ynthesis of novel activity based probes and combinatorial peptide libraries to profile proteases

SYNTHESIS OF NOVEL ACTIVITY BASED PROBES AND COMBINATORIAL PEPTIDE LIBRARIES TO PROFILE PROTEASES RESMI CHANDRASEKHARA PANICKER NATIONAL UNIVERSITY OF SINGAPORE 2007 SYNTHESIS OF NOVEL ACTIVITY BASED PROBES AND COMBINATORIAL PEPTIDE LIBRARIES TO PROFILE PROTEASES RESMI CHANDRASEKHARA PANICKER (M Sc - Mahatma Gandhi University, Kerala, India) A THESIS SUBMITTED FOR THE DOCTOR OF PHILOSOPHY DEGREE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I wish to thank to my supervisor Associate Professor Yao Shao Qin for his novel ideas, patient guidance and invaluable suggestions during the course of my research I am grateful to my project partners Liau Minglee, Eunice, Haung Xuan and Wang Gang as well as other lab-mates Aparna, Souvik, Mahesh, Dawn, Wang Jun, Hong Yan, Elaine, Grace and Hu Yi for all their help during the course of the project Special thanks to my fellow-colleague plus life-partner Raja for his kind support and encouragement in all ways possible I appreciate the support of the laboratory staff from the NMR and the MS labs for providing me the necessary training and technical assistance I am also grateful to the National University of Singapore, for granting the research scholarship Last but not least, I would like to express my sincere thanks to my parents, grandmother, other family members and friends for their constant support and well wishes i TABLE OF CONTENTS Acknowledgements i Table of contents ii Summary vi List of tables viii List of figures ix List of schemes xii Abbreviations xiii Publications xvii Chapter Introduction 1.1 Proteomics 1.2 Conventional techniques for protein profiling 1.3 Activity based protein profiling 1.3.1 Activity-based probes 1.3.2 A few developments from our lab using activity 11 based/affinity based approaches 1.4 Combinatorial peptide libraries 13 1.4.1 Peptide Library on Solid Support 14 ii 1.4.2 Parallel synthesis 15 1.4.3 Split and mix synthesis 16 1.4.4 Reagent mixture synthesis 17 1.4.5 Positional scanning peptide libraries 17 1.5 Bioimaging 18 Chapter Design and Synthesis of activity-based probes targeting caspases 25 2.1 Introduction 25 2.1.1 Caspases 25 2.1.2 Mechanistic details of interaction of caspases with their substrates 27 2.2 Affinity tag approach to develop caspase probe for an in vitro proteomic Experiment 28 2.2.1 Design of the caspase-Cy3 probe 30 2.2.2 Chemical synthesis of the caspase-Cy3probe 33 2.2.3 Results and conclusions of the in vitro experiments 36 2.3 Activity based affinity probes for in vivo labeling of caspases 39 2.3.1 Design of cell permeable caspase probes 39 2.3.2 Chemical synthesis of cell permeable caspase probes 42 2.3.3 Results and conclusions of the in vivo labeling experiments 44 2.4 Experimental details of the synthesis of the caspase probes 47 iii Chapter Positional Scanning Peptide libraries of Fluorogenic substrates to map the substrate specificity of Proteases 58 3.1 Introduction 58 3.2 Design of our fluorogenic peptide library 60 3.3 Synthetic details of the ACC-library 62 3.4 Quality analysis of library 67 3.5 Fingerprinting experiments using ACC library 68 3.6 Data analysis and conclusions 68 3.7 ACC-azide library 71 3.8 Chemical synthesis of the azido-ACC-peptides 71 3.9 Experimental details of Syntheses 72 3.9.1 Experimental details of synthesis of ACC-Positional scanning library 72 3.9.2 Experimental details of synthesis of azido-ACC peptides 81 Chapter Fingerprinting of metalloproteases and cysteine proteases using positional scanning peptide libraries 84 4.1 Affinity based fingerprinting of metalloproteases using a positional scanning inhibitor library of peptidyl hydroxamates 84 4.1.1 Synthesis of the peptidyl hydroxamate library 86 4.1.2 Gel-based inhibition experiments using the peptidyl hydroxamate library 87 4.2 Activity based fingerprinting of cysteine proteases using a positional scanning library of peptide vinyl sulfone probes 89 iv 4.2.1 Synthesis of the vinyl sulfone library 91 4.2.2 Results and conclusions from the labeling experiments 93 4.3 Experimental details of the synthesis 94 4.3.1 Experimental details of the synthesis of the peptide hydroxamate library 94 4.3.2 Experimental details of the synthesis of the vinyl sulfone library 98 Chapter Synthesis of Molecular probes for potential bioimaging experiments 107 5.1 Introduction 107 5.2 Design of the NTA probes 108 5.3 Chemical synthesis of the probes 109 5.4 Results and conclusions of the labeling experiments 111 5.5 Experimental details of the synthesis of NTA probes 113 Chapter References 114 Chapter Appendices 129 Capase-Fluorescein probe (16) – 1H NMR 129 Capase-Fluorescein probe (16) – ESI-MS 130 Capase-Biotin probe (18) – 1H NMR 131 Capase-Biotin probe (18) – ESI-MS 132 AVLQ-ACC-Lys(N3) – ESI-MS 133 NTA-TMR (57) - ESI-MS 134 NTA-FL (59) - ESI-MS 135 v SUMMARY The work presented in this thesis focus mainly on two areas (i) designing specific probes to target a particular class of proteases using activity based affinity tag approach (ii) collection of substrate specificity/binding data of proteases using positional scanning peptide libraries Chapter of the thesis present our efforts towards the design and synthesis of small molecule probes to target caspases, enzymes which play a key mediating role in apoptosis or programmed cell death At first, we synthesized a fluoromethyl ketone containing activity-based probe that specifically target caspases in an in vitro proteomic experiment Later on, we extended this approach to the in vivo labeling of caspases in apoptotic HeLa cells by the use of modified probes which are cell permeable The attractive feature of our strategy is that it allows for the large scale identification of novel enzyme-associating proteins Chapter 3, and mainly focus on the synthesis of positional scanning combinatorial libraries of peptide substrates/inhibitors to profile proteases These works concentrate on the studies of the substrate specificity or “fingerprinting” of various classes of proteases For example, a positional scanning library of 7-amino-4 carbamoylmethylcoumarin (ACC) conjugated peptides were synthesized and assayed against different classes of proteases The substrate specificity profiles of various classes of proteases were successfully obtained using this library Other efforts include the activity-based profiling of cysteine proteases using a twenty member library of vinyl sulfone-containing peptides with varying P1 position and the synthesis of a positional vi scanning combinatorial library of peptidyl hydroxamates to investigate the substrate specificity of metalloproteases at the P2-P4 positions A brief attempt for bioimaging using small molecular probes also has been done as illustrated in chapter Selected NMR and MS spectra are listed in the Appendices vii LIST OF TABLES Table Proteins identified by mass spectrometry 47 Table Optimization of conditions for ACC coupling 65 Table ESI-MS data of the ACC-conjugated peptide azides 83 Table ESI-MS data for vinyl sulfone library 105 viii 56 Shaw, E., Mares-Guia, M., Cohen, W Biochemistry 1965, 4, 2219 57 Lo, L.C., Pang, T.L., Kuo, C.H., Chiang, Y.L., Wang, H.Y., Lin, J.J J Proteome Res 2002, 1, 35 58 Srinivasan, R., Uttamchandani, M., Yao, S.Q Org Lett 2006, 8, 713 59 Zhou, H., Ranish, J.A., Watts, J.D., Aebersold, R Nat Biotechnol 2002, 19, 512 60 Merrifield, R B J Am Chem Soc 1963, 85, 2149 61 Geysen, H M., Meloen, R H and Barteling, S J Proc Natl Acad Sci USA 1984, 81, 3998 62 Houghten, R A Proc Natl Acad Sci USA 1984, 82, 5131 63 Graven, A., St Hilaire, P M., Sanderson, S J., Mottram, J C., Coombs, G H and Meldal, M J Comb Chem 2001, 3, 441 64 Lam, K S., Salmon, S E., Hersh, E M., Hruby, V J., Kazmierski, W M and Knapp, R J Nature, 1991, 354, 82 65 Christensen, C., Groth, T., Schiodt, C B., Foged, N T and Meldal, M QSAR Comb Sci 2003, 22, 737 66 Ostresh, J M., Winkle, J H., Hamashin, V T and Houghten, R A Biopolymers 1994, 34, 1681 67 Lecaille, F., Kaleta, J., Broemme, D Chem Rev 2002, 102, 4459 68 Higaki, J N., Chakravarty, S., Bryant, C M., Cowart, L R., Harden, P., Scardina, J M., Mavunkel, B., Luedtke, G R., Cordell, B J Med Chem 1999, 42, 3889 69 Whittaker, M Curr Opin Chem Biol 1998, 2, 386 70 Maly, D J., Huang, L., Ellman, J A ChemBiochem 2002, 3, 16 71 Mason, D E., Ek, J., Peters, E C., Harris, J L Biochemistry 2004, 43, 6535 121 72 Humet, M., Carbonell, T., Masip, I., Sanchez-Baeza, F., Mora, P., Canton, E., Gobernado, M., Abad, C., Perez-Paya, E., Messeguer, A J Comb Chem 2003, 5, 597 73 Richardson, P L Curr Pharm Design 2002, 8, 2559 74 Nazif, T., Bogyo, M Proc Natl Acad Sci U.S.A 2001, 98, 2967 75 Hermanson, G.T Bioconjugate Techniques, Academic Press, San Diego, CA (1996) 76 Srinivasan, R., Yao, S.Q., Yeo, D.S.Q Comb Chem High Throughput Screening, 2004, 7, 597 77 Yeo, D.S.Y., Srinivasan, R., Chen, G.Y.J., Yao, Y.S.Q Chem Eur J., 2004, 10, 4664 78 Griffin, B.A., Adams, S.R., Tsein, R.Y Science 1998, 28, 269 79 Gaietta, G., Deerinck, T.J., Adams, S.R., Bouwer, J., Tour, O., Laird, D.W., Sosinsky, G.E., Tsien, R.Y., Ellisman, M.H Science, 2002, 296, 503 80 Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H.,Vogel, H.,Johnsson, K Nat Biotechnol., 2003, 21, 86 81 Yeo, D.S.Y., Srinivasan, R., Uttamchandani, M., Chen, G.Y.J., Zhu, Q., Yao, S.Q Chem Commun 2003, 2870 82 Tan, E.L.P., Panicker, R.C., Chen, G.Y.J., Yao, S.Q Chem Commun 2005, 596 83 Guignet, E.G., Hovius, R., Vogel, H Nat Biotechnol 2004, 22, 440 84 Llopis, J., McCaffery, J.M., Miyawaki, A., Farquhar, M.G., Tsien, R.Y Proc Natl Acad Sci USA, 1998, 95, 6803 85 Farinas, J., Verkman, A.S J Biol Chem., 1999, 274, 7603 122 86 Miller, L.W., Sable, J., Goelet, P., Sheetz, M.P., Cornish, V.W Angew Chem Intl Ed 2004, 43, 1672 87 Muir, T.M Annu Rev Biochem 2003, 72, 249 88 Giriat, I., Muir, T.W J Am Chem Soc., 2003, 125, 7180 89 Ellman, J.A., Mendal, D., Schultz, P.G Science 1992, 255, 197 90 Zhang, Z., Smith, B.A., Wang, L., Brock, A., Cho, C., Schultz, P.G Biochemistry, 2003, 4, 6735 91 Grutter, M G Curr Opin Struct Biol 2000, 10, 649 92 Nicholson, D W., Thornberry, N A Trends Biochem Sci 1997, 22, 299 93 Thornberry, N A., Lazebnik, Y Science 1998, 281, 1312 94 Cortese, J The Scientist 2001, 15, 24 95 Hengartner, Nature 2000, 407, 770 96 Earnshaw, W C., Martins, L M., Kaufmann, S H.Annu Rev Biochem 1999, 68, 383 97 Wilson, K P et al., Nature, 1994, 370, 270 98 Cryns, V.L., Byun, Y., Rana, A., Mellor, H., Lustig, K.D., Ghanem, L., Parker, P.J., Kirschner, M.W., Yuan, Y.J., J Biol Chem., 1997, 272, 29449 99 Wu, Y.H., Shih, S.F., Lin, Y.J J Biol Chem., 2004, 279, 19264 100 Wan, J.H., Wang, J.L., Cheng, H.P., Yu, Y.T., Xing, G.C., Qiu, Z.Y., Qian, X.H., He, F.C Electrophoresis, 2001, 22, 3026 101 MacBeath, G., Schreiber, S L Science 2000, 289, 1760 102 Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Dinlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R A., Gerstein, M., Snyder, M Science 2001, 293, 2101 123 103 http://www.ciphergen.com 104 Powers, J C., Gupton, B F., Harley, A D., Nishino, N., Whitley, R Biochem Biophys Acta 1917, 485, 156 105 Kettner, C., Shaw, E Methods in Enzymology, (1981), Vol 80, Part C, 826, Academic Press, New York 106 Leary, R., Larsen, D., Watanabe, H., haw, E Biochemistry 1977, 16, 5857 107 Green, G D J., haw, E J Biol Chem.1981, 256, 1923-1928 108 Shaw, E., Wikstrom, P., Ruscica, J Arch Biochem Biophys 1983, 222, 424 109 Otto, H H Chem Rev 1997, 97, 133 110 Rasnick, D Anal Biochem 1985, 149, 461 111 Mujumdar, R.B., Ernst, L A., Mujumdar, S R., Waggoner, A S Cytometry 1989, 10, 11 112 Southwick, P L., Ernst, L A., Tauriello, E W., Parker, S R., Mujumdar, R B., Mujumdar, S R., Clever, H A., Waggoner, A S Cytometry 1990, 11, 418 113 Imperiati, B., Abeles, R H Tetrahedron Lett.1986, 27,135 114 Revesz, L., Briswalter, C., Heng, R., Leutwiler, A., Mueller, R., Wuethrich, H J Tetrahedron Lett 1994, 35, 9693 115 Linderman, R J., Jamois, E A J Fluorine Chem 1991, 53, 79 116 Walter, M W., Adlington, R M., Baldwin, J E., Schofield, C J J Org Chem 1998, 63, 5179 117 Valerio, R M., Alewood, P F., Johns, R B Synthesis 1988, 10, 786 118 Mancuso, A J., Swern, D Synthesis 1981, 3, 165 119 Mozingo, R Org Synth 1941, 21, 15 124 120 Beer, P D., Cadman, J., Lloris, J M., Manez-Martinez, R., Soto, J., Pardo, T., Marcos, M D J Chem Soc., Dalton Trans 2000, 1805 121 Korbel, G A., Lalic, G., Shair, M D J Am Chem Soc 2001, 123, 361 122 N Jessani and B F Cravatt, Curr Opin Chem Biol., 2004, 8, 54 123 Bogyo, M., Verhelst, S., Bellingard-Dubouchaud, V., Toba, S., Greenbaum, D., Chem Biol 2000, 7, 27 124 Michalet, X., Kapanidis, A N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M., Weiss, S Ann Rev Biophy Biomol 2003, 32, 161 125 Zhang, J., Campbell, R E., Ting, A Y., Tsien, R Y Nat Rev Mol Cell Biol 2002, 3, 906 126 Miyawaki, A., Sawano, A., Kogure, T Nat Cell Biol 2003, S1 127 Tsien, R Y Ann Rev Biochem 1998, 57, 509 128 Wu, M M., Llopis, J., Adams, S R., McCaffery, J M., Teter, K., Kulomaa, M S., Machen, T E., Moore, H P H., Tsien, R Y Methods Enzymol 2000, 327, 546 129 Patricelli , M.P., Giang, D K., Stamp, L M., Burbaum, J J Proteomics 2001,1, 1067 130 Sun, W C., Gee, K R., Klaubert, D H., Haugland, R P J Org Chem 1997, 62, 6469 131 Latt, S A., Auld, D S., Vallee, B L Anal Biochem 1972, 50, 56 132 St Hilaire, P M., Willert, M., Juliano, M A., Juliano, L., Meldal, M J Comb Chem 1999, 1, 509 133 Meldal, M., Svendsen, I., Breddam, K., Auzanneau, F I Proc Natl Acad Sci U.S.A 1994, 91, 3314 125 134 Ludeman, J P., Pike, R N., Bromfield, K M., Duggan, P J., Cianci, J., Le Bonniec, B., Whisstock, J C., and Bottomley, S P Int J Biochem Cell Biol 2003, 35, 221 135 Wu, P., Brand, L Anal Biochem 1994, 218, 136 Meldal, M., Breddam, K Anal Biochem 1991, 195, 141 137 Geenbaum, D C.; Arnold, W D.; Lu, F.; Hayrapetian, L.; Baruch, A.; Krumrine, J.; Toba, S.; Chehade, K.; Bromme, D.; Kuntz, I D.; Bogyo, M Chem Biol 2002, 9, 1085-1094 138 Rano, T A., Timkey, T., Peterson, E P., Rotonda, J., Nicholson, D W., Becker, J W., Chapman, K T., Thornberry, N A Chem Biol 1997, 4, 149 139 Harris, J L., Backes, B J., Leonetti, F., Mahrus, S., Ellman, J A., Craik, C S Proc Natl Acad Sci U.S.A 2000, 97, 7754 140 Backes, B J., Harris, J L., Leonetti, F., Craik, C S., Ellman, J A Nat Biotechnol 2000, 18, 187 141 Knight, C G Methods Enzymol 1995, 248, 18 142 Zimmerman, M., Ashe, B., Yurewicz, E., Patel, G Anal Biochem, 1977, 78, 47 143 Thornberry, N A., Rano, T.A., Peterson, E.P., Rasper, D M., Timkey, T., GarciaCalvo, M , Houtzager, V.M., Nordstrom, P A., Roy, S , Vaillantcourt, K T , Chapman, K T., Nicholson, D W J Biol Chem.1997, 272, 17907 144 Maly, D J., Leonetti, F., Backes, B.J., Dauber, D S., Harris, J L., Craik, C S., Ellman, J A J Org Chem 2002, 67, 910 145 Takeuchi, T., Harris, J L., Huang, W., Yan, K W., Coughlin, S R., Craik, C S J Biol Chem 2000, 275, 34 126 146 Salisbury, C.M, Maly, D.J.,Ellman, J.A., J Am Chem Soc 2002, 124, 14868 147 Bolin, D R., Sytwu, I.-I., Humiec, F., Meienhofer, J Int J Pept Protein Res 1989, 33, 353 148 Xiao, X.-Y., Nova, M P Comb Chem 1997, 135 149 Nicolaou, K C., Xiao, X.-Y., Parandoosh, Z., Senyei, A., Nova, M P Angew Chem., Int Ed Engl 1995, 34, 2476 150 Moran, E J., Sarshar, S., Cargill, J F., Shahbaz, M J M., Lio, A., Mjalli, A M M., Armstrong, R W J Am Chem Soc 1995, 117, 10787 151 Goddard, J.P., Reymond, J.L Curr Opin Biotechnol 2004, 15, 314 152 Eisen, M B., Spellman, P T., Brown, P O., Botstein, D Proc Natl Acad Sci U.S.A 1998, 95, 14863 153 http://merops.sanger.ac.uk/ 154 Soellner, M.B., Nilsson, B.L., Raines, R.T J Am Chem Soc 2006, 128, 8820 155 Lundquist, J.T et al, Org Lett 2002, 4, 3219 156 Bode, W., Maskos, K Biol Chem 2003, 384, 863 157 Turner, A J Biochem Soc Trans 2003, 31, 723 158 Martel-Pelletier, J., Welsch, D J., Pelletier, J P Best Pract Res Clin Rheumatol 2001, 15, 805 159 Hooper, N M., Turner, A J Curr Med Chem 2002, 9, 1107 160 Vihinen, P., Kahari, V M Int J Cancer 2002, 99, 157 161 Sierevogel, M J., Pasterkamp, G., de Kleijn, D P., Strauss, B H Curr Pharm Des 2003, 9, 1033 162 Whittaker, M., Floyd, C D., Brown, P., Gearing, A J H Chem Rev 1999, 99, 2735 127 163 Skiles, J W., Gonnella, N C., Jeng, A Y Curr Med Chem 2001, 8, 425 164 Leung, D., Hardouin, C., Boger, D L., Cravatt, B F Nat Biotechnol 2003, 21, 687 165 Mellor, S L., McGuire, C., Chan, W C Tetrahedron Lett 1997, 38, 3311 166 Beynon, R J., Beaumont, A Thermolysin In Handbook of Proteolytic Enzymes, Barrett, A J., Rawlings, N D., Woessner, J F., Eds., Academic Press: 1998, p 1037 167 Palmer, J T., Rasnick, D., Klaus, J L., Bromme D J Med Chem 1995, 38, 3193 168 Hochuli, E., Dobeli, H., Schacher A J Chromatogr 1987, 411, 177 169 Kapanidis, A.N., Ebright, Y W., Ebright, R H J Am Chem Soc 2001, 123, 12123 128 Chapter Appendices: Capase-Fluorescein probe (16) – 1H NMR 129 Capase-Fluorescein probe (16) – ESI-MS 130 Capase-Biotin probe (18) – 1H NMR 131 Caspase-Biotin probe (18) – ESI-MS 132 AVLQ-ACC-Lys(N3) – ESI-MS 133 NTA-TMR (57) 134 NTA-FL (59) 135 ...SYNTHESIS OF NOVEL ACTIVITY BASED PROBES AND COMBINATORIAL PEPTIDE LIBRARIES TO PROFILE PROTEASES RESMI CHANDRASEKHARA PANICKER (M Sc - Mahatma Gandhi University, Kerala,... mainly focus on the synthesis of positional scanning combinatorial libraries of peptide substrates/inhibitors to profile proteases These works concentrate on the studies of the substrate specificity... representation of activity- based profiling strategy 1.3.1 Activity- based probes Activity- based probes are designed based on their potential application in proteomics The design of these probes depends