Integrated computational screen and validation of thalidomide-based PROTACs targeting SARS-CoV-2 main protease

Abstract

The persistent evolution of SARS-CoV-2 underscores the need for antiviral strategies. The viral main protease (M^pro) represents a conspicuous target due to its essential role in viral replication and high conservation across SARS-CoV-2. Conventional M^pro inhibitors face challenges such as drug resistance and toxicity. Proteolysis-targeting chimeras (PROTACs) offer an event-driven mechanism to degrade rather than inhibit the target protein, overcoming key limitations of occupancy-driven pharmacology. Here, we designed a series of thalidomide-based PROTACs targeting SARS-CoV-2 M^pro and explored their effectiveness through computational simulations and experimental validation. Molecular docking revealed that PROTACs A, B, and C exhibit favorable binding free energies (ΔG < −8.0 kcal mol⁻¹). These findings were further supported by molecular dynamics simulations, which demonstrated consistently stable binding over 10 ns, with backbone RMSD values maintained within the range of 0.18–0.30 Å. Cell experiments indicated that PROTACs A, B, and C effectively induced dose-dependent M^pro degradation in HEK293 stable cells, with DC₅₀ values ranging from 0.530 to 0.985 μM and exhibited high selectivity indices (CC₅₀/DC₅₀ > 10). Mechanistically, PROTACs-induced degradation of M^pro via the ubiquitin–proteasome system was evidenced by enhanced K48-linked polyubiquitination and suppression of degradation upon proteasome inhibition. The PROTACs (A, B and C) exhibit comparable effects and share similar mechanisms in degrading M^pro. Our work develops effective degraders targeting SARS-CoV-2 M^pro and highlights the therapeutic potential of PROTACs in combating drug-resistant viral targets via a catalytic degradation mechanism.