Identification of Direct-Acting nsP2 Helicase Inhibitors with Antialphaviral Activity

Abstract

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.

1

Oxaspiropiperidine nsP2 ATPase Inhibitors.

1. Synthesis of 1a and Analogs (1b–d)­

2. Synthesis of 2a and Sulfonamide (2b–h, 5a–g, 6a–m, 7a-c), Amide (3a–l), and Urea (3p–u) Analogs

3. Synthesis of Tertiary Sulfonamides (2i–m) and Amide (3m)

4. Synthesis of Chloroalkyl Sulfonamides and Amide (2c, 2d, 3e), Cyclic Sulfonamides (2n–2o), Cyclic Amides (3n–o), and Secondary Amine (4c)­

5. Synthesis of Tertiary Alcohol 4e

2

Variable temperature ¹³C NMR of 2n in pyridine-d₅ showing the resonances of the sp³ carbons from 18 to 72 ppm. Numbers above the resonances in the spectra correspond to the carbons labeled in the chemical structure. Individual assignment of the piperidine carbons (P) was not possible. Dashed red lines indicate the respective resonances at the variable temperatures.

3

Antialphaviral activity of 2o. NHDF cells were infected with the alphaviruses and treated with compound 2o. Supernatants were collected at 24 hpi. Viral titers were determined by plaque assay on Vero cells at 48–72 hpi and presented as percentage of antiviral activity, n = 3 ± SD. Absolute viral titers are shown in Figure S10.

4

Correlation of CHIKV antiviral activity with nsP2 ATPase inhibition.

5

Activity of 2o on point mutants of CHIKV-nLuc located in the RecA1 domain of nsP2hel. n = 3 ± SD.

6

¹⁹F NMR spectra of 7b in the absence or presence of the nsP2hel protein (aa 1–464). Relative concentrations of the ligand and protein are indicated.

7

Mechanism of nsP2hel inhibition by 2o. (A,B) Conversion of ³²P-ATP to ³²P-ADP by nsP2 was monitored by TLC at two time points. The IC₅₀ of 2o for inhibition of ADP formation did not change. Panels show two of four independent experiments, with an IC₅₀ = 1.0 ± 0.6 μM (mean ± SD) calculated from all four trials. (C) Rate of conversion of ³²P-ATP to ³²P-ADP by nsP2 at different substrate concentrations. The enzyme K _m was not affected by 2o. Data represent three independent trials fit globally; therefore, standard deviations for individual data points are not shown. (D) 2o inhibited conversion of dsRNA to ssRNA by nsP2. Structure of the forked dsRNA with 5′ and 3′ overhangs is shown. The unwinding assay was performed three times, the panel shows a representative trial. IC₅₀ = 1 ± 1 μM (mean ± SD) calculated from all three trial. (E) 2o did not preclude binding of fluorescein-labeled dsRNA to increasing concentrations of nsP2. Data from three independent trials, each using a different inhibitor concentration as shown in the graph. K _d,app = 22 ± 12 nM (mean ± SD) calculated from all three trials.