Germann, M. Porter, S. Pazhanisamy, M. Fleming, V. Galullo, M. Su and K. Wilson, Protein Sci., 1998, 7, 2249-2255. (d) A. Bishop, C. Kung, K. Shah, L. Wituchi, K. M. Shokat and Y. Liu, J. Am. Chem. Soc., 1999, 121, 627-631. (e) K. Specht and K. M. Shokat, Curr. Opin. Cell Biol., 2002, 14, 155-159. 125. (a) K. Wilson, P. McCaffrey, K. Haiao, S. Pazhanisamy, V. Galullo, G. Bemis, M. Fitzgibbon, P. Caron, M. Murcko and M. Su, Chem. Biol., 1997, 4, 423-431. (b) A. Bridges, Chem. Rev., 2001, 101, 2541-2572. (c) M. Gorre, M. Mohammed, K. 188      Ellwood, N. Hsu, R. Paquette, P. Rao and C. Sawyers, Science, 2001, 293, 876880. 126. (a) M. S. Cohen, C. Zhang, K. M. Shokat and J. Taunton, Science, 2005, 308, 1318-1321. (b) J. A. Blair, D. Rauh, C. Kung, Cai-Hong Yun, Qi-Wen Fan, H. Rode, C. Zhang, M. J. Eck, W. A. Weiss and K. M. Shokat, Nat. Chem. Biol., 2007, 3, 229-238. Appendix 189      11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 7.6811 1.5241 5.5448 1.0558 0.7458 1.6446 0.6519 0.8685 3.5021 1.9125 2.0523 2.0000 O NH 1.0 3.0 0.0003 3.1055 4.1004 4.1234 4.1464 4.2352 4.2697 4.2927 4.3617 2.0 2.0000 1.2588 4.3858 4.4209 3.0 2.5522 4.6148 5.2481 5.4059 5.5895 4.0 0.9498 3.3317 5.6141 7.0922 5.0 4.1441 0.9857 7.1185 7.2226 7.2478 7.2538 7.2719 7.3130 7.3371 7.3612 7.4094 7.4319 7.4483 7.4549 7.4653 7.4954 7.5168 7.5371 7.5639 7.5902 7.6138 6.0 5.1939 O O P O O 2.4987 NH 7.0 2.0196 O 8.0 2.4952 HO 9.0 4.0302 O 7.6286 10.0 7.6549 0.9011 F 7.6833 HO 7.7080 O 8.0509 8.0783 Integral O 2.4843 2.2432 Integral 1.2921 1.3162 1.3397 3.0693 3.0885 3.1159 3.1345 3.1717 3.1871 3.2167 4.1294 4.1858 4.2088 4.2346 4.2559 4.2937 4.3896 4.4143 4.4236 4.4482 4.6767 4.7013 5.3248 5.4820 5.8102 5.8354 7.1338 7.1612 7.2604 7.2828 7.3075 7.3453 7.3699 7.3940 7.5359 7.5611 7.5863 7.7222 7.7474 9.4638   1H normal range AC300, FmocAA *** Current Data Parameters *** NAME EXPNO : PROCNO 2.0 : : BF1 : LOCNUC : NS : O1 : PULPROG : ag16kka *** Acquisition Parameters *** 300.1300000 MHz 1853.43 Hz 2H SFO1 : 300.1318534 MHz SOLVENT : CDCl3 zg30 SW *** Processing Parameters *** : 17.9519 ppm LB : 0.30 Hz PHC0 : 155.201 degree PHC1 : 3.398 degree O O O P O 2-10 (ppm) 0.0 O O2 N F NO 3-7 (ppm) 1.0 190  0.0 10.8   10.4 10.0 9.6 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 4.0 3.6 3.6 3.2 3.2 2.3028 4.4 3.0000 7.50 4.8 3.1010 4.0620 4.0538 4.0433 4.0351 5.2 0.8660 1.9785 7.60 5.6 7.3348 6.0 7.3617 6.4 7.5501 7.5332 6.8 1.0337 O 7.6208 7.6005 7.5945 7.2 0.9959 1.0201 7.6 3.0000 0.8908 1.1449 0.7483 0.9020 N 1.0337 0.9960 H N 0.9959 8.0 7.6268 7.6208 7.6005 7.5945 7.5501 7.5332 7.3617 7.3348 8.4 1.0201 N 0.8546 0.9232 0.8441 0.8409 0.7767 Integral N 1.0547 H N 8.1347 8.1298 7.8947 7.8772 7.8679 7.8509 8.8 1.0179 N 9.2 1.0991 9.6 1.2588 10.0 2.0583 10.4 1.1578 9.3575 9.2194 8.8842 8.8661 8.8480 8.8053 8.7784 8.6091 8.5916 10.8 1.2760 Integral 2.3042 3.3101 3.3046 3.7363 4.8402 7.2426 7.2152 7.1522 7.1445 7.1248 7.1171 7.4809 7.4634 8.2835 8.2775 7.9800 7.9537 7.9334 8.5667 8.5492 8.8155 9.0439 9.0171 9.5260   1H normal range AC300, B3-217 H N O N 4-8 (ppm) 1H normal range AC300, W2 N H N 4-13 (ppm) 7.40 2.8 2.4 2.8 2.4 2.0 1.6 1.2 0.8 0.4 0.0 (ppm) 2.0 1.6 1.2 0.8 0.4 0.0 191    10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 4.50 5.0 N 4.5 3.6 4.0 3.2 2.8 3.5 2.4 N 3.0 2.0 2.5 2.2296 4.0 2.5068 2.5002 0.4731 2.3321 1.1620 2.7854 0.5090 1.0000 0.8991 1.0083 Integral H 2N 2.7383 (ppm) 4.4 3.3592 3.3406 3.3220 4.5026 4.4851 4.4653 4.8 2.6663 (ppm) 5.2 2.0000 7.4 5.6 3.3592 3.3406 3.3220 6.0 2.0000 4.5026 4.4851 4.4653 6.4 1.9956 0.9010 6.8 1.9956 0.9010 1.9118 1.6698 1.1286 7.2 1.9118 7.6 1.6698 (ppm) 7.6 1.1286 0.9577 8.0 0.9577 7.8 0.9829 0.9829 7.7503 7.6873 7.6708 7.6604 7.6440 7.5530 7.5470 7.5256 7.5191 7.5032 7.4785 7.4610 7.3750 7.3635 7.3520 7.3394 7.2276 7.1997 8.4 0.9705 8.60 0.8693 0.9705 8.8 1.7833 9.2 0.9593 9.6 0.9347 8.70 1.7833 9.3270 9.0333 8.7616 8.6465 8.6203 8.5693 8.5583 8.5414 8.1562 8.1502 8.0006 7.9809 7.9612 7.7760 7.7503 7.6873 7.6708 7.6604 7.6440 7.5530 7.5470 7.5256 7.5191 7.5032 7.4785 7.4610 7.3750 7.3635 7.3520 7.3394 7.2276 7.1997 10.3268 10.0 0.9160 8.6465 8.6203 8.5693 8.5583 8.5414 8.7616 10.4 0.8714 0.9593 0.9347 10.8 0.8833 Integral 1.9454 6.0406 6.0225 4.6496 4.6326 4.6151 4.4951 4.4864 4.4781 4.4710 4.4622 4.4535 4.4365 4.4201 4.4113 4.3981 4.3072 4.2990 4.2453 4.2371 4.2289 4.2201 4.2053 4.1976 4.1900 4.1812 4.1713 4.1620 4.1527 4.1423 4.1319 4.1231 4.1133 2.7601 8.3131 8.4961   1H Water suppression N N H N O P N O HO 1.6 1.2 H N N 2.0 0.8 0.4 1.5 O O OH O O P OH OH 4-4 (ppm) 0.0 1H normal range AC300, W1-SA20 HN S O O N N N W1-SA20 O 3.30 N (ppm) (ppm) 1.0 192  0.5 0.0   12.0 11.0 10.0 9.0 8.0 7.0 8.0 6.0 7.8 5.0 7.6 7.4 4.0 4.5 3.0 4.0 2.0 3.5 N 1.0 5.6473 2.0000 0.9849 3.8151 0.9369 0.9356 1.7927 0.9069 0.9367 0.7542 0.8463 7.3500 7.3237 7.5242 7.5045 7.4842 7.4536 7.6420 7.6157 7.8239 7.7987 7.7806 7.9724 7.9532 7.9258 8.5684 8.5860 8.7388 8.7120 8.8336 8.9459 8.9267 N 3.0 0.0669 5.0 1.2305 1.1965 1.1730 1.1489 5.5 2.0894 1.9124 7.6 2.6319 2.5399 2.5004 2.3870 2.2544 1.9865 6.0 1.0932 6.5 3.9500 4.2409 7.8 2.0073 0.9849 8.0 2.8672 8.2 7.7280 7.7105 7.7006 7.6842 7.5423 7.5363 7.5154 7.5083 7.5007 7.4837 7.2744 7.2459 7.0 1.1354 1.1388 8.1794 3.8151 0.9369 (ppm) 1.2469 20.701 8.4 0.9802 7.5 1.0932 0.9819 1.0247 0.9356 1.7927 0.8791 0.9069 0.9367 0.7542 0.8463 0.8261 0.6884 8.6 2.0073 8.6 1.1354 8.8 1.8824 0.8950 9.0719 8.8374 8.7892 8.7728 8.7541 8.6900 8.6742 8.5777 8.5602 8.4145 8.3860 8.2918 8.2868 8.2633 8.2583 8.0 1.1388 0.9802 9.0 0.8937 0.8676 8.5 0.9819 9.2 1.0247 1.0108 9.0 1.8824 9.4 0.8950 9.3644 9.5 0.8937 1.0000 10.0 8.7 0.8676 10.5 8.8 1.0108 Integral 10.5675 10.5610 9.3644 9.0719 8.8374 8.7892 8.7728 8.7541 8.6900 8.6742 8.5777 8.5602 8.4145 8.3860 8.2918 8.2868 8.2633 8.2583 8.1794 7.7280 7.7105 7.7006 7.6842 7.5423 7.5363 7.5154 7.5083 7.5007 7.4837 7.2744 7.2459 Integral 8.9 1.0000 2.6700 Integral 2.5 H N N 7.2 N (ppm) 2.2939 2.5004 3.2307 3.2110 3.1907 3.1710 4.4946 4.4765 4.3944 4.3741 4.3544 9.3261 9.1711 8.9640 8.9459 8.9267 8.8336 8.7388 8.7120 8.5860 8.5684 8.1449 7.9724 7.9532 7.9258 7.8239 7.7987 7.7806 7.6420 7.6157 7.5242 7.5045 7.4842 7.4536 7.3500 7.3237   1H normal range AC300, W2-SA13 H N O N N H 2.0 H N N N 1.5 NAME 6-7 O1 : DATE_t : DATE_d : : N H N O 1.0 EXPNO : PROCNO : INSTRUM : LOCNUC : NS O : NUCLEUS : off *** 1D NMR Plot Parameters *** NUCLEUS : off (ppm) 0.0 193  S F W2-SA13 (ppm) 0.5 1H Imatinib-NDA dialdehyde final (prep) in DMSO-d6 AC300 Bruker *** Current Data Parameters *** jl17ssb *** Acquisition Parameters *** Jul 17 2009 CHO 01:35:43 DBPNAM0 : spect CHO 2H SFO1 : 300.1318534 MHz 1853.43 Hz SOLVENT : DMSO O N (ppm) 7.4 0.0   N N 210 200 H N 190 H N N 180 170 160 150 140 130 120 110 100 90 80 3.0 70 60 50 40 17.5984 14.1073 4.0 33.9844 5.0 37.9409 6.0 3.0000 4.0745 0.8980 7.30 1.0934 7.40 1.0064 7.50 54.4652 7.0 1.0857 7.60 (ppm) 2.0 30 20 3.0000 4.0745 1.0286 1.0351 1.3880 1.6362 2.1113 6.1393 7.70 1.0286 1.0351 0.9563 0.9679 0.9956 0.8774 0.9289 8.4 71.2295 8.0 1.3880 1.6362 2.1113 (ppm) 83.6809 9.0 6.1393 0.9713 0.9563 8.6 149.4215 146.4105 142.9195 137.7338 137.2320 136.6719 135.2101 133.0427 132.5700 131.0063 130.2281 129.5735 129.4353 128.5335 127.2752 124.7224 116.1694 115.8712 107.7399 10.0 0.9679 8.8 0.9956 0.8774 0.9289 0.8752 9.0 170.4550 169.6332 160.9856 160.8547 159.5892 158.0182 195.5760 Integral 2.3075 7.2694 7.2628 7.2425 7.2354 7.1790 7.1510 7.5559 7.5318 7.5060 7.4764 7.4704 7.4529 7.7186 7.7016 7.6912 7.6781 7.6534 7.6260 8.3837 8.3568 8.5437 8.5267 8.6587 8.6319 8.7694 8.9858 3.1868 3.1681 3.1413 3.1238 3.0197 2.9890 2.9747 2.9441 2.6866 2.6784 2.3075 2.1990 4.7958 4.7668 4.7492 4.7202 10.0678 9.3189 8.9858 8.7694 8.6587 8.6319 8.5437 8.5267 8.3837 8.3568 7.9306 7.9246 7.7186 7.7016 7.6912 7.6781 7.6534 7.6260 7.5559 7.5318 7.5060 7.4764 7.4704 7.4529 7.2694 7.2628 7.2425 7.2354 7.1790 7.1510   1H normal range AC300, Photo CL-2 *** Current Data Parameters *** 1.0 O HN 10 NAME : EXPNO : PROCNO : ag25kka9 *** Acquisition Parameters *** BF1 : 300.1300000 MHz BF2 : 300.1300000 MHz BF3 : 300.1300000 MHz DATE_t : 06:01:12 DATE_d : Aug 25 2009 NS : 80 SOLVENT : DMSO 7.20 (ppm) (ppm) 0.0 13C Standard AC300, CL-2 *** Current Data Parameters *** NAME : EXPNO : PROCNO : ag13kka9 *** Acquisition Parameters *** BF1 : BF2 : 300.1300000 MHz BF3 : DATE_t : DATE_d : Aug 13 2009 NS : 15360 SOLVENT : DMSO 75.4677490 MHz 75.4677490 MHz 06:49:55 O 6-13 O (ppm) 194    210 200 190 180 170 160 150 140 130 120 110 5.0 100 4.5 90 4.0 80 3.5 70 3.0 60 2.5 50 2.0 40 30 21.8588 5.5 27.4663 6.0 37.2122 6.5 44.3034 42.9506 7.0 73.2936 7.5 78.7265 8.0 100.5164 8.5 120.6554 9.0 125.3465 9.5 131.0849 129.4703 129.1212 10.0 139.5725 10.5 142.9472 4.3404 0.8915 1.6814 1.7098 1.7245 1.1978 0.8260 3.8349 7-9 148.3001 1.0000 N 153.4203 164.1844 163.7553 0.8505 2.0004 2.5071 2.4080 2.3784 2.3565 3.1662 3.1437 3.1213 3.8335 4.4328 4.4115 4.3901 5.2952 5.8967 7.2855 7.1524 8.1768 10.6685   1H normal range AC300, B3-57 N NH N NH (ppm) 1.5 1.0 20 0.5 (ppm) 10 195  0.0 13C Standard AC300, B3-57 [...]... while the potential interaction partner (termed prey) is expressed with the activation domain If the two proteins (the bait and the prey) interact, it functionally reconstitutes the GAL4 which in tern induces the expression of reporter genes On the other hand if the proteins do not interact, the transcription of the reporter gene does not take place The Y2H system also allows the screening of the protein. .. 4.2 Synthesis of the two warheads (W1 & W2) for Imatinib -based click library 5.1 84 Scheme showing the three-component cross-linking reaction of kinase with its pseudosubstrate and NDA-AD 116 5.2 Synthesis of the cross-linker, OPA-AD 118 5.3 Synthesis of the cross-linker, NDA-AD 118 6.1 Synthesis of the Abl-directed probes 135 7.1 Scheme for constructing a protein -based PTP-probe using Expressed Protein. .. labeling of Abl with 6-13 in the presence of the generic kinase inhibitor Staurosporine 6.9 Evaluation of detection limit of pure Abl with compound 6-13 145 146 6.10 Labeling of different amounts of spiked Abl in the presence of K-562 mammalian proteome 7.1 Domain structure of c-Src kinase 147 156 7.2 Chemical structures of the peptide ligation partners for the construction of the protein -based PTP-probes... levels of the proteins, relatively newer approaches such as Activity- Based Protein Profiling (ABPP) provide more insights into the functional states of these proteins, which are of more relevance in the cellular physiology and pathology This dissertation reports certain chemical approaches developed towards betterunderstanding and manipulations of some important members in these two classes of proteins... Summary The reversible phosphorylation of proteins catalyzed by the opposing actions of protein kinases (PKs) and protein phosphatases (PPs) has been identified as one of the major post-translational modes (PTMs) of cellular signal transduction These two classes of enzymes and their extremely intricate protein interaction networks and associated signal cascades play the most crucial roles in maintaining the. .. post-translational protein modifications (PTMs) have long been known to play key roles in regulating the activities and functions of many proteins.3 Of the different PTMs, the reversible protein phosphorylation catalyzed by the opposing actions of protein kinases (PKs) and protein phosphatases (PPs) has been identified as one of the most important modes of cellular signal transduction.4 Hence, 1      detailed investigations. .. PTP-probes (A) the peptide for pTRAP probe and (B) the peptide for the ppCAP probe 7.3 LC-MS profiles of the purified pTRAP and pCAP peptides 158 160 xxviii      List of Tables Table Page 2.1 The 11 probes and their AA sequences and substrate preferences 2.2 34 Comparison of the substrate specificity of PTP1B obtained from the probe-mediated inactivation of the enzyme (Ki values) and that from the Malachite... List of Schemes Scheme 2.1 Synthesis of the unnatural amino acid, 2-FMPT 2.2 Page 31 Solid-phase synthesis of 10 phosphopeptides and 11 peptide -based ABPs 33 2.3 Schematic representation of different PTP constructs used 36 3.1 Proposed mechanism for light-mediated uncaging of o-nitrobenzyl caged molecules 60 3.2 Synthesis of caged 2-FMPT 65 3.3 Synthesis of caged peptide -based ABPs 67 4.1 Synthesis of. .. aspects; such as the expression levels, sub-cellular localizations, catalytic activities phosphorylation- mediating and enzymes are functional of roles paramount of these importance reversible in our understanding of their roles in maintaining the normal cellular physiology Together with several conventional proteomic techniques, relatively newer approaches such as activity- based protein profiling (ABPP)... Being the key mediators of several cellular communications, the activities of members in these two classes of enzymes are tightly controlled by a variety of mechanisms and in many cases imbalances in such a control and the resultant aberrant activities of some of these proteins have been identified as the root causes of several pathological conditions in humans Hence detailed investigations of individual .   DEVELOPING CHEMICAL BIOLOGY APPROACHES FOR THE ACTIVITY- BASED INVESTIGATIONS OF REVERSIBLE PROTEIN PHOSPHORYLATION- MEDIATING ENZYMES KARUNAKARAN NAIR A Wee Liang for helping me in the synthesis of the NDA-AD cross-linker, Derek for helping me in the synthesis of the dialdehyde 7 and Liquian and Hongyan for their help in the peptide synthesis. regarding the expression levels of the proteins, relatively newer approaches such as Activity- Based Protein Profiling (ABPP) provide more insights into the functional states of these proteins,