Discovery of a tetrahydrobenzothiophen-2-yl-pyrazolo[1,5-a]pyrimidine-3-carboxamide-based PROTAC as degrader of SARS-CoV-2 main protease

Targeted protein degradation has emerged as a new strategy in drug development, particularly in the anti-cancer field. Although still limited, in recent years, the application of Proteolysis Targeting Chimera (PROTAC) technology against viral infections has been investigated, establishing its broad therapeutic potential. Here, we present the design, synthesis, and biological characterization of a series of PROTACs targeting SARS-CoV-2 Main Protease (M^Pro) based on a previously identified M^Pro inhibitor linked to ligands of either Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligase. Among the synthesized compounds, the VHL-addressing PROTAC 6 emerged as the most potent M^pro degrader, effectively reducing SARS-CoV-2 M^pro protein levels in infected 293T-hACE2 cells with a DC₅₀ value of 0.9 μM and a D_max of almost 90% at 25 μM. In parallel, it displayed antiviral activity against SARS-CoV-2 in the low micromolar range, also confirmed in the disease-relevant model of human lung Calu-3 cells, and retained activity against the human endemic coronavirus HCoV-OC43. PROTAC 6 resulted inactive in inhibiting SARS-CoV-2 M^pro catalytic activity in vitro and mechanistic studies confirmed that its antiviral activity is due to the binding and successive induction of degradation of SARS-CoV-2 M^pro. Kinetic solubility experiments showed that PROTAC 6 exhibits very low solubility in phosphate-buffered saline solution but significant higher solubility in two biorelevant dissolution media mimicking intestinal conditions under fasted and fed states, as FaSSIF and FeSSIF. In conclusion, we developed SARS-CoV-2 M^pro degraders based on a novel warhead and confirmed the significant role of targeted protein degradation technology in advancing the identification of new, effective anti-coronavirus PROTACs.