Repositioning niclosamide and alectinib to inhibit the hyperphosphorylation of SARS‐CoV‐2's nucleocapsid protein
Song Chen, Zunyu He, Jean Kanyo, TuKiet Lam, Grace Ha, Brett Lindenbach, Ya HaBackground and purpose
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.
Experimental approach
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.
Key results
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.
Conclusions and implications
This work provides the biochemical basis for a novel approach to treat COVID‐19 by synergistically repositioning two FDA‐approved drugs.