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

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 à . Cell experiments indicated that PROTACs A, B, and C effectively induced dose-dependent M(pro) degradation in HEK293 stable cells, with DC(50) values ranging from 0.530 to 0.985 μM and exhibited high selectivity indices (CC(50)/DC(50) > 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.