Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells

Abstract

Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2'-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound's triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.

Keywords: NS5; ProTides; RNA-dependent RNA polymerase; Zika virus; antiviral agents; neural stem cells; nucleoside analogues; prodrugs.

Scheme 1

Synthesis of 2′-C-ethenyluridine ProTide. (i) H₂ (g), Pd/BaSO₄, quinoline, benzene, 78%; (ii) bovine serum albumin (BSA), uracil, SnCl₄, ACN, 60 °C to 80 °C, 55%; (iii) NaOMe, MeOH, 0 °C to reflux, 94%; (iv) tBuMgCl, N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphynyl]-L-alanine 1-methylethyl ester, THF, −70 °C to room temperature (RT), 45%.

Figure 1

Design and structure of nucleotide analogue inhibitors tested against Zika virus (ZIKV) in this study.

Figure 2

Protection from ZIKV-induced cytopathic effect by 2′-C-methyluridine aryloxyl phosphoramidate ProTide and sofosbuvir in a ZIKV-sensitive glioblastoma stem cell model. DMSO-treated GSC 387 cells [-ZIKV and +ZIKV, H/PAN multiplicity of infection (MOI) 10], 10 or 30 µM sofosbuvir-treated cells or 10 or 30 µM 2′-C-methyluridine aryloxyl phosphoramidate ProTide (2′-CMU ProTide)-treated cells were incubated for 72 h at 37 °C and 5% CO₂, and cell foci were subsequently imaged in bright-field.

Figure 3

Reduction in ZIKV titers by 2′-C-methyluridine aryloxyl phosphoramidate ProTide and sofosbuvir in neural stem cells as observed by plaque assay. Viral infections were conducted in human fetal neural stem cells (H/PAN ZIKV, MOI 0.1) using vehicle-untreated (ONLY ZIKV), DMSO vehicle-treated, 10 or 30 µM sofosbuvir (SOF)-treated, and 10 or 30 µM 2′-C-methyluridine aryloxyl phosphoramidate ProTide (2′CMU)-treated samples. The dotted line represents the limit of detection of the assay. Virus was titered as described in the methods section and plaque forming units (PFU) per ml were calculated for each sample, ± standard error (SE), n = 4.

Figure 4

Kinetic characterization of nucleotide and nucleotide analogue incorporation by ZIKV RdRP. (A) ZIKV RdRP and primer/template (P/T) substrate (n) was mixed with excess UTP, 2′-F-2′-C-methyluridine triphosphate (2′-F-2′-C-MeUTP), or 2-C-methyluridine triphosphate (2′-C-MeUTP), and the reaction was quenched at the indicated timepoints. The substrate and the single nucleotide extension product (n + 1) were separated by using a 20% polyacrylamide denaturing gel, and bands were quantified to determine percent incorporation and plotted against time. (B) A single exponential fit was used to determine an observed rate of incorporation, k_obs (% incorporation min⁻¹) of 0.151 ± 0.004, 0.0085 ± 0.001, and 0.016 ± 0.001, for UTP (dotted black line), 2′-F-2′-C-MeUTP (dashed red line), and 2′-C-MeUTP (solid blue line), respectively. SE is reported as deviation from the fit, n = 2.

Figure 5

Structural superpositioning of apo and bound ZIKV RdRP and hepatitis C virus (HCV) RdRP. The ZIKV RdRP model gc-o3 [34] in complex with two manganese ions (green), ATP (red/CPK coloring), and RNA (orange/CPK coloring) is shown in red, and a crystal structure of apo ZIKV RdRP is shown in magenta (PDB 5WZ3 [38]). A crystal structure of HCV RdRP complexed with sofosbuvir diphosphate (cyan/CPK coloring), RNA (orange/CPK coloring), and a manganese ion (purple) is shown in cyan (PDB 4WTG [36]), and an apo structure of HCV RdRP is shown in blue (PDB 3MWV [39]). A comparison of the homologous residue D225 (HCV RdRP) and D1141 (ZIKV RdRP) is highlighted in stick form.