A Biocompatible “Split Luciferin” Reaction and Its Application for Non‐Invasive Bioluminescent Imaging of Protease Activity in Living Animals

Aurélien Godinat1, Ghyslain Budin1, Alma R. Morales1, Hyo Min Park2, Laura E. Sanman3, Matthew Bogyo4, Allen Yu2, Andreas Stahl2, Elena A. Dubikovskaya1

1 Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology of Lausanne, Lausanne, 2 Department of Nutritional Science and Toxicology, University of California Berkeley, Berkeley, California, 3 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, 4 Department of Pathology, Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140047
Online Posting Date:  September, 2014
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Abstract

The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real‐time non‐invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the “split luciferin” biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide‐specific substrates. In this unit, we describe several applications of this “split luciferin” reaction for quantification of protease activities in test tube assays and living animals. Curr. Protoc. Chem. Biol. 6:169‐189 © 2014 by John Wiley & Sons, Inc.

Keywords: protease activity; bioocompatible reaction; non‐invasive; bioluminescent imaging; in vivo

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro (Cell‐Free) Bioluminescence Imaging of Protease Activity Using “Split Luciferin” Reaction
  • Alternate Protocol 1: In Vitro (Cell‐Free) Imaging of Thrombin Protease Activity
  • Basic Protocol 2: Real‐Time Non‐Invasive Imaging of Caspase‐3/7 Activities in Transgenic Reporter Mice (FVB‐Luc+)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro (Cell‐Free) Bioluminescence Imaging of Protease Activity Using “Split Luciferin” Reaction

  Materials
  • Caspase buffer (see recipe)
  • 200 nM of purified caspase‐3 solution (see recipe)
  • 800 μM D‐cysteine solution (see recipe)
  • 800 μM DEVD‐(D‐Cys) peptide solution (see recipe)
  • 400 μM 6‐amino‐2‐cyanobenzothiazole (NH 2‐CBT; see recipe)
  • 60 μg/ml Firefly luciferase enzyme solution (see recipe)
  • V‐shaped 96‐well plate (Vitaris, cat. no. 3897‐COR)
  • 37°C incubator
  • Black 96‐well plate (BD Falcon, cat. no. 353219)
  • Bioluminescence plate reader or imager (Spectramax M5, Molecular Devices)

Alternate Protocol 1: In Vitro (Cell‐Free) Imaging of Thrombin Protease Activity

  Materials
  • Thrombin stock solution (see recipe)
  • Thrombin buffer (see recipe)
  • D‐cysteine solution (see recipe)
  • 500 μM GGR‐(D‐Cys) peptide solutions (see recipe)
  • 500 μM NH 2‐CBT solution (see recipe)
  • 60 μg/ml Firefly luciferase enzyme solution (see recipe)
  • V‐shaped 96‐well plate (Vitaris, cat. no. 3897‐COR)
  • 37°C incubator
  • Black 96‐well plate (BD Falcon, cat. no. 353219)
  • Bioluminescence plate reader (Spectramax Gemini, Molecular Devices) or camera‐like IVIS Spectrum (Perkin Elmer)

Basic Protocol 2: Real‐Time Non‐Invasive Imaging of Caspase‐3/7 Activities in Transgenic Reporter Mice (FVB‐Luc+)

  Materials
  • 3.1 mg/ml D‐luciferin potassium salt (Intrace Medical) solution (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 20012‐068), sterile
  • FVB‐Luc+ mice [FVB‐Tg(CAG‐luc,‐GFP)L2G85Chco/J, The Jackson Laboratory; Cao et al., ]
  • Isoflurane for anesthesia (Phoenix)
  • 50 μg/ml lipopolysaccharide (LPS) (from Salmonella typhosa, Sigma‐Aldrich, cat. no. L7895) solution (see recipe)
  • 133.5 mg/ml D‐(+)‐galactosamine hydrochloride (D‐GalN) solution (see recipe)
  • 5.65 mg/ml DEVD‐(D‐Cys) peptide solution (see recipe)
  • 8.5 mg/ml 6‐amino‐2‐cyanobenzothiazole (NH 2‐CBT) solution (see recipe)
  • Dimethyl sulfoxide (DMSO), sterile
  • 0.5‐ml insulin syringes (U‐100 insulin syringe, 28‐G × ½‐in. needle; BD, cat. no. 329465)
  • Clear, Plexiglas box
  • Bioluminescence imaging system for small laboratory animals (IVIS Spectrum, PerkinElmer) or similar device
  • 37°C heating pad
NOTE: The following procedure is described for an average 25 g FVB‐luc+ mouse. If the mice used have different weight, it is advised to adjust the concentrations of solutions in order to keep the doses constant.
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Figures

Videos

Literature Cited

Literature Cited
  Agard, N.J., Baskin, J.M., Prescher, J.A., Lo, A., and Bertozzi, C.R. 2006. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 1:644‐648.
  Biserni, A., Martorana, F., Roncoroni, C., Klaubert, D., Maggi, A., and Ciana, P. 2010. Identification of Apoptotic Cells in Reporter Mice Using Modified Luciferin, Promega Corporation Web site. http://www.promega.com/resources/articles/pubhub/identification‐of‐apoptotic‐cells‐in‐reporter‐mice‐using‐modified‐luciferin. Accessed November 2012.
  Blackman, M.L., Royzen, M., and Fox, J.M. 2008. Tetrazine ligation: Fast bioconjugation based on inverse‐electron‐demand Diels‐Alder reactivity. J. Am. Chem. Soc. 130:13518‐13519.
  Budin, G., Yang, K.S., Reiner, T., and Weissleder, R. 2011. Bioorthogonal probes for polo‐like kinase 1 imaging and quantification. Angew. Chem. Int. Ed. 50:9378‐9381.
  Cali, J.J., Ma, D., Sobol, M., Simpson, D.J., Frackman, S., Good, T.D., Daily, W.J., and Liu, D. 2006. Luminogenic cytochrome P450 assays. Exp. Opin. Drug Metab. Toxicol. 2:629‐645.
  Cao, Y., Wagers, A.J., Beilhack, A., Dusich, J., Bachmann, M.H., Negrin, R.S., Weissman, I.L., and Contag, C.H. 2004. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc. Natl. Acad. Sci. U.S.A. 101:221‐226.
  Chang, P.V., Prescher, J.A., Hangauer, M.J., and Bertozzi, C.R. 2007. Imaging cell surface glycans with bioorthogonal chemical reporters. J. Am. Chem. Soc. 129:8400‐8401.
  Chang, P.V., Prescher, J.A., Sletten, E.M., Baskin, J.M., Miller, I.A., Agard, N.J., Lo, A., and Bertozzi, C.R. 2010. Copper‐free click chemistry in living animals. Proc. Natl. Acad. Sci. U.S.A. 107:1821‐1826.
  Cohen, A.S., Dubikovskaya, E.A., Rush, J.S., and Bertozzi, C.R. 2010. Real‐time bioluminescence imaging of glycans on live cells. J. Am. Chem. Soc. 132:8563‐8565.
  Conley, N.R., Dragulescu‐Andrasi, A., Rao, J., and Moerner, W.E. 2012. A selenium analogue of firefly D‐luciferin with red‐shifted bioluminescence emission. Angew. Chem. Int. Ed. 51:3350‐3353.
  Cosby, N., Scurria, M., Daily, W., Ugo, T., Promega Corp., and Promega Biosciences Inc. 2007. Custom enzyme substrates for luciferase‐based assays. Cell Notes 18:9‐11.
  Devaraj, N.K., Weissleder, R., and Hilderbrand, S.A. 2008. Tetrazine‐based cycloadditions: Application to pretargeted live cell imaging. Bioconjugate Chem. 19:2297‐2299.
  Devaraj, N.K., Thurber, G.M., Keliher, E.J., Marinelli, B., and Weissleder, R. 2012. Reactive polymer enables efficient in vivo bioorthogonal chemistry. Proc. Natl. Acad. Sci. U.S.A. 109:4762‐4767.
  Dragulescu‐Andrasi, A., Liang, G., and Rao, J. 2009. In vivo bioluminescence imaging of furin activity in breast cancer cells using bioluminogenic substrates. Bioconjugate Chem. 20:1660‐1666.
  Dube, D.H., Prescher, J.A., Quang, C.N., and Bertozzi, C.R. 2006. Probing mucin‐type O‐linked glycosylation in living animals. Proc. Natl. Acad. Sci. U.S.A. 103:4819‐4824.
  Fedele, M., Gualillo, O., and Vecchione, A. 2012. Animal models of human pathology 2012. J. Biomed. Biotechnol. 2012:404130.
  Geiger, G.A., Parker, S.E., Beothy, A.P., Tucker, J.A., Mullins, M.C., and Kao, G.D. 2006. Zebrafish as a “biosensor”? Effects of ionizing radiation and amifostine on embryonic viability and development. Cancer Res. 66:8172‐8181.
  Godinat, A., Park, H.M., Miller, S.C., Cheng, K., Hanahan, D., Sanman, L.E., Bogyo, M., Yu, A., Nikitin, G.N., Stahl, A., and Dubikovskaya, E.A. 2013. A biocompatible in vivo ligation reaction and its application for non‐invasive bioluminescent imaging of protease activity in living mice. ACS Chem. Biol. 8:987‐999.
  Goun, E.A., Pillow, T.H., Jones, L.R., Rothbard, J.B., and Wender, P.A. 2006. Molecular transporters: Synthesis of oligoguanidinium transporters and their application to drug delivery and real‐time imaging. Chembiochem 7:1497‐1515.
  Hangauer, M.J. and Bertozzi, C.R. 2008. A FRET‐based fluorogenic phosphine for live‐cell imaging with the Staudinger ligation. Angew. Chem. Int. Ed. Engl. 47:2394‐2397.
  Harwood, K.R., Mofford, D.M., Reddy, G.R., and Miller, S.C. 2011. Identification of mutant firefly luciferases that efficiently utilize aminoluciferins. Chem. Biol. 18:1649‐1657.
  Henkin, A.H., Cohen, A.S., Dubikovskaya, E.A., Park, H.M., Nikitin, G.F., Auzias, M.G., Kazantzis, M., Bertozzi, C.R., and Stahl, A. 2012. Real‐time noninvasive imaging of fatty acid uptake in vivo. ACS Chem. Biol. 7:1884‐1891.
  Hickson, J., Ackler, S., Klaubert, D., Bouska, J., Ellis, P., Foster, K., Oleksijew, A., Rodriguez, L., Schlessinger, S., Wang, B., and Frost, D. 2010. Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z‐DEVD‐aminoluciferin. Cell Death Differ. 17:1003‐1010.
  Jewett, J.C., Sletten, E.M., and Bertozzi, C.R. 2010. Rapid Cu‐free click chemistry with readily synthesized biarylazacyclooctynones. J. Am. Chem. Soc. 132:3688‐3690.
  Laferla, F.M. and Green, K.N. 2012. Animal models of Alzheimer disease. Cold Spring Harb. Perspect. Med. 2:a006320.
  Lang, K., Davis, L., Wallace, S., Mahesh, M., Cox, D.J., Blackman, M.L., Fox, J.M., and Chin, J.W. 2012. Genetic encoding of bicyclononynes and trans‐cyclooctenes for site‐specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels‐Alder reactions. J. Am. Chem. Soc. 134:10317‐10320.
  Langdon, S.P. 2012. Molecular pathology in cancer therapeutics: Where are we now and where are we going? Animal modeling of cancer pathology and studying tumor response to therapy. Curr. Drug Targets 13:1535‐1547.
  Li, Z., Zhao, G., Qian, S., Yang, Z., Chen, X., Chen, J., Cai, C., Liang, X., and Guo, J. 2012. Cerebrovascular protection of β‐asarone in Alzheimer's disease rats: A behavioral, cerebral blood flow, biochemical and genic study. J. Ethnopharmacol. 144:305‐312.
  Liang, G., Ren, H., and Rao, J. 2010. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. J. Nat. Chem. 2:54‐60.
  Liang, Y., Mackey, J.L., Lopez, S.A., Liu, F., and Houk, K.N. 2012a. Control and design of mutual orthogonality in bioorthogonal cycloadditions. J. Am. Chem. Soc. 134:17904‐17907.
  Liang, Y., Walczak, P., and Bulte, J.W. 2012b. Comparison of red‐shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells. J. Biomed. Opt. 17:016004.
  Lin, F.L., Hoyt, H.M., van Halbeek, H., Bergman, R.G., and Bertozzi, C.R. 2005. Mechanistic investigation of the Staudinger ligation. J. Am. Chem. Soc. 127:2686‐2695.
  Liu, J.J., Wang, W., Dicker, D.T., and El‐Deiry, W.S. 2005. Bioluminescent imaging of TRAIL‐induced apoptosis through detection of caspase activation following cleavage of DEVD‐aminoluciferin. Cancer Biol. Ther. 4:885‐892.
  Massoud, T.F. and Gambhir, S.S. 2003. Molecular imaging in living subjects: Seeing fundamental biological processes in a new light. Genes Dev. 17:545‐580.
  McCaffrey, A., Kay, M.A., and Contag, C.H. 2003. Advancing molecular therapies through in vivo bioluminescent imaging. Mol. Imaging 2:75‐86.
  McCutcheon, D.C., Paley, M.A., Steinhardt, R.C., and Prescher, J.A. 2012. Expedient synthesis of electronically modified luciferins for bioluminescence imaging. J. Am. Chem. Soc. 134:7604‐7607.
  Merrifield, R.B. 1965. Automated synthesis of peptides. Science 150:178‐185.
  Merrifield, R.B. and Stewart, J.M. 1965. Automated peptide synthesis. Nature 207:522‐523.
  Neves, A.A., Stöckmann, H., Harmston, R.R., Pryor, H.J., Alam, I.S., Ireland‐Zecchini, H., Lewis, D.Y., Lyons, S.K., Leeper, F.J., and Brindle, K.M. 2011a. Imaging sialylated tumor cell glycans in vivo. FASEB J. 25:2528‐2537.
  Neves, A.A., Stöckmann, H., Stairs, S., Ireland‐Zecchini, H., Brindle, K.M., and Leeper, F.J. 2011b. Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells. Chem. Sci. 2:932‐936.
  Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., and Chin, J.W. 2011. Genetically encoded 1,2‐aminothiols facilitate rapid and site‐specific protein labeling via a bio‐orthogonal cyanobenzothiazole condensation. J. Am. Chem. Soc. 133:11418‐11421.
  Nilsson, B.L., Kiessling, L.L., and Raines, R.T. 2000. Staudinger ligation: A peptide from a thioester and azide. Org. Lett. 2:1939‐1941.
  Ning, X., Guo, J., Wolfert, M.A., and Boons, G. 2008. Visualizing metabolically labeled glycoconjugates of living cells by copper‐free and fast huisgen cycloadditions. Angew. Chem., Int. Ed. Engl. 47:2253‐2255.
  O'Brien, M.A., Daily, W.J., Hesselberth, P.E., Moravec, R.A., Scurria, M.A., Klaubert, D.H., Bulleit, R.F., and Wood, K.V. 2005. Homogeneous, bioluminescent protease assays: Caspase‐3 as a model. J. Biomol. Screening 10:137‐148.
  Peters, M., Trembovler, V., Alexandrovich, A., Parnas, M., Birnbaumer, L., Minke, B., and Shohami, E. 2012. Carvacrol together with TRPC1 elimination improve functional recovery after traumatic brain injury in mice. J. Neurotrauma 29:2831‐2834.
  Prescher, J.A. and Contag, C.H. 2010. Guided by the light: Visualizing biomolecular processes in living animals with bioluminescence. Curr. Opin. Chem. Biol. 14:80‐89.
  Prescher, J.A., Dube, D.H., and Bertozzi, C.R. 2004. Chemical remodeling of cell surfaces in living animals. Nature 430:873‐877.
  Reddy, G.R., Thompson, W.C., and Miller, S.C. 2010. Robust light emission from cyclic alkylaminoluciferin substrates for firefly luciferase. J. Am. Chem. Soc. 132:13586‐13587.
  Ren, H., Xiao, F., Zhan, K., Kim, Y., Xie, H., Xia, Z., and Rao, J. 2009. A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins. Angew. Chem. Int. Ed. 48:9658‐9662.
  Rogers, A.B. 2012. Gastric Helicobacter spp. in animal models: Pathogenesis and modulation by extragastric coinfections. Methods Mol. Biol. 921:175‐188.
  Saxon, E. and Bertozzi, C.R. 2000. Cell surface engineering by a modified Staudinger reaction. Science 287:2007‐2010.
  Saxon, E., Armstrong, J.I., and Bertozzi, C.R. 2000. A “traceless” Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2:2141‐2143.
  Scabini, M., Stellari, F., Cappella, P., Rizzitano, S., Texido, G., and Pesenti, E. 2011. In vivo imaging of early stage apoptosis by measuring real‐time caspase‐3/7 activation. Apoptosis 16:198‐207.
  Shah, K., Tung, C., Breakefield, X.O., and Weissleder, R. 2005. In vivo imaging of S‐TRAIL‐mediated tumor regression and apoptosis. Mol. Ther. 11:926‐931.
  Sletten, E.M. and Bertozzi, C.R. 2009. Bioorthogonal chemistry: Fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. Engl. 48:6974‐6998.
  Sletten, E.M., Nakamura, H., Jewett, J.C., and Bertozzi, C.R. 2010. Difluorobenzocyclooctyne: Synthesis, reactivity, and stabilization by beta‐cyclodextrin. J. Am. Chem. Soc. 132:11799‐11805.
  Stennicke, H.R. and Salvesen, G.S. 1999. Caspases: Preparation and characterization. Methods 17:313‐319.
  Van Berkel, S.S., van Eldijk, M.B., and van Hest, J.C. 2011. Staudinger ligation as a method for bioconjugation. Angew. Chem. Int. Ed. 50:8806‐8827.
  Van de Bittner, G.C., Dubikovskaya, E.A., Bertozzi, C.R., and Chang, C.J. 2010. In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter. Proc. Natl. Acad. Sci. U.S.A. 107:21316‐21321.
  Van de Bittner, G.C., Bertozzi, C.R., and Chang, C.J. 2013. Strategy for dual‐analyte luciferin imaging: In vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation. J. Am. Chem. Soc. 135:1783‐1795.
  Wehrman, T.S., von Degenfeld, G., Krutzik, P.O., Nolan, G.P., and Blau, H.M. 2006. Luminescent imaging of beta‐galactosidase activity in living subjects using sequential reporter‐enzyme luminescence. Nat. Methods 3:295‐301.
  White, E.H., McCapra, F., and Field, G.F. 1963. The structure and synthesis of firefly luciferin. J. Am. Chem. Soc. 85:337‐343.
  Wilson, J.W., Schurr, M.J., LeBlanc, C.L., Ramamurthy, R., Buchanan, K.L., and Nickerson, C.A. 2002. Mechanisms of bacterial pathogenicity. Postgrad. Med. J. 78:216‐224.
  Woodroofe, C.C., Shultz, J.W., Wood, M.G., Osterman, J., Cali, J.J., Daily, W.J., Meisenheimer, P.L., and Klaubert, D.H. 2008. N‐alkylated 6′‐aminoluciferins are bioluminescent substrates for Ultra‐Glo and QuantiLum luciferase: New potential scaffolds for bioluminescent assays. Biochemistry 47:10383‐10393.
  Yang, J., Šečkutė, J., Cole, C.M., and Devaraj, N.K. 2012. Live‐cell imaging of cyclopropene tags with fluorogenic tetrazine cycloadditions. Angew. Chem., Int. Ed. Engl. 51:7476‐7479.
  Yao, H., So, M., and Rao, J. 2007. A bioluminogenic substrate for in vivo imaging of beta‐lactamase activity. Angew. Chem. Int. Ed. Engl. 46:7031‐7034.
  Ye, D., Liang, G., Ma, M.L., and Rao, J. 2011. Controlling intracellular macrocyclization for the imaging of protease activity. Angew. Chem. Int. Ed. 50:2275‐2279.
  Yuana, Y. and Liang, G. 2014. A biocompatible, highly efficient click reaction and its applications. Org. Biomol. Chem. 12:865‐871.
  Zhou, W., Shultz, J.W., Murphy, N., Hawkins, E.M., Bernad, L., Good, T., Moothart, L., Frackman, S., Klaubert, D.H., Bulleit, R.F., and Wood, K.V. 2006a. Electrophilic aromatic substituted luciferins as bioluminescent probes for glutathione S‐transferase assays. Chem. Commun. 4620‐4622.
  Zhou, W., Valley, M.P., Shultz, J., Hawkins, E.M., Bernad, L., Good, T., Good, D., Riss, T.L., Klaubert, D.H., and Wood, K.V. 2006b. New bioluminogenic substrates for monoamine oxidase assays. J. Am. Chem. Soc. 128:3122‐3123.
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