Development of Novel Chemical Tools to Measure Protease Activation
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Proteases are enzymes that cleave peptide bonds of proteins. To prevent cleavage at the wrong place or time, and thus protect the body from aberrant proteolysis, most proteases are synthesised as inactive proteins called zymogens (Figure 1). They become activated in response to a conformational change, which can be mediated by alterations in pH or cleavage by other proteases. Once activated, proteases are also subject to spatial and temporal regulation by endogenous inhibitors such as cystatins. As a result of these complex modes of post-translational modification, traditional biochemical methods that survey total protein levels rarely reflect the pool of active, functional enzymes. The ability to specifically measure and modulate the activity of a protease in its native environment is therefore required to define its precise proteolytic functions during health and disease.
To achieve this, efforts from our team and others have focussed on developing activity-based probes (ABPs) for diverse cysteine and serine proteases. These tools capitalise on the catalytic mechanism of proteolysis, combining a protease recognition sequence with a reactive functional group called a warhead. When the catalytic residue of an active protease attacks this warhead, a covalent, irreversible bond forms (Figure 2).
To detect the formation of this bond, and thus measure protease activity, ABPs are tagged with fluorophores that emit light only after protease cleavage. This fluorescence can be visualised using a number of optical imaging applications, including whole animal and tissue imaging, flow cytometry, confocal microscopy, and SDS-PAGE (in-gel fluorescence). The identity of the probe’s targets can then be confirmed by immunoprecipitation with protease-specific antibodies or proteomic methods (Figure 3).
This project will involve collaboration with chemists to develop novel activity-based probes for cysteine and serine proteases.
Bridget Halloran, PhD Student (at Monash University - co-supervised with Philip Thompson)
Professor Philip Thompson, Monash Institute of Pharmaceutical Sciences
NHMRC Peter Doherty Early Career Fellowship (2015-2018)
Grimwade Research Fellowship (2018-2022)
ARC DECRA Fellowship (2019-2021)
Edgington-Mitchell LE, Bogyo M, Verdoes M. Live cell imaging and profiling of cysteine cathepsin activity using a quenched activity-based probe. Methods in Molecular Biology 2017; In Press.
Duivenvoorden HM, Rautela J, Edgington-Mitchell LE, Spurling A, Greening DW, Nowell CJ, Lolloy TJ, Robbins E, Huang P, Brockwell NK, Faou P, Lee CS, Chen M, Holliday A, Selinger CI, Hu M, Britt KL, Stroud DA, Bogyo M, Moller A, Polyak K, Sloane BF, O’Toole SA, Parker BS. Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion. J Path 2017; 243(4): 496-509.
Edgington-Mitchell LE, Barlow N, Aurelio L, Samha A, Szabo M, Graham B, Bunnett NW. Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation. Bioorg Med Chem Lett 2017; 27(2): 254-260.
Edgington-Mitchell LE, Wartmann T, Fleming AK, Gocheva V, van der Linden WA, Withana NP, Verdoes M, Aurelio L, Edgington-Mitchell D, Lieu T, Parker BS, Graham B, Reinheckel T, Furness JB, Joyce JA, Storz,P., Halangk W, Bogyo M, Bunnett NW. Legumain is activated in macrophages during pancreatitis. Am J Physiol Gastrointest Liver Physiol 2016; 311(3): G548-60.
Lieu TM, Savage E, Zhao P, Edgington-Mitchell LE, Barlow N, Bron R, Poole D, McLean P, Lohman R Fairlie D, Bunnet NW. Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor 2. British J Pharmacol 2016; 173(18): 2752-65.
Edgington-Mitchell LE. Pathophysiological roles for proteases in gastrointestinal disease. Am J Physiol Gastrointest Liver Physiol 2016; 310(4): G234-9.
Edgington-Mitchell LE, Rautela J, Duivenvoorden HM, Jayatilleke KM, van der Linden W, Verdoes M, Bogyo M, Parker BS. Cysteine Cathepsin Activity Suppresses Osteoclastogenesis of Myeloid-Derived Suppressor Cells in Breast Cancer. Oncotarget 2015; 6(29): 27008-22.
Edgington LE, Verdoes M, Ortega A, Withana NP, Lee J, Syed S, Bachmann MH, Blum G, Bogyo,M. Functional imaging of legumain in cancer using a new quenched activity-based probe. J Am Chem Soc 2013; 135(1): 174-82.
Edgington LE, Bogyo M. In vivo imaging and biochemical characterization of protease function using fluorescent activity-based probes. Curr Protoc Chem Biol 2013; 5(1): 25-44.
Edgington LE, van Raam BJ, Verdoes M, Wierschem C, Salvesen GS, Bogyo M. An optimized activity-based probe for the study of caspase-6 activation. Chem Biol 2012; 19; 340-352.
Verdoes, M, Edgington LE, Scheeren FA, Leyva M, Blum G, Weiskopf K. Bachmann MH, Ellman JA., and Bogyo, M. A nonpeptidic cathepsin s activity-based probe for noninvasive optical imaging of tumor-associated macrophages. Chem Biol 2012; 9: 619-628. 22.25.
Edgington LE, Verdoes M, Bogyo M. Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes. Curr Opin Chem Biol 2011; 15: 798-805.
Edgington LE, Berger B, Blum G, Albrow VE, Paulick, MG, Lineberry N, Bogyo M. Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes. Nat Med 2009; 15: E967-973.
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