ABSTRACT
We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry. Utilizing analogues of halofuginone and small molecule inhibitors of the PRS, we establish that inhibition of HS presentation and viral replication is dependent on proline tRNA synthesis opposed to PRS activation of the integrated stress response (ISR). Moreover, we provide evidence that these effects are mediated by the depletion of proline tRNAs. In line with this, we find that SARS-CoV-2 polyproteins, as well as several HS proteoglycans, are particularly proline-rich, which may make them vulnerable to halofuginone translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a promising clinical trial candidate for the treatment of COVID-19.