From Inhibition to Protein-Targeted Degradation: Structural Insights into SARS-CoV-2 and CVB3 Proteins for Antiviral Drug Development

The COVID-19 pandemic has highlighted the urgent need for innovative antiviral strategies capable of addressing both existing and emerging viral threats. This PhD research focused on two complementary approaches for the development of antiviral therapeutics: the inhibition of viral proteases through small molecules and the exploitation of the targeted protein degradation (TPD) strategy. In particular, the work centred on the 3-Chymotrypsin-like Protease (3CLPro) of SARS-CoV-2, the 3C Protease (3CPro) of Coxsackievirus B3 (CVB3), and the Nsp13 helicase of SARS-CoV-2, enzymes that play essential roles in viral replication and represent validated drug targets. The first part of this work investigated the inhibitory potential of gold-based metallodrugs such as Auranofin and its derivatives against SARS-CoV-2 3CLPro. Kinetic assays yielded Ki values in the low micromolar range (0.4–2.1 μM), while ESI-MS and X-ray crystallography confirmed adduct formation and identified gold coordination sites. The second part explored the PROTAC (PROteolysis TArgeting Chimera) strategy as an innovative means to induce the degradation of viral proteases. Two PROTAC classes were designed and characterized, starting from respectively the Nirmatrelvir and Ibuzatrelvir nitrile inhibitors and the peptidomimetic inhibitor GC-376. Solution NMR spectroscopy, fluorescence binding assays, and analytical size-exclusion chromatography (SEC) were employed to monitor binary and ternary complex formation involving the viral protease, the PROTAC molecule, and the Celebron (CRBN) E3 ubiquitin ligase (CRBN-TBD and CRBN-midi). Evidence of productive ternary complex formation was obtained for the CVB3 3CPro:GC-376 PROTAC:CRBN-midi system, while steric hindrance likely prevented similar assembly with SARS-CoV-2 3CLPro. A third research axis focused on the optimization of SARS-CoV-2 Nsp13 expression and purification. The implementation of zinc and iron supplementation increased soluble protein yield up to 0.5 mg/L of culture, enabling future studies on the enzyme’s interaction with novel inhibitors within the PNRR Spoke 4 collaborative framework. Finally, the study outlined the prospective use of cellular experiments, X-ray crystallization, computational modelling to characterize PROTAC-mediated ternary complexes. Collectively, the results provide molecular insights into inhibition and targeted degradation mechanisms, advancing the understanding of viral protease druggability and paving the way towards structure-guided antiviral design for future pandemics.