Enantioselective Chemical Probe for Chikungunya nsP2 Helicase with Antialphaviral Activity

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated a high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to the nsP2hel protein was confirmed by ¹⁹F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.