Modified Peptides as Viral Protease Inhibitors
Creators: name: Ullrich, Sven
Publisher: name: The Australian National University
Year: 2024

description: Peptides have experienced a renaissance as promising therapeutics. This development has been driven by modified peptides, including peptidomimetics and constrained peptides. Modifications often endow peptides with improved target binding affinity and metabolic stability, addressing limitations of their canonical counterparts. Valued for these beneficial therapeutic qualities, modified peptides have become a focal point of pharmaceutical research (Chapter 1). In the pursuit of drug candidates for disease-relevant targets, encoded peptide libraries have emerged as reliable discovery platforms. Among these, the RaPID (random non-standard peptides integrated discovery) platform, a variation of mRNA display using genetic code reprogramming, contributes to the identification of cyclic peptide ligands for a wide range of targets. Most recently, viral proteins have been the focus of several RaPID campaigns (Chapter 2). Viral proteases are particularly attractive targets for the investigation of peptide-derived drugs. These enzymes play a critical role in the replication of many medically relevant viruses. While protease inhibitors are indispensable in treating chronic viral infections, the development of antiviral drug candidates for the proteases of acute viruses like Zika and SARS-CoV-2 represents a promising frontier in antiviral drug discovery (Chapter 3). In the wake of the COVID-19 pandemic, the SARS-CoV-2 main protease became a key antiviral target. Previous drug discovery efforts focused mainly on covalently binding, substrate-inspired peptidomimetics. Out of several substrate-derived cyclic and linear peptides without electrophilic reactive groups, only a longer linear variant showed moderate inhibition (Chapter 4). Surveying a greater chemical space with the RaPID system, several high-affinity peptide macrocycles were identified, notable for inhibiting SARS-CoV-2 main protease with non-covalent binding modes (Chapter 5). The emergence of SARS-CoV-2 variants has drawn attention to the potential effect of their mutations on vaccines and antivirals. Despite numerous changes in the spike protein, the main protease of the virus has accumulated only single amino acid substitutions across various lineages. Six circulating main protease mutants, including one of the Omicron variant, maintained comparable in vitro catalytic competence to the ancestral strain. Additionally, the clinically relevant inhibitor nirmatrelvir exhibited similar potency in vitro against these variants (Chapter 6). Enhancing the preparedness for future viral adaptations, a combined computational and experimental workflow revealed five currently non-abundant main protease mutations that confer potential resistance against nirmatrelvir and a non-covalent cyclic peptide inhibitor (Chapter 7). Developing diverse cyclic peptide topologies is important to broaden the chemical space of drug discovery. The selective reactivity of aminothiols and dicyanopyridine embedded in non-canonical amino acids enabled a catalyst-free biocompatible synthesis of bicyclic peptides in aqueous buffer at physiological pH. A bicyclic peptide inhibitor of Zika virus protease demonstrated the potential of this method for the development of therapeutics (Chapter 8). The structural analysis of proteins using NMR spectroscopy can be improved with modified and unnatural amino acid side chains. To facilitate the chelation of paramagnetic lanthanoid ions for NMR purposes, a modular dicyanopyridine-based tag was installed on proteins (Chapter 9). Additionally, the incorporation of non-canonical trimethylsilyl and trifluoromethyl amino acids helped evaluate SARS-CoV-2 main protease inhibitors with NMR analysis complementary to activity-based assays (Chapter 10).