Site-Selective Chemical Modification and Inhibition of Cysteine Proteases

This Thesis is divided into three different parts. In thesis part I, fluorobenzene probes with accurately tunable reactivity for protein profiling through site-selective cysteine labeling are reported. The reactivity profile of an array of fluorobenzenes was investigated. It was demonstrated that reactivity can be tuned by selection of a strongly electron-withdrawing substituent combined with variation of the number of fluorine atoms for fine-tuning. The optimal probes were completely chemo-selective for arylation of cysteine over all other nucleophilic amino acid residues under aqueous conditions. These probes linked to azide, a fluorophore or biotin were used for labeling of eGFP and albumin. These probes were also tuned for use in selective labeling among several cysteine residues and were therefore applicable for activity-based protein profiling in cell lysates and for selective inhibition of cysteine proteases over serine proteases. In thesis part II, the synthesis of fluoroaryl peptides as novel substrate mimetics for selective cysteine protease inhibition is presented. In particular, selectivity is a major challenge in developing covalent drugs targeting closely related cysteine proteases. Caspase-1(interleukin converting enzyme) an overexpressed cysteine protease in inflammatory diseases and a validated target in drug discovery, was selectively targeted by a fluoroaryl tetrapeptide mimetic. We have demonstrated that mimicking substrate structure and using mild reactive fluoroaryl warhead is a successful approach for selective inhibition of caspase-1. The inhibition kinetics of other synthesized substrate mimetics are currently investigated in details to determine the influence of the tunable reactivity of these warheads and/or the recognition peptide on the potency and selectivity of the inhibition.
In thesis part III, evolutionary principles of stepwise selection and variation offered by combinatorial methods guided by molecular modelling were used to develop catalytic metallopeptides that mimic metalloproteases. These metallo-peptides show efficiency and selectivity in peptide bond cleavage in water at room temperature and neutral conditions. These small and versatile organozymes take advantage of metal coordinating building blocks that are strategically positioned centrally in a peptide backbone or in a peptide macrocycle. This approach provided peptide-metal complexes with scaffolds capable of utilizing the peptide functionality for productive binding of fluorogenic FRET peptide substrates. This subsequently led to highly selective peptide bond cleavage. The in-solution cleavage kinetics of these organozymes are currently investigated