show Abstracthide AbstractRespiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, young children, the elderly and the immunocompromised. Effective directly acting antivirals are not available.To address this need, we screened the ReFRAME drug-repurposing library consisting of 12,993 small molecules. Around 68% of these compounds are either licensed for use in humans or in advanced stages of clinical development. Disease annotations of these molecules are dominated by cancer (43%), central nervous system (CNS, 23%), and cardiovascular/respiratory (20%). Thus, 86% of molecules target physiological processes in humans, making the library a unique resource for identification of repurposing candidates and for discovery of host factors suitable for antiviral therapies.Interrogating the library with a RSV GFP reporter virus, we identified primary candidates mapping to 61 distinct target categories. Expectedly, RSV F protein inhibitors accumulated the largest number of candidates, followed by HSP90, and NF-kappaB inhibitors. We selected lonafarnib, a farnesyltransferase inhibitor used for the treatment of Hutchinson-Gilford Progeria Syndrome and in phase three trials for the treatment of hepatitis delta virus infections (HDV), for further follow up.Dose-response analyses and plaque assays confirmed the antiviral activity against RSV A and B strains (RSV A IC50 of 0.02 uM, CC50 of 13.74 uM). Tipifarnib, another farnesyltransferase inhibitor, was antiviral against HDV but did not inhibit RSV, suggesting that lonafarnib blocks RSV independently of interfering with the farnesyltransferase. Passaging of RSV with lonafarnib selected for a phenotypically resistant virus population accumulating non-synonymous mutations in the RSV F protein (T335I and T400A). Lentiviral pseudotypes programmed with parental or variant RSV F proteins confirmed that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance revealed RSV F protein binding of lonafarnib, but not tipifarnib. Oral administration of lonafarnib modestly reduced RSV virus dose in a murine infection model.Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide F protein inhibitor.