Identification of Direct-Acting nsP2 Helicase Inhibitors with Antialphaviral Activity

Alphaviruses are mosquito-borne RNA viruses that pose
a significant
public health threat, with no FDA-approved antiviral therapeutics
available. The nonstructural protein 2 helicase (nsP2hel) is an enzyme
involved in unwinding dsRNA essential for alphavirus replication.
This study reports the discovery and optimization of first-in-class
oxaspiropiperidine inhibitors targeting nsP2hel. Structure–activity
relationship (SAR) studies identified potent cyclic sulfonamide analogs
with nanomolar antiviral activity against chikungunya virus (CHIKV).
Biochemical analyses of nsP2hel ATPase and RNA unwindase activities
showed these compounds act in a noncompetitive mode, suggesting that
they are allosteric inhibitors. Viral resistance mutations mapped
to nsP2hel and a fluorine-labeled analog exhibited direct binding
to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers
of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis
virus (VEEV). These findings support nsP2hel as a viable target for
the development of broad-spectrum, direct-acting antialphaviral drugs.