Beyond binding affinity – elucidating protease inhibition mechanisms from a biophysical perspective
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Abstract
Proteases play essential roles in nature and are involved in protein maturation, digestion, forming extracellular matrices and their degradation, and immune escape. Inhibition of such proteases often represents a promising strategy to counter related diseases. Detailed knowledge beyond binding affinity is a prerequisite to developing more potent inhibitors. Characterizing the modes of action relies on biophysical techniques that can provide structural information on binding modes and determine binding kinetics or thermodynamics. Furthermore, they can trace conformational changes. In the course of this work, inhibitors for several disease-related proteases were characterized using a toolset of biophysical techniques. The target proteases were selected for investigation based on their distinct functions and catalysis mechanisms. In this regard, the flaviviral NS3 proteases feature an NS2B cofactor, can adopt at least two conformations, and are essential for polyprotein processing and maturation. Staphylococcus aureus sortase A is a transpeptidase that relies on a Ca2+ cofactor and is vital to forming extracellular matrices. The human matriptase displays a roughly symmetric substrate binding site and degrades extracellular matrices by activating matrix metalloproteases. Thereof, the following projects were derived: Project 1. Two strategies were examined to elucidate the mechanistic basis of entropic inhibitor optimization: The macrocyclization of Zika virus NS2B/NS3 protease inhibitors and the introduction of higher ligand symmetry of human matriptase inhibitors to enable multiple equivalent binding modes. The various opportunities to derive data from well-designed ITC experiments prompted us to develop a set of teaching experiments, demonstrating the versatility of ITC experiment execution and analysis. To guide medicinal scientists through ITC-assisted ligand development, we implemented the webserver ITCcalc. Project 2. The Dengue virus and Zika Virus NS2B/NS3 proteases display an allosteric binding site. Several biophysical methods, such as X-ray co-crystallization, MST, ITC, DSF, CD, EPR, fluorometric assays, smFRET, and 19F-NMR, were utilized to give in-depth information on conformational changes that are induced upon ligand binding.Project 3. During virus maturation, the NS2B/NS3 protease is autocatalytically cleaved at the NS2B/NS3 junction. To date, the biological significance of this highly conserved process is unknown. In this project, we investigated the mechanisms of this process to understand the regulatory functions of autocatalytic cleavage. To this end, we used fluorometric assays, CD, extensive site-directed mutagenesis, analytical SDS-PAGE, and protein MS. Project 4. Due to their antibacterial properties, metal ions are widely applied as additives in surface disinfectants, alloys of medicinal instruments, textiles, and wound dressings. SrtA constitutes an exciting anti-virulence target to counter antibiotic-resistant Staphylococcus aureus lineages. In this work, we found several metal cations to activate, inhibit, or modulate SrtA activity in FRET-based transpeptidase assays. Furthermore, their mode of action behind the activity modulation of SrtA was elucidated. Tb3+ FRET assays, ITC, DSF, MST, and X-ray crystallography were used for this purpose.