Enantioselective Chemical Probe for Chikungunya nsP2 Helicase with Antialphaviral Activity

Abstract

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.

Keywords: allosteric; alphavirus; chemical probe; enantioselective; helicase; inhibitor.

1

Racemic oxaspiropiperidine nsP2hel inhibitor 1. The chiral center at C-4 is indicated.

2

Assignment of absolute configuration at C-4 of the active enantiomer. (i) 1 M KOH solution, rt; then 4-methoxybenzoyl chloride, Et₃N, dichloromethane (DCM), 63% yield (over two steps); (ii) 1 M KOH solution, rt; then 4-chloro-1-butylsulfonyl chloride, Et₃N, dimethylformamide (DMF), 91% yield (over two steps); (iii) 1,8-diazabicyclo[5.4.0]­undec-7-ene (DBU), acetonitrile (ACN), 70 °C, yield 74%; (iv) HCl in dioxane, 99% yield; and (v) 1-phenylcyclopentane-1-carboxylic acid, (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimde) (EDC), hydroxybenzotriazole (HOBT), diisopropylethylamine (DIPEA), DMF. 56% yield.

3

Conformational isomerism and thermal motion of the oxaspiropiperidine ring. (A) (R)-1 was isolated as a stable noncrystalline powder. (B) ¹H NMR spectrum of (R)-1 in CD₃CN at 25 °C showing line broadening of the piperidine protons (P). (C) ¹³C NMR spectrum of (R)-1 in pyridine-d ₅ at 25 °C showing line broadening of the piperidine carbons (red arrows) and cyclopentane carbons (blue arrows). (D) Refined X-ray structure of (R)-2 showing disorder in the piperidine ring and Boc group. (E) Depiction of the thermal motion in the piperidine ring and the amide rotamers seen in the SC-XRD structure of (R)-2.

4

Inhibition of nsP2 helicase by (R)-1 (closed circles) and (S)-1 (open squares). (A) ATPase assay. (B) RNA unwinding assay. Error bars represent the average of triplicate determinations.

5

Target engagement was determined by ¹⁹F NMR. (A) Difluorinated analogs (R)-9 and (S)-9. (B) ¹⁹F NMR peak areas of (R)-9 and (S)-9 in the absence or presence of the nsP2hel protein (aa 1–464). Relative concentrations of the ligand and protein are indicated.