Phenothiazines inhibit SARS-CoV-2 cell entry via a blockade of spike protein binding to neuropilin-1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells using angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP-1) as the primary receptor and entry co-factor, respectively. Cell entry is the first and major step in initiation of the viral life cycle, representing an ideal target for antiviral interventions. In this study, we used a recombinant replication-deficient vesicular stomatitis virus-based pseudovirus bearing the spike protein of SARS-CoV-2 (SARS2-S) to screen a US Food and Drug Administration-approved drug library and identify inhibitors of SARS-CoV-2 cell entry. The screen identified 24 compounds as primary hits, and the largest therapeutic target group formed by these primary hits was composed of seven dopamine receptor D2 (DRD2) antagonists. Cell-based and biochemical assays revealed that the DRD2 antagonists inhibited both fusion activity and the binding of SARS2-S to NRP-1, but not its binding to ACE2. On the basis of structural similarity to the seven identified DRD2 antagonists, which included six phenothiazines, we examined the anti-SARS-CoV-2 activity of an additional 15 phenothiazines and found that all the tested phenothiazines shared an ability to inhibit SARS2-S-mediated cell entry. One of the phenothiazines, alimemazine, which had the lowest 50% effective concentration of the tested phenothiazines, exhibited a clear inhibitory effect on SARS2-S–NRP-1 binding and SARS-CoV-2 multiplication in cultured cells but not in a mouse infection model. Our findings provide a basis for the development of novel anti-SARS-CoV-2 therapeutics that interfere with SARS2-S binding to NRP-1. •A screen of a US FDA-approved drug library using a VSV-based pseudovirus bearing the SARS-CoV-2 spike protein (SARS2-S).•Phenothiazines are potent inhibitors of SARS2-S-mediated cell entry.•Phenothiazines inhibit SARS2-S binding to neuropilin-1, but not its binding to ACE2.•The phenothiazine alimemazine strongly inhibits SARS-CoV-2 multiplication in cultured cells, but not in mice in vivo.