Vinyl Sulfone-Based Inhibitors of Nonstructural Protein 2 Block the Replication of Venezuelan Equine Encephalitis Virus

Abstract

Emerging infectious diseases like those caused by arboviruses such as Venezuelan equine encephalitis virus (VEEV) pose a serious threat to public health systems. Development of medical countermeasures against emerging infectious diseases are of utmost importance. In this work, an acrylate and vinyl sulfone-based chemical series was investigated as promising starting scaffolds against VEEV and as inhibitors of the cysteine protease domain of VEEV's nonstructural protein 2 (nsP2). Primary screen and dose response studies were performed to evaluate the potency and cytotoxicity of the compounds. The results provide structural insights into a new class of potent nonpeptidic covalent inhibitors of nsP2 cysteine protease represented by compound 11 (VEEV TrD, EC₅₀ = 2.4 μM (HeLa), 1.6 μM (Vero E6)). These results may facilitate the evolution of the compounds into selective and broad-spectrum anti-alphaviral drug leads.

Scheme 1. Synthesis of Target Compounds 1–16

Reagents and conditions: (a) CH₂Cl₂:CH₃OH, Et₃N, Boc₂O, 23°C, 2 h, > 90%; (b) H₂O, NaIO₄, RT, 1 h, 64%; (c) NaH, THF, methyl diethylphosphonoacetate, diethyl((methylsulfonyl)methyl) phosphonate or diethyl((phenylsulfonyl)methyl) phosphonate, 0°C, 25 min, 40–70%; (d) 33% TFA in DCM, 0°C, 1.5 h. Then ACN, R-COOH, Et₃N, HBTU, RT, 16 h, 30-40%; (e) DMP, H₂O–DCM, 23 °C, 1 h, 40–60%.

Figure 1

Inhibition of VEEV nsP2 protease by compounds 12 and 13 in a gel discontinuous assay. V12: CFP-YFP FRET substrate; nsP2: tag-free nonstructural protease 2. For both gels, Lane 1 is V12 alone, Lane 2 is V12 + nsP2, Lanes 3–10 are V12 + nsP2 + 12 or 13 at 3.6, 1.8, 0.9, 0.45, 0.22, 0.11, 0.06, and 0.03 μM, respectively. The molecular weights (MW) of uncut V12, the two V12 fragments (cut v12), and tag-free nsP2 are 58.3, 30.9, 27.4, and 38.29 kDa, respectively. The reactions were carried out in 50 mM HEPES buffer pH 7.4 for 24 h at room temperature. The concentrations of VEEV nsP2 and V12 were 1 μM and 10 μM, respectively.

Figure 2

Modeled complex of VEEV nsP2 and compound 11. The highlighted residues are predicted to have significant van der Waals interactions (piecewise linear potential) with compound 11. The blue dash depicts H-bond interaction. The figure was prepared using Molecular Operating Environment (MOE).

Figure 3

Modeled complex of VEEV nsP2 and compound 11. The left panel is a stick model representation of the predicted network of H-bond interactions (depicted as the blue dash lines) stabilizing the nsP2–11 complex. Asn 545, His 510, and His 546 residues are predicted to have direct H-bond interactions with compound 11. Carbon atoms are depicted as yellow in 11. The right panel is a 2D depiction of the proximity (gray contour) of active site residues to compound 11 as well as the extent of exposure (purple hue) of the compound’s atoms to solvent. The figure was prepared using Molecular Operating Environment (MOE).