Identification and Characterization of a Metal Binding-Independent Rift Valley Fever Virus Endonuclease Inhibitor

The viral cap-snatching mechanism, facilitated by the endonuclease (EndoN) domain of viral polymerases, is critical for viral gene transcription and translation and is therefore an attractive target for antiviral development. The successful development of EndoN inhibitor XOFLUZA (Baloxavir marboxil) has opened the possibility that the EndoN domain of negative-sense RNA viruses (NSVs) can be targeted in a similar fashion. Rift Valley Fever Virus (RVFV) belongs to the <i>Bunyaviricetes</i> class, which includes other pathogens with pandemic potential, such as severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV). Here, we identify MBXC-4522 as a RVFV EndoN inhibitor using a high-throughput FRET-based assay. We screened a library of >20,000 compounds and identified those that target the RVFV EndoN domain. MBXC-4522, a spiro piperidine pyrido pyridine, directly binds the RVFV EndoN domain, increases protein stability, and inhibits EndoN activity. MBXC-4522 acts in a metal binding-independent mechanism, while XOFLUZA's mode of action is metal binding-dependent. Infectious assays also support the ability of MBXC-4522 to inhibit pathogenic RVFV (ZH501), suggesting that hit-to-lead optimization and future <i>in vivo</i> validation are warranted.