Repositioning niclosamide and alectinib to inhibit the hyperphosphorylation of SARS-CoV-2's nucleocapsid protein

In the past two decades, three pathogenic coronaviruses, including SARS-CoV-2, spilled over from natural animal reservoirs into the human population. Upon entry into infected cells, the nucleocapsid (N) protein of coronavirus becomes heavily phosphorylated within its central Ser-Arg domain by host kinase GSK-3, which triggers genomic RNA unpackaging and facilitates its recruitment to the replication transcription complex. Although initially considered a promising antiviral strategy, inhibiting N hyperphosphorylation requires high concentrations of kinase inhibitor and is impractical to achieve by conventional pharmacological means.

We investigated the effect of GSK-3 inhibition on SARS-CoV-2 replication in Calu-3 and Vero E6 cells. The dose-dependent effect of the GSK-3 inhibitor to block N hyperphosphorylation was examined in 293T cells heterologously expressing the viral protein. Liquid chromatography-tandem mass spectrometry analysis was performed to evaluate the phosphorylation profile of the purified N protein.

GSK-3 inhibition delays the release of progeny virus from infected cells. N hyperphosphorylation relies on a complex, redundant priming mechanism and exhibits strong cooperativity, which together contribute to resistance against GSK-3 inhibitors. The N^R203M and N^R203K/G204R mutations-present in the delta and omicron variants of concern (VOCs), respectively-reduce the efficiency of GSK-3-mediated phosphorylation. We discovered a novel pharmacological tool combining niclosamide and alectinib that partially degrades GSK-3 and restores the sensitivity of N hyperphosphorylation to the clinically tested GSK-3 inhibitor, enzastaurin.

This work provides the biochemical basis for a novel approach to treat COVID-19 by synergistically repositioning two FDA-approved drugs.