Discovery of thiacyanine dyes as a new class of potent coronavirus inhibitors that suppress viral RNA synthesis

Topoisomerase poisons are clinically used anticancer drugs that can induce DNA cleavage complexes to block replication. TOP3B is the only topoisomerase that can catalyze topological changes on either DNA or RNA and induce cleavage complexes on both nucleic acids. We proposed that TOP3B poisons may inhibit coronavirus RNA genome replication and tested this hypothesis by using mouse hepatitis coronavirus (MHV). We found that one of the two types of reported TOP3B poisons, thiacyanine dyes, possess potent inhibitory activities for MHV. Interestingly, the antiviral activity of the thiacyanine dyes is unaltered in Top3b-KO cells, suggesting that these dyes inhibit viral replication independent of TOP3B. Subsequent screening revealed that multiple members of the thiacyanine dye family have antiviral activity comparable to or stronger than remdesivir, the U.S. Food and Drug Administration (FDA)-approved drug for coronavirus, in an MHV-infected cell line model. One thiacyanine dye (NSC93472) significantly inhibits MHV replication in mouse lungs, showing its potential as an anticoronavirus drug. Mechanistic studies showed that NSC93472 preferentially binds two RNA fragments derived from SARS-CoV-2 genome over random ssRNA, interferes with assembly of an elongation-competent complex between the viral RNA-dependent RNA polymerase and the RNA template, and inhibits the RNA synthesis mediated by the RNA-dependent RNA polymerase. Moreover, NSC93472 can inhibit RNA synthesis by the reverse transcriptase of Moloney murine leukemia virus. Our studies demonstrate that thiacyanine dyes represent a new family of coronavirus inhibitors and suggest that TOP3B poisons and anti-RNA virus drugs share common characteristics in RNA binding and inhibition of enzymatic reactions on RNA.