Fluorogenic peptide-based probes for detection of proteases in inflammation
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Rodriguez-RiosM_2023.pdf (11.72Mb)
Date
17/07/2023Item status
Restricted AccessEmbargo end date
17/07/2024Author
Rodriguez-Rios, Maria
Metadata
Abstract
Proteases can be great biomarkers of disease as these enzymes cleave amide bonds in peptides and proteins and regulate a variety of cellular process. Indeed, their dysregulation has been associated with a variety of diseases. Monitoring proteolytic activity using fluorescent probes has shown great application in disease diagnostics and for intraoperative imaging, but the current toolbox is still limited, and compounds often lack specificity for their target or provide limited signal amplification. In inflammatory diseases, overactivation of neutrophils and macrophages can lead to chronic and acute disease states in pathologies such as COVID-19 infection, pneumonia, lung injury or fibrosis. The overactivation of these immune cells is often associated with a higher proteolytic activity (to fight invading pathogens), which can be monitored using fluorogenic probes to provide information on the inflammatory response during infection and be used for diagnostic application.
In this thesis, a series of fluorogenic probes for the detection of neutrophil and macrophage proteases implicated in inflammatory processes are developed. In the first chapter a major review of proteases and fluorogenic probes is presented. In the two subsequent chapters a series of probes that provide an OFF/ON fluorescent signal for the detection of the serine protease human neutrophil elastase (hNE) is reported. Two generations of probes were developed, the first generation consists of a series of probes with fluorescence emission in the green region of the spectrum based on previous successful designs. The probes allowed detection of elastase in activated neutrophils and in so-called neutrophil extracellular traps (NETs) which have been implicated in the pathogenesis of acute and chronic inflammatory diseases. Limitations of its in vivo application led to the development of a second generation of probes with fluorescence emission in the NIR, where tissue penetration and fluorescence background are reduced. A novel synthetic approach was implemented to synthesise the second generation of probes with NIR emission. The NIR probes were validated in vitro and are currently being evaluated in cells. The optimised NIR probe could serve as an excellent tool for in vivo imaging. The fourth chapter focuses on the synthesis of a fluorogenic, pH stable, water-soluble FRET probe for the specific detection of CatD, an aspartic protease present in macrophages that is upregulated in macrophages when fighting bacterial infections. The probe was designed to serve as a tool to understand the mechanism of apoptosis associated bacterial killing by macrophages, where the activity of CatD was detected in macrophages exposed to bacteria.