Peptide-based targeted covalent inhibitors of cysteine proteases

Targeted covalent inhibitors (TCIs) can modulate dysregulated enzymes by covalently engaging with nucleophilic residues in active sites. Cysteine cathepsins, implicated in diseases such as cancer, osteoporosis, and viral infections, are promising targets for TCI development. The nucleophilic cysteine residue in the active site reacts with inhibitory electrophilic molecules, but structural similarity between cathepsins has resulted in a lack of specificity of inhibition. This work explores the synthesis of peptide-based isothiazolones, a class of reactive heterocycles, as selective covalent inhibitors of cathepsins. These compounds exhibit a reactive sulfur–nitrogen bond that can be targeted by thiol nucleophiles, forming labile disulfide linkages and have previously been shown to interact with biological thiols. Cysteine-containing peptides were synthesised via solid-phase peptide synthesis and modified to incorporate isothiazolone moieties by using a Norrish Type II transformation of a phenacylsulfide derivative. Optimisation of reaction conditions led to successful isothiazolone formation, alongside other interesting side products, for several peptide candidates. The scaffold of a known peptidic inhibitor of cathepsin B, a protease strongly implicated in tumour progression, was selected for TCI design and showed promising isothiazolone conversion rates. One candidate was evaluated in vitro and showed potential inhibitory activity under non-reducing conditions, supporting the scaffold’s suitability for further biological investigation. C-terminal carboxylate analogues were also shown to undergo isothiazolone formation, providing constructs relevant for interactions with the occluding loop hidtidine residues of cathepsin B. Ongoing work is directed toward the improved design, isolation and purification of these peptide isothiazolones in sufficient quantities to permit expanded biological evaluation as TCIs.

Funder

Research Ireland

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

cbn

Collections

University of Galway Theses (Research Masters Theses)