Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication.

Abstract

Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406. Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.