Activity of selected nucleoside analogue protides against zika virus in human neural stem cells

Article abstract of DOI:10.3390/v11040365

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

Full text of DOI:10.3390/v11040365

viruses
Article
Activity of Selected Nucleoside Analogue ProTides
against Zika Virus in Human Neural Stem Cells
Jean A. Bernatchez 1,2,† , Michael Coste 3,†, Sungjun Beck 1,†, Grace A. Wells 3, Lucas A. Luna 3,
Alex E. Clark 1,4, Zhe Zhu 5,6 , David Hecht 3,7 , Jeremy N. Rich 5,6, Christal D. Sohl 3,
* ,
Byron W. Purse 3,8,
*
and Jair L. Siqueira-Neto 1,2,
*
1
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego,
La Jolla, CA 92093, USA; jbernatchez@ucsd.edu (J.A.B.); sjbeck@ucsd.edu (S.B.);
alexclark@ucsd.edu (A.E.C.)
Center for Discovery and Innovation in Parasitic Diseases, University of California, San Diego,
La Jolla, CA 92093, USA
Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182, USA;
michael.coste1@gmail.com (M.C.); graceawells@gmail.com (G.A.W.); luluna@ad.ucsd.edu (L.A.L.);
dhecht@swccd.edu (D.H.)
2
3
4
5
6
Department of Cellular and Molecular Medicine, University of California, San Diego,
La Jolla, CA 92093, USA
Sanford Consortium for Regenerative Medicine, La Jolla, CA 92093, USA;
zhz504@ucsd.edu (Z.Z.); drjeremyrich@gmail.com (J.N.R.)
Department of Medicine, Division of Regenerative Medicine, School of Medicine, University of California,
San Diego, La Jolla, CA 92093, USA
Department of Chemistry, Southwestern College, Chula Vista, CA 91910, USA
The Viral Information Institute, San Diego State University, San Diego, CA 92182, USA
Correspondence: csohl@sdsu.edu (C.D.S.); bpurse@sdsu.edu (B.W.P.); jairlage@ucsd.edu (J.L.S.-N.);
Tel.: +1-619-594-2053 (C.D.S.); +1-619-594-6215 (B.W.P.); +1-858-822-5595 (J.L.S.-N.)
These authors contributed equally to this paper.
7
8
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 March 2019; Accepted: 18 April 2019; Published: 20 April 2019
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 20-C-methyluridine and 20-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking
group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate
ProTide group of 20-C-methyluridine completely abolished antiviral activity of the compound.
The aryloxyl phosphoramidate ProTide of 20-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: Zika virus; nucleoside analogues; antiviral agents; NS5; prodrugs; ProTides; neural stem
cells; RNA-dependent RNA polymerase
Viruses 2019, 11, 365; doi:10.3390/v11040365
www.mdpi.com/journal/viruses

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1. Introduction
The explosive spread of Zika virus (ZIKV) during the 2015–2016 epidemics in Latin America
attracted worldwide attention to this previously neglected disease. The lack of effective vaccines or
small molecules to prevent or treat this infection remains a cause for concern and emphasizes the urgent
need for new therapeutic options [
neurodevelopmental anomalies in the fetus, including microcephaly, congenital ZIKV syndrome (CZS),
and even fetal demise [ ]. While most cases of ZIKV infection are asymptomatic, reports of rash,
conjunctivitis, pain in the muscles or joints, and fever have been recorded in clinical manifestations of
1]. ZIKV, an emerging flavivirus infection, causes several serious
2
the disease, and in rare cases, ZIKV infection has been linked to the neuroinflammatory Guillain-Barr
é
syndrome [2].
Viral polymerases remain attractive drug targets for the development of selective antiviral
therapies [ ]. Generally, clinically-approved inhibitors that target these proteins fall into two broad
classes [ ]. The first class consists of nucleoside analogues that mimic the natural substrate of the
3–5
6,7
enzyme. Upon analogue incorporation by the virally-encoded polymerase, DNA or RNA synthesis is
abrogated by preventing further nucleotide incorporation (chain termination), thereby arresting viral
replication. The second class is known as non-nucleoside inhibitors, which bind allosterically and
arrest viral nucleic acid synthesis by distorting the polymerase active site geometry to interfere with
nucleotide binding or nucleotide incorporation.
A common prodrug strategy used for antiviral ribonucleoside analogues involves the
chemical synthesis of nucleoside analogue monophosphates with metabolically-removable masking
groups [8–10]. These masking groups neutralize the negative charge of the phosphate on the nucleoside
and allow for better membrane penetrance of these compounds. Chemical addition of the first
phosphate group is crucial for improving intracellular levels of the active triphosphate metabolite form
of ribonucleoside analogues. Metabolic addition of the first phosphate group is relatively slow for this
class of compounds and represents a rate-limiting step for the triphosphorylation of ribonucleoside
analogues to active drug molecules.
This “ProTide” strategy has been successfully deployed in the synthesis of bioactive ribonucleoside
analogues [11
(HCV) drug, sofosbuvir [13]. Sofosbuvir has recently been shown by multiple groups to also be active
against ZIKV in vitro and in vivo 14 17], demonstrating that ribonucleoside analogue ProTides are an
,12], including the Food and Drug Administration-(FDA) approved hepatitis C virus
[
–
attractive avenue for the development of novel, selective antivirals against ZIKV.
Numerous nucleoside analogues have recently been explored as antiviral agents against
ZIKV [18–23]. However, many of these compounds were tested in either cell-free systems or using
cells that are not clinically relevant for the disease (such as Vero cells). Further, sofosbuvir is the
only FDA approved inhibitor tested against ZIKV thus far in ProTide form. In addition, recent work
by our group and others has suggested that sofosbuvir and ProTides in general have differential
activity depending on the cell line used, which may be linked to the cell-specific metabolism of
ProTides [17,24]. In this work, we chemically synthesized a library of 13 ProTides and tested them for
activity against ZIKV in human neural stem cells, a disease-specific cell model of infection. Interestingly,
the activity of 20-C-modified aryloxyl phosphoramidate ProTides was strongly biased towards uridylate
derivatives, with only modest activity observed for the 20-C-modified adenylate and cytidylate aryloxyl
phosphoramidate ProTides we tested. Changing the aryloxyl phosphoramidate masking group to
a 2-(methylthio)ethyl phosphoramidate abolished the activity of the 20-C-methyluridine ProTide,
a representative hit from our library. In a head-to-head comparison between the 20-C-methyluridine
aryloxyl phosphoramidate ProTide and sofosbuvir, better suppression of viral titers was observed
for the former compound. In addition, better protection from ZIKV-induced cytopathic effect was
observed for the 20-C-methyluridine aryloxyl phosphoramidate ProTide over sofosbuvir in a highly
ZIKV-susceptible stem cell model of infection with strong phenotypic changes during ZIKV infection.
These results represent the first broad study of nucleoside analogue ProTides against ZIKV.

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2. Materials and Methods
2.1. Chemistry
All nucleoside analogue ProTides, including McGuigan aryloxy phosphoramidite ProTides
and 2-(methylthio)ethyl tryptamine ProTides, were synthesized using established phosphorus
chemistry [25
ribose precursor
(Scheme 1) [27]. The ethenyl group of compound
alkyne using 5% Pd/BaSO4 in (1:1) quinoline/benzene [28]. Ribosylation of uracil to compound
using N,O-bis(trimethylsilyl)acetamide (BSA) and SnCl4 in ACN afforded benzoyl-protected
20-C-ethenyluridine
. The deprotection of compound was performed using NaOMe in MeOH
to afford 20-C-ethenyluridine
, which was then treated with tBuMgCl in THF followed by
,
26]. The 20-C-ethenyluridine ProTide was synthesized using a known ethynyl
1, which was obtained by following a previously reported synthetic route
2
was obtained by hydrogenation of the
2
3
3
4
N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphinyl]-l-alanine 1-methylethyl ester and to afford
20-C-ethenyluridine aryloxy phosphoramidate 5.
2.1.1. General Synthetic Experimental
All reagents, chemicals, and nucleoside analogues precursors were purchased from Acros Organics,
Carbosynth, Chem-Impex International, AK Scientific, and Fisher Chemical at ACS reagent grade or
higher purity and used as received. The (3R,4R,5R)-5-(benzoyloxy)methyl)-3-ethynyltetrahydrofuran-2,
3,4-triyl tribenzoate
1 was synthesized by following a previously reported procedure [27].
Synthetic details for aryloxy phosphoramidate ProTides other than for 20-C-ethynyluridine and
20-C-ethenyluridine were published previously by our group [24]. All reactions were carried out
in either an oven-dried round bottom flask or a Schlenk tube under a nitrogen atmosphere using
commercially available anhydrous solvents and monitored by thin-layer chromatography with detection
by UV light. The 1H NMR and the 31P NMR spectra were acquired on a Varian 400-MHz spectrometer
and recorded at 298 K. Chemical shifts were referenced to the residual protio solvent peak and are given
in parts per million (ppm). Splitting patterns are denoted as s (singlet), d (doublet), dd (doublet of
doublet), dq (doublet of quartet), ddd (doublet of doublet of doublet), ddt (doublet of doublet of triplet),
t (triplet), td (triplet of doublet), tdd (triplet of doublet of doublet), q (quartet), and m (multiplet).
20-C-Ethynyluridine Aryloxy Phosphoramidate
To a stirred suspension of 20-C-ethynyluridine (0.09 mmol, dried under vacuum at 50 ◦C overnight)
in dry THF (1 mL) was added a 2.0 M solution of tert-butyl magnesium chloride in THF (96
µ
L,
◦
0.19 mmol). The mixture was stirred at 0 C for 30 min and then allowed to warm to room
temperature and stirred for an additional 30 min. The reaction mixture was then cooled to 0 ◦C and
N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphinyl]-L-alanine 1-methylethyl ester (46 mg, 0.10
mmol) was added. The reaction mixture was stirred for 18 h as the temperature was allowed to warm
to room temperature. The solvent was removed by rotary evaporation. The reaction mixture was
purified first using flash chromatography (a gradient from 0 to 30% methanol in dichloromethane)
and then using preparative, normal-phase HPLC (10 to 40% MeOH in dichloromethane gradient) to
1
afford 25.5 mg of the product (54.3%) in
≥
95% purity. H NMR (400 MHz, CD3OD):
δ
= 7.65 (d, 1H,
J = 8.1 Hz), 7.40–7.34 (m, 2H), 7.27 (dq, 2H, J = 7.7, 1.2 Hz), 7.20 (ddd, 1H, J = 8.2, 7.1, 1.1 Hz), 6.04
(s, 1H), 5.61 (d, 1H, J = 8.1 Hz), 4.97 (m, 1H), 4.49 (ddd, 1H, J = 11.8, 6.0, 2.1 Hz), 4.36 (ddd, 1H, J = 11.8,
6.1, 3.7 Hz), 4.16 (d, 1H, J = 9.1 Hz), 4.08 (ddt, 1H, J = 9.0, 3.9, 2.1 Hz), 3.92 (dq, 1H, J = 9.9, 7.1 Hz), 3.08
(s, 1H), 1.35 (dd, 3H, J = 7.2, 1.0 Hz), 1.22 (dd, 6H, J = 6.3, 1.8 Hz). 31P NMR (162 MHz, CD3OD)
δ= 3.78.
5-((Benzoyloxy)methyl)-3-vinyltetrahydrofuran-2,3,4-triyl tribenzoate 2
To a stirring solution of
(2 mL) under H2 (g), we added 5% palladium on barium sulfate (20.8 mg, 10 wt%) followed by
quinolone (22 L) and stirred at room temperature for 2 h. The mixture was diluted in ethyl acetate,
1 (208.7 mg, 0.362 mmol, 1 equivalent) in benzene (2 mL) and ethanol
µ

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washed 3 times with water, and dried over anhydrous sodium sulfate. The reaction mixture was
purified using flash chromatography (0 to 30% ethyl acetate in hexane gradient) to afford purified
product; (162.7 mg, 0.281 mmol, 77.7%) 1H NMR (400 MHz, CDCl3):
δ = 8.23–8.09 (m, 4H), 8.07–8.03
(m, 2H), 7.92–7.88 (m, 2H), 7.69–7.39 (m, 10H), 7.18–7.12 (m, 2H), 6.46 (dd, 1H, J = 17.6, 11.2 Hz), 6.25
(d, 1H, J = 8.3 Hz), 4.54 (dd, 2H, J = 12.2, 4.8 Hz), 4.81 (ddd, 2H, J = 8.4, 4.7, 3.9 Hz), 4.73 (dd, 1H,
J = 12.2, 3.9 Hz), 4.54 (dd, 1H, J = 12.2, 4.8 Hz)
5-((Benzoyloxy)Methyl)-2-(2,4-Dioxo-3,4-Hihydropyrimidin-1(2H)-yl)-3-Vinyltetrahydrofuran-3,4-diyl
Benzoate 3
Uracil (63.0 mg, 0.562 mmol, 2 equivalent) and
2 (162.7 mg, 0.281 mmol, 1 equivalent) were dried
under high vacuum in separate round bottom flasks for 2 h. Under N2 (g) and stirring, we added
dry acetonitrile (2 mL) to uracil followed by the addition of bis(trimethylsilyl)acetamide (550.1 µL,
2.250 mmol, 8 equivalent). The reaction mixture was refluxed at 80 ◦C for 1 h then cooled to 0 ◦C.
Then, compound 2 in dry acetonitrile (2 mL) was added to the reaction mixture followed by tin (IV)
chloride (229.9 µ
L, 1.968 mmol, 7 equivalent) and heated to 60 ◦C for 3 h. The reaction mixture was
poured into a separatory funnel containing ice cold water, extracted 3 times with ethyl acetate, and the
combined organic layer was dried over anhydrous sodium sulfate. The reaction mixture was purified
using flash chromatography (0 to 100% ethyl acetate in hexane gradient) to afford purified product;
(89.4 mg, 0.153 mmol, 54.6%) 1H NMR (400 MHz, CDCl3): δ= 9.22 (s, 1H), 8.09 (m, 4H), 7.86–7.82
(m, 2H), 7.63–7.56 (m, 2H), 7.63–7.56 (m, 6H), 7.29–7.21 (m, 2H), 6.65 (s, 1H), 6.12 (dd, 1H, J = 17.5,
11.1 Hz), 6.04 (d, 1H, J = 5.2 Hz), 5.64 (dd, 1H, J = 8.2, 2.1 Hz), 5.46–5.40 (dd, 2H), 4.94 (dd, 1H, J = 12.3,
3.2 Hz), 4.81 (dd, 1H, J = 12.3, 5.7 Hz), 4.66 (td, 1H, J = 5.5, 3.2 Hz)
1-(3,4-dihydroxy-5-(hydromethyl)-3-vinyltetrahydrofuran2-yl)pyrimidine-2,4(1H,3H)-dione 4
Compound
3 (90.5 g, 0.155 mmol, 1 equivalent) was dried overnight on high vacuum. Under
N2 (g), methanol (1.5 mL) was added, then the reaction mixture was cooled to 0 ◦C followed by the
dropwise addition of sodium methoxide (86.5 µL, 1.553 mmol, 10 equivalent). The reaction mixture
was raised to room temperature and stirred for 1.5 h. The reaction mixture was cooled to 0 ◦C followed
by the addition of formic acid until pH = 4. The reaction mixture was dried in vacuo then purified
using flash chromatography (0 to 40% methanol in dicholoromethane gradient) to afford a purified
product; (30.2 mg, 0.146 mmol, 93.8%) 1H NMR (400 MHz, CDCl3): δ= 8.13 (d, 1H, J = 8.1 Hz), 5.95
(s, 1H), 5.74–5.65 (m, 2H), 5.44 (dd, 1H, J = 17.3, 1.3 Hz), 5.26 (dd, 1H, J = 10.8, 1.3 Hz), 4.22 (d, 1H,
J = 9.2 Hz), 4.03–3.97 (m, 2H), 3.84–3.79 (m, 1H)
isopropyl(((5-(2,4-dioxo-3,4-dihydropyridimin-1(2H)-yl)-3,4-dihydroxy-3-inyltetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate 5
To a stirring solution of
4
(36.4 mg, 0.176 mmol, 1 equivalent) in dry tetrahydrofuran (1 mL) at
L, 0.369 mmol, 2.1 equivalent). The reaction
0 ◦C was added tert-butyl magnesium chloride (184.5
µ
mixture was raised to room temperature and allowed to react for 30 min. The reaction mixture was
cooled to 0 ◦C, then N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphinyl]-L-alanine 1-methylethyl
ester (95.5 mg, 0.211 mmol, 1.2 equivalent) was added and gradually warmed to room temperature
overnight. The reaction mixture was purified using flash chromatography (0 to 30% methanol [MeOH]
in dichloromethane gradient) to afford the purified product; (42.5 mg, 0.0788 mmol, 44.8%) 1H NMR
(400 MHz, CD3OD): δ= 7.76 (d, 1H, J = 8.1 Hz), 7.38 (dd, 2H, J = 8.6, 7.2 Hz), 7.31–7.25 (m, 2H), 7.25–7.16
(m, 2H), 5.94 (s, 1H), 5.68 (dd, 1H, J = 17.3, 10.8 Hz), 5.60 (d, 1H, J = 8.1 Hz), 5.48 (d, 1H, J = 1.4 Hz), 5.44
(d, 1, J = 1.4 Hz), 5.27 (dd, 1H, J = 10.8, 1.4 Hz), 4.96 (1H, m), 4.58–4.49 (m, 1H), 4.45–4.38 (m, 1H), 4.17
(s, 2H), 4.00–3.87 (m, 1H), 1.38 (dd, 3H, J = 7.1, 1.0 Hz), 1.21 (d, 6H, J = 6.3 Hz). 31P NMR (162 MHz,
CD3OD) δ = 3.78.

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(5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl
(2-(methylthio)ethyl) (2-(1H-indol-3-yl)ethyl)phosphoramidate (20-C-methyluridine methylthioethyl
tryptamine ProTide)
The 20-C-methyluridine (45.3 mg, 0.1790 mmol, 1 equivalent) and the triethylammonium
2-(methylthio)ethyl phosphonate (92.1 mg, 0.3579 mmol, 1.5 equivalent) were added to an oven-dried
schlenk tube and dried overnight on the high vacuum. To the flask under N2 (g) was added dry
pyridine (2 mL) followed by the dropwise addition of trimethylacetyl chloride (60.6 µL, 0.4923 mmol,
2.75 equivalent), and the reaction was stirred for 3 h at room temperature. The reaction was quenched
by saturated sodium bicarbonate and extracted 3 times with dicholormethane. The organic layer was
dried over anhydrous sodium sulfate and concentrated in vacuo at room temperature and dried for 2 h
on high vacuum. The dried residue was dissolved in pyridine (2 mL) under N2 (g). To the reaction
mixture was simultaneously added trimethylamine (49.9
(114.7 mg, 0.7158 mmol, 4 equivalent, dissolved in dry pyridine 2 mL), and carbon tetrachloride
(26.0 L, 0.2685 mmol, 1.5 equivalent) and stirred for 25 min at room temperature. The reaction
µL, 0.3579 mmol, 2 equivalent), tryptamine
µ
mixture was diluted with methanol and purified using flash chromatography (0 to 30% methanol in
dichloromethane gradient) to afford a purified product as a mixture of two epimers at phosphorus;
(8.0 mg, 0.01443 mmol, 7.3%) 1H NMR (400 MHz, CD3OD):
δ= 7.72 (dd, 1H, J = 21.4, 8.2 Hz), 7.55–7.50
(m, 1H), 7.33–7.29 (m, 1H), 7.06 (t, 2H, J = 7.3 Hz), 6.97 (tdd, 1H, J = 7.5, 4.6, 2.4 Hz), 5.95 (d, 1H,
J = 5.2 Hz), 5.64 (dd, 1H, J = 8.1, 3.8 Hz), 4.37–4.29 (m, 1H), 4.17 (ddd, 1H, J = 11.7, 5.0, 3.1 Hz), 4.04
(dt, 1H, J = 10.4, 6.8 Hz), 4.00–3.90 (m, 1H), 3.77 (dd, 1H, J = 12.4, 9.3 Hz), 3.21 (td, 2H, J = 10.9, 5.6 Hz),
2.96 (dt, 2H, J = 7.3, 4.4 Hz), 2.66 (td, 2H, J = 6.7, 2.3 Hz), 2.07 (d, 3H, J = 1.4 Hz), 1.13 (s, 3H). 31P NMR
(162 MHz, CD3OD) δ= 10.52 and 10.41.
Scheme 1. Synthesis of 20-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%.
2.2. Cell Culture
Human fetal NSCs were purchased from Clontech (human neural cortex; catalog number Y40050).
Maintenance of hNSCs was performed in Neurobasal medium without phenol red (Thermo Fisher,
Waltham, MA, USA) and in the presence of B-27 supplement (1:100; catalog number 12587010;
Thermo Fisher), N-2 supplement (1:200; catalog number 17502-048; Invitrogen, San Diego, CA, USA),
20 ng/mL fibroblast growth factor (catalog number 4114-TC-01M; R&D Systems, Minneapolis, MN,
USA, 20 ng/mL epidermal growth factor (catalog number 236-EG-01M; R&D Systems), GlutaMAX

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(catalog number 35050061; Thermo Fisher), and sodium pyruvate. Cells were prepared in 6-well plates
precoated with laminin (10 mg/mL; catalog number L2020; Sigma-Aldrich, St. Louis, MO, USA) and
were grown to near confluence (80 to 90%) prior to passage. For passaging, cells were rinsed gently
with phosphate-buffered saline (PBS) (without calcium and magnesium) and then treated with TryPLE
for 5 min at 37 ◦C for cell detachment (catalog number 12563029; Thermo Fisher). The GSC 387 cell line
was prepared and maintained similarly to what has been previously described using the media and
detachment conditions used for hNSCs described above [29]. Vero cells (ATCC CCL-81) were purchased
from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA)
with high glucose (4.5 g/L), 10% fetal calf serum (FCS) (Sigma), and 1% Penicillin-Streptomycin (PS)
(Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦C and 5% CO2.
2.3. Viruses
Viral strains used in this study were provided through BEI Resources, NIAID, NIH: Zika
virus H/PAN/2016/BEI-259634, NR-50210 (GenBank accession number KX198135), Zika virus
PRVABC59/Human/2015/Puerto Rico, NR-50240 (GenBank accession number KU501215).
Vero cells were used to expand viral cultures for 2 to 3 serial passages in order to amplify the
titers. Centrifugation of infected cell supernatants was performed to remove cell debris, and then
viral stocks were concentrated through use of a sucrose cushion. Neural maintenance medium base
[50% DMEM–F-12 medium–GlutaMAX, 50% Neurobasal-A medium, 1
supplement (Life Technologies)] supplemented with 1% DMSO (Sigma) and 5% fetal bovine serum
(FBS) (Gibco) was used to resuspend concentrated viral stocks, and aliquots were stored at
80 ◦C.
×
N-2 supplement, 1
×
B-27
−
Plaque assay on Vero cells was used to determine viral titers, which were greater than or equal to
2 × 108 PFU/mL.
2.4. CellTiter-Glo Activity Assay
NSCs (5000 cells/well) were seeded into 1536-well black, clear bottom plates containing 20-point,
2-fold serial dilutions of ProTides or DMSO vehicle controls and were either mock-infected, infected
with ZIKV H/PAN/2016/BEI-259634 at the multiplicity of infection (MOI) of 10, or infected with ZIKV
PRVABC59/Human/2015/Puerto Rico at MOI of 10. Cell viability was measured using the CellTiter-Glo
assay (Promega, Madison, WI, USA) 72 h post-infection. An EnVision plate reader (PerkinElmer)
was used for readouts of luminescence intensity. All data were normalized to DMSO vehicle controls
and were expressed as % relative luminescence intensity. Normalized activity and toxicity data were
plotted against ProTide concentration and fit to a sigmoidal dose-response curve with variable slope
using Prism 6 (GraphPad Software) to generate EC50 and CC50 curves.
2.5. Bright-Field Microscopy
GSC 387 cells (5000 cells/well) were seeded into 1536-well black, clear bottom plates with wells
containing either 10 or 30 µM sofosbuvir, 10 or 30 µ
M 20-C-methyluridine aryloxyl phosphoramidate
ProTide, or DMSO vehicle controls, and were either mock-infected or infected with ZIKV
H/PAN/2016/BEI-259634 at MOI of 10. Cell foci images were acquired 72 h post-infection in bright-field
using an ImageXpress Micro automated microscope (Molecular Devices, San Jose, CA, USA).
2.6. Plaque Assay
Vero cells were used for the titering experiments for drug-treated and vehicle-treated hNSC
cultures infected with ZIKV H/PAN/2016/BEI-259634. Vero cells were seeded at a density of 30,000 cells
per well in 96-well plates and incubated in 5% CO2 at 37 ◦C in high-glucose DMEM with 1% FBS and 1%
penicillin/streptomycin for 24 h before the addition of the viral inoculum from ZIKV-infected hNSCs.
Inocula were collected from ZIKV H/PAN/2016/BEI-259634-infected NSCs (MOI 0.1) in the presence of
10
mock-infected cultures, infected cultures without compound treatment, and infected cultures with the
µM or 30 µ µM or 30 µM sofosbuvir,
M 20-C-methyluridine aryloxyl phosphoramidate ProTide, 10

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DMSO vehicle at 48 h post-infection. Collected inocula were diluted 10-fold in high-glucose DMEM
containing 1% FBS and 1% penicillin-streptomycin in 96-well plates. These inocula were added to the
pre-prepared Vero cells for 1 h in a volume of 100 µ
L and at a temperature of 37 ◦C, covered with an
agarose overlay, and lastly incubated for 72 h at 37 ◦C and 5% CO2. Fixation with 4% formaldehyde
(final concentration) for 24 h was then performed for the samples. Following this, the overlay was
removed, and crystal violet staining was performed to count the ZIKV plaques.
2.7. ZIKV RdRP Expression and Purification
The RdRP domain of ZIKV NS5 (amino acids 2772-3423 of accession number AMB18850) was fused
to an N-terminal 6 -histidine tag containing a TEV cleavage site, and the cDNA was codon-optimized
×
and synthesized by IDT (San Diego, CA, USA). This construct was then inserted into a pET-17b
vector (Invitrogen) and heterologously expressed in the Rosetta strain of Escherichia coli. Cultures
transfected with the RdRP construct were grown at 37 ◦C in LB media containing 1% ethanol and
100
with 100
Cells were then harvested by centrifugation at 9000 RPM for 30 min at 4 ◦C, and pellets were stored at
80 ◦C. For purification, pellets were resuspended in lysis buffer (50 mM HEPES at pH 7.5, 20 mM
NaCl, 20% glycerol, 0.1% octyl-glucoside, 5 mM -mercaptoethanol) supplemented with a protease
µ
g/mL amplicillin until an OD600 of 0.6–0.7 was reached. Protein expression was then induced
µ
M IPTG prior to an additional 5 h incubation at 20 ◦C and lower speed shaking (130 RPM).
−
β
inhibitor tablet (Thermo Fisher). Cells were lysed using a microfluidizer (Microfluidics International
Corporation, Newton, MA, USA) and clarified via centrifugation at 12,000 RPM for 30 min at 4 ◦C.
The supernatant was loaded onto TALON cobalt affinity beads (Clontech, Mountain View, CA, USA) at
4 ◦C. The unbound protein was washed with 50 mL each of the following buffers: wash buffer (50 mM
HEPES at pH 7.5, 5 mM β-mercaptoethanol) containing 100 mM NaCl and 10 mM imidazole, wash
buffer containing 500 mM NaCl and 20 mM imidazole, and finally wash buffer containing 200 mM
NaCl and 30 mM imidazole. The protein was eluted in wash buffer containing 200 mM NaCl and a
linear imidazole gradient of 30–300 mM. Protein fractions were pooled and dialyzed overnight against
50 mM HEPES at pH 7.0, 100 mM NaCl, 10% glycerol, 0.01% CHAPS, and 5 mM dithiothreitol at 4 ◦C.
The protein was concentrated to ≥6 µM and stored in single-use aliquots at −80 ◦C.
2.8. Single Nucleotide Incorporation Assay
To prepare the double-stranded RNA substrate, primer (50-AGUUGUUGAUC30) was 32P-labeled
at the 50 position upon incubation with [ -32P] ATP (PerkinElmer, Waltham, MA, USA) and T4
γ
polynucleotide kinase (NEB, Ipswich, MA, USA) at 37 ◦C for 30 min. Excess [ -32P] ATP was removed
γ
with a Bio-Spin 6 column (Bio-Rad, Hercules, CA, USA). After heat inactivation, the 50-32P labeled
primer was combined with an excess template (50-AUUCACUCAGAUCAACAACU-30). The primer
and the template were annealed via step incubation at 95 ◦C (5 min), 55 ◦C (15 min), and 37 ◦C (10 min)
to generate the primer/template substrate (P/T substrate), where the next correct nucleotide to be
incorporated was UTP.
Single nucleotide incororation assays were modeled from previous work [19]. Briefly, ZIKV RdRP
(1
37 ◦C with the addition of 150
µ
M) and P/T substrate (200 nM) were pre-incubated at 37 ◦C for 20 min. Reactions were initiated at
µM UTP or analog in 50 mM HEPES pH 7.5, 15% glycerol, 15 mM NaCl,
7.5 mM MnCl2, and 10 mM dithiothreitol. At specified time points, reaction aliquots were quenched
with ethylenediamine tetra-acetic acid (EDTA) to a final concentration of 0.3 M.
Prior to loading equi-volume samples on a 20% polyacrylamide, 8 M urea, TBE (89 mM Tris-Borate,
2 mM EDTA) denaturing gel, the quenched samples were combined with dye (formamide, 0.01%
xylene cyanol, 0.01% bromophenol blue) and 1 µL of 5 µ
M D10 (50-AGTTGTTGAT-30) trap, followed by
incubation at 95 ◦C for 5 min. The trap was used to bind to RNA template to reduce gel smearing [30].
Oligos were separated via electrophoresis at a maximum of 1900 V and 75 W for 3–6 h. Phosphorimaging
(FLA 9500 imager, GE, Marlborough, MA, USA) was used to visualize the bands, and bands were
quantified using ImageQuant software (GE). Plots of percent incorporation versus time were fit to a

Viruses 2019, 11, 365
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single exponential equation using Prism (Graphpad Software, San Diego, CA, USA) to compare relative
incorporation. While single-turnover conditions were used, non-physiologically relevant rates were
obtained, as incorporation occurred during a steady-state time scale. This has been a common issue in
the field when measuring ZIKV RdRP incorporation [19,20,31] due to measuring incorporation into
a double-stranded template instead of physiologically preferred de novo RNA replication (an issue
seen in HCV incorporation assays [32]), and perhaps the presence of purification tags [33]. However,
since here we were interested in comparing relative incorporation efficiency rather than obtaining rate
constants, we and others [19,31] have found this experimental set-up ideal and better suited to scaling
up the number of analogues to be assessed.
2.9. Structural Alignment
The following model and structures were aligned to HCV RdRP complexed with RNA, Mn2+
,
and sofosbuvir-DP (PDB 4WTD [34]) using Pymol [35]: 1) a model of ZIKV RdRP in complex with
RNA, Mn2+ ions, and an incoming ADP modified to an ATP (gc-o3 [36]) generated from ZIKV RdRP
stucture PDB 5TFR [37] (160 atoms aligned, RMS = 1.650); 2) a crystal structure of apo ZIKV RdRP
(PDB 5WZ3 [38]) (154 atoms aligned, RMS = 2.860); and 3) a crystal structure of apo HCV RdRP
(PDB 3MWV [39]) (530 atoms aligned, RMS = 0.874).
3. Results
3.1. ProTide Library Design
To investigate the activity of a set of nucleotide analogue ProTides against ZIKV and obtain basic
structure-activity relationships, we selected a set of nucleotides designed to inhibit viral RdRPs by
three different mechanisms: obligate chain termination, nonobligate chain termination, and lethal
mutagenesis (Figure 1) [40,41]. Past work—and the successful results of drug development against
HCV—shows that nucleosides alone lack adequate potency owing to slow phosphorylation in the
target cells [42]. McGuigan’s nucleotide aryloxy phosphoramidates include amino acid esters and
are well established; sofosbuvir includes this functional group, and we chose it as our starting
point [9,43]. One potential drawback of the McGuigan design is that unmasking is initiated by the
action of carboxyesterases, which are highly active in the hepatocytes targeted by HCV but less
active in the neural cell targets of ZIKV [42]. For that reason, we chose to investigate a recently
developed methylthioethyl tryptamine ProTide from Wagner, which is unmasked by chemical cleavage
of methylthioethanol followed by tryptamine removal by HINT1 [26]. HINT1 also catalyzes P-N bond
cleavage in the classic McGuigan ProTide. Our hypothesis was that the carboxyesterase-independent
unmasking of the methylthioethyl tryptamine ProTide would provide greater potency against ZIKV in
human neural stem cells.
The selection of nucleoside analogues for testing focused on ribose modifications known to induce
chain termination in vitro when used as a nucleoside triphosphate analogue with the viral RdRP. Some
of the selected compounds, e.g., the 30-deoxy and the 30-O-methyl, are obligate chain terminators,
but phosphorylation of this class of compounds in cells is often inefficient [18,19]. Better success
in antiviral drug development has been found when using nonobligate terminators, especially 20
modifications that alter the nucleoside conformation and prevent extension after incorporation during
RNA synthesis [14
of the 20-C-methyl ribosides, which have known potency against the Flaviviridae family [19
Also included were new ProTides of 20-C-ethynyl- and 20-C-ethenyluridine. The 20-C-ethynyl ribosyl
triphosphates are known sub- M inhibitors of flavivirus RdRPs [31 45], and we prepared the new
20-C-ethenyluridine ProTide to probe the active site of the RdRP and the metabolic consequences of
further modification of the 20-C-
position. A 5-fluorouridine ProTide was included in our set of
,
31]. We included sofosbuvir in this set as a point of reference, along with ProTides
,
31,44].
µ
,
β
analogues to test inhibition by lethal mutagenesis, although toxicity of mutagenic nucleoside analogues
is typically prohibitive of clinical applications.

Viruses 2019, 11, 365
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O
P
O
N
obligate chain terminators
R4
=
H
3'-deoxy (R1 = H, R2 = OH, R3 = H)
O
O
classic McGuigan
aryloxy
phosphoramidate
ProTide
3'-O-methyl (R1 = H, R2 = OH, R3 = OCH3)
3'-deoxy-3'-fluoro (R1 = H, R2 = OH, R3 = F)
nonobligate chain terminators
base
R4O
H3CS
O
2'-C-methyl (R1 = CH3, R2 = OH, R3 = OH)
2'-C-ethynyl (R1 = C CH, R2 = OH, R3 = OH)
2'-C-ethenyl (R1 = CH CH2, R2 = OH, R3 = OH)
2'-O-methyl (R1 = H, R2 = OCH3, R3 = OH)
O
R1
O
P
R4
=
R3 R2
NH
2'-deoxy-2'-fluoro-2'-methyl (R1 = CH3, R2 = F, R3 = OH)
mutagens
fluorinated nucleobase (R1 = H, R2 = OH, R3 = OH)
N
H
methylthioethyl tryptamine
ProTide
Figure 1. Design and structure of nucleotide analogue inhibitors tested against Zika virus (ZIKV) in
this study.
3.2. Activity against ZIKV and Toxicity of Nucleoside Analogue ProTides in Human Neural Stem Cells
To profile the activity and toxicity profiles of our library of ProTides, we screened compounds
in a 20-point, two-fold dose response with a highest concentration of 50
µM against ZIKV
PRVABC59/Human/2015/Puerto Rico and ZIKV H/PAN/2016/BEI-259634 in neural stem cells (MOI of 10)
using a luminescent cell-viability assay (CellTiter Glo) and determined EC50 (effective concentration 50)
and CC50 (cytotoxic concentration 50) values from these data (Table 1). We observed that the compounds
with the best selectivity indexes (SIs) were the 20-C-methyluridine aryloxyl phosphoramidate ProTide
(EC50PRVABC59: 1
20-C-ethynyluridine aryloxyl phosphoramidate ProTide (EC50PRVABC59: 0.3
0.8 M, CC50: >50 M, SIs: >166, >62, respectively). These outperformed sofosbuvir in terms
of anti-ZIKV activity (EC50PRVABC59: 35 M, EC50H/PAN: 46 M, CC50: >50 M, SIs: >1.4,
>1.1, respectively). Activity was observed for the 20-C-methyladenosine aryloxyl phosphoramidate
ProTide (EC50PRVABC59: 10 M, EC50H/PAN: 18 M, CC50: 42 M, SIs: >4, >2, respectively) and
20-C-methylcytidine aryloxyl phosphoramidate ProTide against PRVABC59 strain (EC50PRVABC59:
48 M, CC50PRVABC59: >50 M, SI: >1), albeit at lower levels. None of the other ProTides tested
µM, EC50H/PAN: 2
µM, CC50: 47
µM, SIs: >47, >23, respectively) and the
µM, EC50H/PAN:
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
displayed activity. Interestingly, the 20-C-ethenyluridine aryloxyl phosphoramidate ProTide was
completely inactive and quite toxic to the host cells, suggesting that this functional group may be more
promiscuous in terms of target binding or reactive with cellular components [46]. Replacement of
the classic McGuigan ProTide masking group with methylthioethyl tryptamine phosphoramidate for
the 20-C-methyluridine ProTide completely abolished activity. This result was not expected because
the classic McGuigan ProTides are unmasked with the involvement of both a carboxyesterase and
HINT1, whereas the methylthioethyl tryptamine phosphoramidate requires only HINT1 in addition to
spontaneous, chemical steps [26]. Clearly, cell line specificity for ProTide strategies must be considered
in the design of nucleotide analogue inhibitors of ZIKV.
Table 1. Anti-ZIKV activity and toxicity of nucleoside analogue ProTides in neural stem cells.
EC50
PRVABC59
ZIKV, µM
EC50
H/PAN
ZIKV, µM
SI 1
PRVABC59
ZIKV
Nucleoside Analogue
ProTide
CC50
µM
,
SI 1
H/PAN ZIKV
20-C-Methylcytidine
aryloxyl phosphoramidate
30-Deoxyadenosine aryloxyl
phosphoramidate
50-Fluorocytidine aryloxyl
phosphoramidate
48 ± 1
>50
>50
>50
>50
>50
>50
>1
>1
>1
>1
>50
10 ± 1
>0.2
>0.2

Viruses 2019, 11, 365
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Table 1. Cont.
EC50
PRVABC59
ZIKV, µM
EC50
H/PAN
ZIKV, µM
SI 1
PRVABC59
ZIKV
Nucleoside Analogue
CC50
µM
,
SI 1
H/PAN ZIKV
ProTide
30-O-Methyluridine aryloxyl
>50
>50
>50
>50
>50
>50
>1
>1
>1
>1
phosphoramidate
20-O-Methylcytidine
aryloxyl phosphoramidate
20-C-Methyluridinearyloxyl
1 ± 1
10 ± 1
>50
2 ± 1
18 ± 1
>50
47 ± 1
42 ± 1
>50
>47
>4
>23
>2
phosphoramidate
20-C-Methyladenosine
aryloxyl phosphoramidate
30-Deoxycytidine aryloxyl
phosphoramidate
>1
>1
30-Deoxy-30-Fluoroguanosine
aryloxyl phosphoramidate
>50
>50
>50
>1
>1
20-C-Ethenyluridine aryloxyl
>50
>50
10 ± 1
>50
>0.1
>166
>0.1
>62
phosphoramidate
20-C-Ethynyluridine
aryloxyl phosphoramidate
0.3 ± 1
0.8 ± 1
20-C-Methyluridine
2-(methylthio)ethyl
phosphoramidate
>50
>50
>50
>50
>1
>1
Sofosbuvir
35 ± 1
46 ± 1
>1.4
>1.1
1 SI = selectivity index: CC50/EC50 for a given viral strain. Data values are the represented
±
standard error (SE), n = 4.
3.3. Protection from ZIKV-Induced Cytopathic Effect by 20-C-Methyluridine Aryloxyl Phosphoramidate
ProTide and Sofosbuvir in a ZIKV-Sensitive Glioblastoma Stem Cell Line
We and others have previously shown that certain lines of brain tumors with stem cell-like genetic
programs, such as glioblastomas, are highly susceptible to ZIKV infection [29,47–49]. These cells display
a striking phenotypic change upon infection. To highlight the ability of a representative ProTide hit from
our library, 20-C-methyluridine aryloxyl phosphoramidate, to protect against ZIKV-induced cytopathic
effect, we treated the glioblastoma stem cell line GSC 387 with either 10 or 30
aryloxyl phosphoramidate and compared this to 10 or 30 M sofosbuvir or the DMSO vehicle control
µ
M 20-C-methyluridine
µ
treatment. Samples were prepared in both ZIKV-infected (MOI of 10) and uninfected conditions. After
72 h post-infection, bright-field microscopy images were obtained for each sample and are shown in
Figure 2. While both compounds were able to protect against the cytopathic effect at 30
µM (intact cell
foci were seen), the 20-C-methyluridine aryloxyl phosphoramidate ProTide was better able to protect
against ZIKV-induced cytopathic effect at the lower concentration of 10 µM than sofosbuvir, which is
in line with our EC50 data in neural stem cells. These data suggest that monosubstituted 20-C-modified
uridylate aryloxyl phosphoramidate ProTides may be an attractive chemical series to pursue for more
potent and selective ZIKV polymerase inhibitors than sofosbuvir.

Viruses 2019, 11, 365
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Figure 2.
Protection from ZIKV-induced cytopathic effect by 20-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 µ
M 20-C-methyluridine aryloxyl phosphoramidate ProTide (20-CMU
ProTide)-treated cells were incubated for 72 h at 37 ◦C and 5% CO2, and cell foci were subsequently
imaged in bright-field.
3.4. Repression of ZIKV Titers by 20-C-Methyluridine Aryloxyl Phosphoramidate ProTide and Sofosbuvir in
Neural Stem Cells
To further assess the antiviral activity of the 20-C-methyluridine aryloxyl phosphoramidate ProTide
against ZIKV, we performed plaque assays at 48 h post-infection to titer ZIKV (H/PAN, MOI of 0.1)
levels during treatment with either 10 or 30
ProTide or 10 or 30
µ
M of 20-C-methyluridine aryloxyl phosphoramidate
M of sofosbuvir in human fetal neural stem cells (Figure 3). At 30 µM compound,
µ
both 20-C-methyluridine aryloxyl phosphoramidate ProTide and sofosbuvir were able to repress viral
titers to undetectable levels. However, only 20-C-methyluridine aryloxyl phosphoramidate ProTide
was able to reduce ZIKV titers to undetectable levels at the 10
superior antiviral activity compared to sofosbuvir.
µM compound, again highlighting its
Figure 3. Reduction in ZIKV titers by 20-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 µ
M 20-C-methyluridine aryloxyl
phosphoramidate ProTide (20CMU)-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.
3.5. Enzymatic Incorporation of the Active Triphosphate Metabolites of 20-C-Methyluridine and Sofosbuvir
Over Time Reveals Higher Levels of Incorporation for the Former Compound
Single nucleotide incorporation assays were used to compare the relative preference of ZIKV RdRP
for UTP versus the UTP analogs 20-C-methyluridine triphosphate and 20-fluoro-20-C-methyluridine
triphosphate (the active form of sofosbuvir) (Figure 4). Both analogues appeared to serve as chain
terminators of ZIKV RdRP polymerization, as previously reported [19,31]. Unsurprisingly, UTP

Viruses 2019, 11, 365
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was most efficiently incorporated, with 50% incorporation by 5 min and full incorporation by
~30 min, followed by misincorporating extension. The 20-fluoro-20-C-methyluridine triphosphate had
significantly less efficient incorporation by ZIKV RdRP, as previously reported [19,31,50]. Incorporation
plateaued at 15%, and half maximal incorporation was only reached after nearly 90 min. In contrast,
20-C-methyluridine triphosphate reached half of its eventual maximal incorporation of 35% after
~45 min, showing improved incorporation over the active form of sofosbuvir. These trends are supported
by previous work focusing on biochemical characterization of 20-C-methyluridine triphosphate and
20-fluoro-20-C-methyluridine triphosphate incorporation [19
,20]. Importantly, here we verify the
preference of 20-C-methyluridine over sofosbuvir in both biochemical and cell-based assays, suggesting
a robust inhibitor testing pipeline that supports the hypothesis that cellular efficacy occurs as a result
of RdRP inhibition by nucleoside analogs.
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, 20-F-20-C-methyluridine triphosphate (20-F-20-C-MeUTP), or 2-C-methyluridine triphosphate
(20-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, kobs (% incorporation min−1
)
of 0.151
±
0.004, 0.0085
±
0.001, and 0.016
±
0.001, for UTP (dotted black line), 20-F-20-C-MeUTP (dashed
red line), and 20-C-MeUTP (solid blue line), respectively. SE is reported as deviation from the fit, n = 2.
3.6. Molecular Superpositioning of the ZIKV NS5 Active Site: Consequences for 20-C-Derivatitized
Nucleoside Analogues
A previously prepared model (gc-o3 [36]) of ZIKV RdRP in complex with RNA and ATP generated
from PDB 5TFR [37] and PDB 4WTD [34], an apo structure of ZIKV RdRP (PDB 5WZ3 [38]), and an
apo structure of HCV RdRP (PDB 3MWV [39]) were aligned with a crystal structure of HCV RdRP
complexed with RNA and sofosbuvir diphosphate (PDB 4WTG [34]), shown in Figure 5. ZIKV RdRP
is shown in magenta (apo form) or in red (in complex with ligands), and HCV RdRP is shown in dark
blue (apo form) or in cyan (in complex). The crystal structure of inhibited HCV RdRP (cyan) shows
that residue D225 moved upward to accommodate sofosbuvir diphosphate, while in the ZIKV model
(red) [36], the corresponding residue D1141 in ZIKV RdRP pointed down in the opposite direction
to accommodate the nucleotide (Figure 5). While the position of the aspartic acid in both the apo
forms of the viral RdRPs was similar, it is interesting that the nucleoside ligands were accommodated
in different ways, with ZIKV RdRP displaying a larger change in positioning in this residue. While
modeling was used to generate the holo-structure of ZIKV RdRP, this suggests some evidence that the
ZIKV binding pocket is poised to accommodate a variety of nucleoside analogues.

Viruses 2019, 11, 365
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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.
4. Discussion
In this work, we demonstrated that ProTides other than sofosbuvir have promise as potential
antiviral drugs to treat ZIKV infections. Interestingly, an apparent bias towards 20-C-modified
uridylate ProTides (with the exception of the ethenyl derivative) was observed, suggesting a possible
nucleobase bias to compound activity. Recent biochemical data examining levels of off-target
incorporation of nucleoside analogue triphosphates by the human mitochondrial RNA polymerase

Viruses 2019, 11, 365
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may help explain the phenotypic relationship we observed; both 20-C-methyluridine triphosphate and
20-C-methyl-20-C-fluorouridine triphosphate (the active metabolite of sofosbuvir) show much lower
levels of incorporation by this host enzyme than 20-C-modified analogues with other nucleobases [51].
We propose that this may account for the better safety profile observed for sofosbuvir compared to
other 20-C-modified compounds that were tested in clinical trials. This may also suggest that there
is greater selectivity for 20-C-modified uridylate incorporation by flavivirus polymerases over host
polymerases, though the exact mechanism by which this occurs should be examined in future studies.
We also observed that changing the ProTide functionality of 20-C-methyluridine from an aryloxyl
phosphoramidate masking group to a 2-(methylthio)ethyl phosphoramidate resulted in a complete
loss of activity, suggesting that the local metabolic environment of the host cell is critical for processing
of the ProTide functionality. Further studies examining the relationship between different ProTide
functionalities and the cell type dependence of their antiviral activity as well as testing the impact of
these effects using in vivo animal model testing are planned in our research groups.
We have demonstrated here for the first time that ProTide technology can be broadly applied to
anti-ZIKV drug development with nucleobase and ProTide group selectivity observed. These data will
guide future work to design highly selective, safe, and bioavailable compounds for the treatment of
ZIKV infections.
Author Contributions: J.A.B., M.C., S.B., G.A.W., L.A.L., A.E.C., Z.Z., J.N.R., C.D.S., B.W.P., and J.L.S.-N. conceived
and designed the experiments; J.A.B., M.C., S.B., G.A.W., L.A.L., D.H., A.E.C., and Z.Z. performed experiments;
J.A.B., M.C., S.B., G.A.W., C.D.S., B.W.P., and J.L.S.N. analyzed the data; J.A.B., M.C., S.B., G.A.W., C.D.S., B.W.P.,
and J.L.S.-N. wrote the original draft of manuscript; J.A.B., M.C., S.B., G.A.W., L.A.L., A.E.C., Z.Z., D.H., J.N.R.,
C.D.S., B.W.P., and J.L.S.-N. reviewed and edited the manuscript.
Funding: This research was supported by Clinical and Translational (CTRI) pilot grant UL1TR001442 (to J.L.S.-N.),
and a CSUPERB New Investigator grant (to C.D.S). B.W.P., M.C., G.A.W., L.A.L., and C.D.S. thank San Diego State
University and the California Metabolic Research Foundation for financial support.
Acknowledgments: The authors acknowledge the access and use of the UCSD Screening Core to perform part of
the experiments presented in this manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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