Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Herein, high-throughput screening of an FDA-approved drug library was performed against LASV entry using a pseudo-type virus enveloping LASV glycoproteins. Two hit drugs, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both drugs inhibited LASV entry by blocking low-pH-induced membrane fusion. Moreover, lacidipine irreversibly bound to the LASV glycoprotein complex (GPC), resulting in virucidal activity. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine without apparent loss of the viral growth profile. Furthermore, lacidipine showed antiviral activity and specificity against both LASV and the Guanarito virus (GTOV), which is also a category A new world arenavirus. Drug-resistant variants indicate that the V36M in ectodomain of SSP mutant and V436A in the transmembrane domain of GP2 mutant conferred GTOV resistance to lacidipine, suggesting that lacidipine might act via a novel mechanism other than calcium inhibition. This study shows that both lacidipine and phenothrin are candidates for LASV therapy, and the membrane-proximal external region of the GPC might provide an entry-targeted platform for inhibitors.

Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors