Tools for the Design and Characterization of Covalent Cysteine Protease Inhibitors
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Abstract
The research of covalent cysteine protease inhibitors is on the cutting edge of drug discovery, as the recent U.S. Food and Drug Administration’s approval of the very first registered inhibitor nirmatrelvir demonstrated that covalent inhibitors of this target class have finally arrived in reality. The repertoire of potential cysteine-reactive inhibitors was yet found to be tremendous and new chemotypes are constantly being discovered and characterized. In this regard, tuning the reactivity of the electrophilic warhead is essential for the successful development of cysteine protease inhibitors, and hence, the search for those is often synonymous with the quest for new covalent warheads.
In this dissertation, novel warheads for addressing the catalytic cysteine residues of S. aureus sortase A and T. brucei rhodesiense cathepsin L (rhodesain) were developed and characterized. Rhodesain and sortase A each represent a drug target with disease relevance; rhodesain for the neglected parasite-borne African sleeping sickness and sortase A for antibiotic-resistant pathogenic Gram-positive bacteria such as S. aureus. However, rhodesain and sortase A are unrelated cysteine proteases that prefer different warhead chemotypes highlighted by their very distinctive catalytic mechanisms.
In the course of this work, mechanistic investigations and optimizations of six warhead chemotypes were performed. Enzyme kinetic, biophysical, and quantum chemical methods were used to elucidate the underlying reaction mechanisms and to find rational approaches to increase biological effectiveness and selectivity. Disulfanylbenzamides and sulfonylpyrimidines were shown to be potent and selective irreversible inhibitors of the S. aureus sortase A, and these were investigated as novel resistance-preventing anti-virulence agents during cell-based biofilm inhibition experiments. Four additional rhodesain-targeting warheads were investigated, namely substituted naphthoquinones, fluorodinitroarenes, fluorovinyl sulfones, and vinylogous arginine Michael acceptors. The first three of which featured a covalent-reversible course of inhibition which proved to be an advantageous property across all chemotypes for avoiding off-target reactivity and cellular toxicity.
Instrumental hurdles arose recurrently during the investigation of the new warhead entities which were addressed by the development of open-source hardware and software that contributed to the solution of synthetic and biochemical questions. The development of a liquid-handling and peptide synthesis robot (FINDUS), a differential scanning fluorometer (openDSF), and a web toolkit for crystallographic analysis of normalized B-factors (BANΔIT) highlight the focus of this work to invent chemical, technical, and conceptual tools for drug discovery.