Nucleoside analogs as a rich source of antiviral agents active against arthropod-borne flaviviruses

Abstract

Nucleoside analogs represent the largest class of small molecule-based antivirals, which currently form the backbone of chemotherapy of chronic infections caused by HIV, hepatitis B or C viruses, and herpes viruses. High antiviral potency and favorable pharmacokinetics parameters make some nucleoside analogs suitable also for the treatment of acute infections caused by other medically important RNA and DNA viruses. This review summarizes available information on antiviral research of nucleoside analogs against arthropod-borne members of the genus Flavivirus within the family Flaviviridae, being primarily focused on description of nucleoside inhibitors of flaviviral RNA-dependent RNA polymerase, methyltransferase, and helicase/NTPase. Inhibitors of intracellular nucleoside synthesis and newly discovered nucleoside derivatives with high antiflavivirus potency, whose modes of action are currently not completely understood, have drawn attention. Moreover, this review highlights important challenges and complications in nucleoside analog development and suggests possible strategies to overcome these limitations.

Keywords

Nucleoside analog antiviral agent arthropod-borne flavivirus antiviral therapy inhibitor

Introduction

The genus Flavivirus belongs to the Flaviviridae family and includes more than 70 single-stranded plus-sense RNA viral species. Flaviviruses of human medical importance are tick- or mosquito-transmitted viruses with typical representatives being tick-borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus (OHFV), Kyasanur Forest disease virus (KFDV), Alkhurma hemorrhagic fever virus (AHFV), Powassan virus (POWV), West Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), or Zika virus (ZIKV).^1,2 The Flaviviridae family also includes some less known or neglected viruses, such as louping ill virus (LIV), Usutu virus, Langat virus, or Wesselsbron virus.^3–6 The flaviviral genome is a single-stranded, plus-sense RNA of about 11 kb in length that encodes a single polyprotein, which is co- and posttranslationally processed into three structural (capsid, premembrane or membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).⁷ Both NS3 and NS5 proteins possess enzymatic activities reported to be important targets for antiviral development. Whereas NS3 acts as a serine protease, a 5′-RNA triphosphatase, a nucleoside triphosphatase (NTPase), and a helicase,^8,9 NS5 consists of a complex containing the RNA-dependent RNA polymerase (RdRp) and the methyltransferase (MTase) activities.^10,11

Flaviviral infections are accompanied by a wide spectrum of distinct clinical manifestations, ranging from relatively mild fevers and arthralgia to severe viscerotropic symptoms (YFV and DENV), hemorrhagic fevers (KFDV and OHFV), encephalitis/myelitis (JEV, WNV, and TBEV), and neuropathic or teratogenic manifestations (ZIKV). More than 200 million clinical cases of flaviviral infections, including numerous deaths, are reported annually worldwide.¹² Currently no specific antiviral therapies are available to treat patients with flaviviral infections, thus the search for safe and effective small-molecule inhibitors that would be active against these viruses represents a high research priority.¹³

Nucleoside analog inhibitors have figured prominently in the search for effective antiviral agents.¹⁴ Nucleoside analogs are synthetic, chemically modified nucleosides that mimic their physiological counterparts (endogenous nucleosides) and block cellular division or viral replication by impairment DNA/RNA synthesis or by inhibition of cellular or viral enzymes involved in nucleoside/tide metabolism (Figure 1).¹⁵ The first antiviral analogs were developed in the late 1960s and currently there are over 25 approved therapeutic nucleosides used for the therapy of viral infections of high medical importance, such as HIV/AIDS (tenofovir),^16,17 hepatitis B (lamivudine/entecavir),^18,19 hepatitis C (sofosbuvir),²⁰ or herpes infections (acyclovir).²¹ So far, numerous nucleoside analogs have been described to inhibit arthropod-transmitted flaviviruses. Since these viruses are closely related to the hepatitis C virus (HCV), for which many potent inhibitors are being currently developed, anti-HCV nucleoside analogs represent promising tools to be repurposed against other viruses within the Flaviviridae family.¹²

Figure 1.

Intracellular uptake and metabolism of nucleoside analogs and nucleoside analog prodrugs. Nucleoside analogs enter cells through specific plasma membrane nucleoside transporters. Inside the cell, the compounds are phosphorylated by cellular nucleoside kinases resulting in formation of nucleoside mono-, di-, and triphosphates. The first kinase phosphorylation is the rate-limiting step of the triphosphate conversion, which can be overcome by the monophosphate prodrug approach based on the introduction of a phosphorylated group into the 5′ nucleoside position. The phosphorylated group includes protecting moieties to increase hydrophobicity and facilitate the cellular uptake of the prodrug. Monophosphate prodrugs enter cells independently of membrane transporters and the protecting groups are removed by intracellular esterases or phosphoramidases after cell penetration. The triphosphates of nucleoside species represent the active forms of nucleoside analogs that act by inhibiting cellular or viral enzymes, such as DNA/RNA polymerases. During DNA/RNA replication, nucleoside analogs are incorporated into nascent DNA or RNA chains resulting in termination of nucleic acid synthesis or in accumulation of mutations in viral genomes to suppress viral replication due to error catastrophe. At normal physiological conditions, intracellular nucleoside concentrations are maintained at low levels due to nucleoside/nucleotide catabolic pathways, such as deamination (oxidation) of heterocyclic base, hydrolysis or phosphorolysis of heterocyclic base, and hydrolysis of phosphomonoester bonds. These catabolic reactions also concern most nucleoside analogs containing the natural N-glycosidic bond and/or the degradable functional groups of the heterocyclic base. Figure created using Servier Medical Art available on www.servier.com.

The aim of this review is to provide an overview of known antiviral agents targeting selected arthropod-borne flaviviruses and to discuss their characteristic properties, modes of action, and advantages or limitations of their therapeutic use. Moreover, the important challenges and complications in antiflavivirus nucleoside analog development are highlighted and possible strategies to overcome their shortcomings are suggested.

Major classes of antiflavivirus nucleosides

Nucleoside analogs active against arthropod-borne flaviviruses are usually classified according to their targets in the viral life cycle. Such antiviral molecules act as inhibitors of flaviviral RdRp,^22–26 MTase,^27,28 and helicase/NTPase.^29,30 Other nucleoside scaffolds suppress host cell enzymes involved particularly in nucleoside biosynthetic pathways^31–34 or in some cases, exhibit multiple modes of action.^32,35 In vitro antiviral effects and cytotoxicity profiles of the most important antiflavivirus nucleoside analogs are summarized in Table 1. An overview of in vivo antiflaviviral activities of selected nucleosides as evaluated in different animal models is presented in Table 2.

Table 1.

In vitro antiviral activities and cytotoxicity profiles of selected nucleoside inhibitors of flaviviral replication.

Structure	Virus	Strain	Cell line	Assay	EC₅₀ (µM)	CC₅₀ (µM)	References
Inhibitors of flaviviral RdRp
GS-441524	AHFV	200300001	Vero	CPE	49.9	ND	Lo et al.⁴⁵
	KFDV	P9605	Vero	CPE	46.3	ND	Lo et al.⁴⁵
	TBEV	Hypr	Vero	CPE	51.2	ND	Lo et al.⁴⁵
	OHFV	Bogoluvovska	Vero	CPE	50.6	ND	Lo et al.⁴⁵
	YFV	ND	ND	Cell based	11	>30	Cho et al.⁴³
	DENV-2	ND	ND	Cell based	9.46	>30	Cho et al.⁴³
	WNV	ND	ND	Cell based	>30	>30	Cho et al.⁴³
GS-5734	AHFV	200300001	Vero	CPE	4.2	ND	Lo et al.⁴⁵
	KFDV	P9605	Vero	CPE	1.8	ND	Lo et al.⁴⁵
	TBEV	Hypr	Vero	CPE	2.1	ND	Lo et al.⁴⁵
	OHFV	Bogoluvovska	Vero	CPE	1.2	ND	Lo et al.⁴⁵
2′-C-methyladenosine	TBEV	Hypr	PS	VTR	1.4	>50	Eyer et al.⁵⁴
	WNV	New York isol.	Vero	CPE	5.1	25	Migliaccio et al.⁴⁹
	DENV-2	New Guinea C	Vero	CPE	4	18	Migliaccio et al.⁴⁹
	YFV	17-D	Vero	CPE	3.2	13	Migliaccio et al.⁴⁹
	ZIKV	MR766	Vero	VTR	5.26	>100	Eyer et al.²³
7-Deaza-2′-C-methyl-adenosine	TBEV	Hypr	PS	VTR	1.1	>50	Eyer et al.⁵⁴
	WNV	New York isol.	Vero	CPE	4.5	250	Olsen et al.⁵⁰
	DENV-2	New Guinea C	Vero	CPE	15	>320	Olsen et al.⁵⁰
	YFV	17-D	Vero	CPE	15	>320	Olsen et al.⁵⁰
	ZIKV	MR766	Vero	CPE	20	>357	Zmurko et al.⁶²
	ZIKV	MR766	Vero	VYR	9.6	>357	Zmurko et al.⁶²
	ZIKV	MR766	Vero	PA	1.3	>357	Zmurko et al.⁶²
	ZIKV	MR766	Vero	IFA	5.7	>357	Zmurko et al.⁶²
2′-C-methylguanosine	TBEV	Hypr	PS	VTR	1.4	>50	Eyer et al.⁵⁴
	WNV	New York isol.	Vero	CPE	30	>100	Migliaccio et al.⁴⁹
	DENV-2	New Guinea C	Vero	CPE	13.6	>60	Migliaccio et al.⁴⁹
	YFV	17-D	Vero	CPE	17	>50	Migliaccio et al.⁴⁹
	ZIKV	MR766	Vero	VTR	22.25	>100	Eyer et al.²³
INX-08189	DENV-2	ND	Huh-7	Replicon	0.0142	>1	Yeo et al.⁵⁸
2′-C-methylcytidine	TBEV	Hypr	PS	VTR	1.8	>50	Eyer et al.⁵⁴
	AHFV	200300001	A549	qRT-PCR	2.5		Flint et al.⁵⁵
	AHFV	200300001	A549	CPE	15.3
	KDFV	P9605	A549	CPE	7.2
	OHFV	Bogoluvovska	A549	CPE	3.2
	POWV	Byers	A549	CPE	5.5
	DENV	ND	Huh-7	Replicon	11.2	–	Lee et al.⁵⁶
	YFV	17-D	Vero	Visual inspection	2.5a	22a	Julander et al.⁶³
	YFV	17-D	Vero	Neutral red uptake	2.1a	22a	Julander et al.⁶³
	YFV	17-D	Vero	VYR	0.7a	22a	Julander et al.⁶³
	ZIKV	MR766	Vero	VTR	10.51	>100	Eyer et al.²³
2′-C-methyluridine	TBEV	Hypr	PS	VTR	11.1	>50	Eyer et al.⁵⁴
2′-C-methyluridine	ZIKV	MR776	Vero	VTR	45.45	>100	Eyer et al.²³
Sofosbuvir	ZIKV	ND	BHK-21, SH-sy5y, Huh-7	CPE	0.12–1.9	>300	Sacramento et al.²⁵
	ZIKV	ND	-	RdRp inhibition	0.38	ND	Sacramento et al.²⁵
	ZIKV ZIKV ZIKV	PRVABC59 Paraiba Dakar 41519	Huh-7, Jan Huh-7, Jan Huh-7, Jan	PA qRT-PCR	1–5 1–5 1–5	>200 >200 >200	Bullard-Feibelman et al.⁶⁹
2′-C-ethynyladenosine	DENV-2	ND	A549	CFI	1.41	40	Chen et al.²²
NITD008	DENV-2 DENV-2 DENV-2 DENV-2 DENV-2	New Guinea C MY10245 MY10340 MY22366 MY22713	A549 PBMC PBMC PBMC PBMC	CFI CFI CFI CFI CFI	0.46–2.61 0.58 0.58 0.58 0.58	>100 >100 >100 >100 >100	Chen et al.⁷² Chen et al.⁷² Chen et al.⁷² Chen et al.⁷² Chen et al.⁷²
	ZIKV	GZ01/2016	Vero	VTR	0.241	ND	Deng et al.⁷⁶
	ZIKV	GZ01/2016	Vero	qRT-PCR	0.137	ND	Deng et al.⁷⁶
	ZIKV	FSS13025/2010	Vero	VTR	0.950	ND	Deng et al.⁷⁶
	ZIKV	FSS13025/2010	Vero	qRT-PCR	0.283	ND	Deng et al.⁷⁶
	TBEV	Hypr	A549	CPE, CFI, VTR	0.9–2.99	>100	Lo et al.⁷⁷
	AHFV	200300001	A549	CPE, CFI, VTR	1.51–9.29	>100	Lo et al.⁷⁷
	KDFV	P9605	A549	CPE, CFI, VTR	1.42–4.01	>100	Lo et al.⁷⁷
	OHFV	Bogoluvovska	A549	CPE, CFI, VTR	0.61–3.04	>100	Lo et al.⁷⁷
NITD449	DENV (1–4)	New Guinea C MY10245 MY10340 MY22366 MY22713	A549	CFI CFI CFI CFI CFI	1.62–6.99	ND ND ND ND ND	Chen et al.⁷² Chen et al.⁷² Chen et al.⁷² Chen et al.⁷² Chen et al.⁷²
NITD449	DENV (1–4)	New Guinea C MY10245 MY10340 MY22366 MY22713	PBMC PBMC PBMC PBMC	CFI CFI CFI CFI CFI	5 5 5 5	ND ND ND ND ND
NITD203	DENV (1–4)	New Guinea C MY10245 MY10340 MY22366 MY22713	A549	CFI CFI CFI CFI CFI	0.54–0.71	ND ND ND ND ND	Chen et al.⁷² Chen et al.⁷² Chen et al.⁷² Chen et al.⁷² Chen et al.⁷²
NITD203	DENV (1–4)	New Guinea C MY10245 MY10340 MY22366 MY22713	PBMC PBMC PBMC PBMC	CFI CFI CFI CFI CFI	<0.1 <0.1 <0.1 <0.1	ND ND ND ND ND
4′-C-azidocytidine	TBEV	Hypr	PS	VTR	2.7	>50	Eyer et al.⁵⁴
Balapiravir	DENV (1–4)	Various	DC	qRT-PCR qRT-PCR qRT-PCR	5.2–6	ND ND ND	Nguyen et al.⁸³ Nguyen et al.⁸³ Nguyen et al.⁸³
			Huh-7		1.9–11
			PHM		1.3–3.2
RO-9187	TBEV	Hypr	PS	VTR	0.3	>50	Eyer et al.⁵⁴
BCX4430	TBEV	Hypr	PS PS PS PS	VTR VTR VTR VTR	1.48	>100 >100 >100 >100	Eyer et al.⁹⁰ Eyer et al.⁹⁰ Eyer et al.⁹⁰ Eyer et al.⁹⁰
	LIV	LI/31			12.33
	KFDV	W-377			11.37
	WNV	Eg101			2.33
	YFV	17-D	ND	ND	14.1	>100 >100	Warren et al.⁸⁹ Warren et al.⁸⁹ Warren et al.⁸⁹
	JEV	SA14			43.6	>100 >100
	DENV-2	New Guinea C			32.8	>296
	ZIKV ZIKV	Various Various	Vero, Huh-7, RD Vero, Huh-7, RD	CPE	3.8–11.7a	>100a >100a	Julander et al.⁹¹ Julander et al.⁹¹
	ZIKV ZIKV	Various Various	Vero, Huh-7, RD Vero, Huh-7, RD	VYR	5.4–18.2b	>100a >100a	Julander et al.⁹¹ Julander et al.⁹¹
T-1106	YFV YFV YFV	17-D 17-D 17-D	Vero Vero Vero	Neutral red uptake	1800	>4000 >4000 >4000	Julander et al.⁹⁴ Julander et al.⁹⁴ Julander et al.⁹⁴
				CPE	2630
				Luciferase based	1080
6-Methyl-7-deazaadenosine	DENV-2 DENV-2	ND ND	Vero Vero	PA	0.062	ND ND	Wu et al.⁹⁶ Wu et al.⁹⁶
6-Methyl-7-deazaadenosine	DENV-2 DENV-2	ND ND	Vero Vero	qRT-PCR	0.039	ND ND	Wu et al.⁹⁶ Wu et al.⁹⁶
N6-(9-antranylmethyl) adenosine	TBEV	Absettarov	PEK	PA	15	>50c	Orlov et al.³⁵
N6-(1-pyrenylmethyl) adenosine	TBEV	Absettarov	PEK	PA	6	>50c
N6-benzyl-5′-O-triisopropylsilyl adenosine	TBEV	Absettarov	PEK	PA	5	>50c
N6-benzyl-5′-O-trityl adenosine	TBEV	Absettarov	PEK	PA	2	>50c
N6-benzyl-5′-O-tert-butyldimethylsilyl-adenosine	TBEV	Absettarov	PEK	PA	20	>50c
2′,5′Di-O-trityluridine	DENV-2	New Guinea C	Vero Vero	CPE CPE	30a	>100a >100a	Saudi et al.¹⁰⁴ Saudi et al.¹⁰⁴
2′,5′Di-O-trityluridine	YFV	17-D	Vero Vero	CPE CPE	1.2a	>100a >100a
3′,5′Di-O-trityluridine	DENV-2	New Guinea C	Vero Vero	CPE CPE	1.75a	>10a
3′,5′Di-O-trityluridine	YFV	17-D	Vero Vero	CPE CPE	1a	>85a
Inhibitors of flaviviral methyltransferase
GRL-002	WNV	NY99	–	N-7 methylation inhibition	33.9d	–	Chen et al.¹¹¹
	WMV	NY99	–	2′-O-methylation inhibition	5.5d	–	Chen et al.¹¹¹
	WMV	NY99	A549	VTR	52	48	Chen et al.¹¹¹
GRL-003	WMV	NY99	–	N-7 methylation inhibitin	17.3d	–	Chen et al.¹¹¹
	WMV	NY99	–	2′-O-methylation inhibition	19.8d	–	Chen et al.¹¹¹
	WMV	NY99	A549	VTR	27	236	Chen et al.¹¹¹
Flex 1, R=H, Ac, Flex 1-TP, R=triphosphate	DENV-3	ND	–	2′-O-methylation inhibition	22d	–	K. Seley-Radtke, manuscript in preparation
Flex 1, R=H, Ac, Flex 1-TP, R=triphosphate	ZIKV	French Polynesisa (2013/PF KJ776791.2)	–	2′-O-methylation inhibition	22d	–	K. Seley-Radtke, manuscript in preparation
Flex 2	DENV-3	ND	–	2′-O-methylation inhibition	3.2d	–	unpublished results, Smee laboratory, Utah
Ribavirin and other nucleoside synthesis inhibitors
Ribavirin	DENV (1–4)	Various	Vero	CPE	19.8–41.9a	>100a^,e	Crance et al.³¹
	JEV	Nakayama	Vero	CPE	134.1a	>100a^,e	Crance et al.³¹
	WNV	E101	Vero	CPE	71.2a	>100a^,e	Crance et al.³¹
	USUV	DakArD 19848	Vero	CPE	62.6a	>100a^,e	Crance et al.³¹
	LGTV	ND	Vero	CPE	33.9a	>100a^,e	Crance et al.³¹
	YFV	17D and FNV	Vero	CPE	42.4; 48.2a	>100a^,e	Crance et al.³¹
	WESSV	ND	Vero	CPE	91.7a	>100a^,e	Crance et al.³¹
	ZIKV	Various	Vero, Huh-7, RD	CPE	3.8–142.9a	>100a^,e	Crance et al.³¹ and Julander et al.⁹¹
	ZIKV	Various	Vero, Huh-7, RD	VYR	9.52–281b	>100a^,e	Crance et al.³¹ and Julander et al.⁹¹
ETAR	DENV-2	ND	Vero	ND	9.5	>1000	McDowell et al.¹³⁵
IM18	DENV-2	ND	Vero	ND	106.1	ND	McDowell et al.¹³⁵
6-Azauridine	DENV (1–4)	Various	Vero	CPE	0.1–0.5a	>100a^,e	Crance et al.³¹
	JEV	Nakayama	Vero	CPE	0.5a	>100a^,e	Crance et al.³¹
	WNV	E101	Vero	CPE	0.2a	>100a^,e	Crance et al.³¹
	USUV	DakArD 19848	Vero	CPE	0.1a	>100a^,e	Crance et al.³¹
	LGTV	ND	Vero	CPE	0.2a	>100a^,e	Crance et al.³¹
	YFV	17D and FNV	Vero	CPE	0.2; 0.2a	>100a^,e	Crance et al.³¹
	WESSV	ND	Vero	CPE	1.3a	>100a^,e	Crance et al.³¹
	ZIKV	DakArB 11514	Vero	CPE	1.5a	>100a^,e	Crance et al.³¹
Rigid amphipathic nucleosides
5-(Perylen-3-yl)ethynyl-arabino-uridine	TBEV	Absettarov	PEK	PA	0.018f	>50c	Orlov et al.¹⁰¹
5-(Perylen-3-yl)ethynyl-2′-deoxy-uridine	TBEV	Absettarov	PEK	PA	0.024f	>50c	Orlov et al.¹⁰¹
5-(Pyren-1-yl)ethynyl-2′-deoxy-uridine	TBEV	Absettarov	PEK	PA	0.98f	>50c	Orlov et al.¹⁰¹

AHFV: Alkhurma hemorrhagic fever virus; CFI: cellular flavivirus immunodetection; CPE: cytopathic effect reduction assay; DC: dendritic cells; DENV: dengue virus; JEV: Japanese encephalitis virus; KFDV: Kyasanur Forest disease virus; LGTV: Langat virus; LIV: louping ill virus; ND: not determined; OHFV: Omsk hemorrhagic fever virus; PA: plaque reduction assay; PBMC: peripheral blood mononuclear cells; PHM: primary human macrophages; POWV: Powassan virus; TBEV: tick-borne encephalitis virus; USUV: Usutu virus; VTR: viral titer reduction assay; VYR: viral yield reduction assay; WESSV: Wesselsbron virus; WNV: West Nile virus; YFV: yellow fever virus; ZIKV: Zika virus.

^aEC₅₀ and CC₅₀ values are expressed as µg/ml.

^bEC₉₀ values, expressed as µg/ml.

^cCC₅₀ (24 h): the cell culture was treated for 24 h with the appropriate compound to obtain the cytotoxicity data.

^dIC₅₀ values obtained from enzyme-based inhibition assays.

^eCC₅₀ values for confluent compound-treated cells.

^fEC₅₀ (sim): the cell culture was TBEV infected and simultaneously treated with the appropriate compound.

Table 2.

Examples of in vivo antiflaviviral activities of selected nucleoside analogs.

Compound	Animal model	Admin. route	Treatment start	Treatment length	Dose	Virus	Infection route	Survival rate (%)	References
7-Deza-2′-C-methyladenosine	BALB/c mouse	i.p.	0 dpi	17 days	25 mg/kg/2× day	TBEV	s.c.	60	Eyer et al.⁶¹
	BALB/c mouse	i.p.	0 dpi	17 days	5–15 mg/kg/2× day	TBEV	s.c.	35–50	Eyer et al.⁶¹
	AG129 mouse	p.o.	−1 h	10 days	50 mg/kg/day	ZIKV	i.p.	25	Zmurko et al.⁶²
2′-C-methylcytidine	ICR suckling mouse	i.c.	1 dpi	5 days	15 or 30 mg/kg/day	DENV	i.c.	60	Lee et al.⁵⁶
	Syrian golden hamster	i.p.	−4 h	4–7 days	120 mg/kg/day	YFV	i.p.	90	Julander et al.⁶³
	Syrian golden hamster	i.p.	3 dpi	4–7 days	120 mg/kg/day	YFV	i.p.	80	Julander et al.⁶³
Sofosbuvir	C57BL/6J mouse	p.o.	1 dpi	7 days	33 mg/kg/day	ZIKV	s.c.	50	Bullard-Feibelman et al.⁶⁹
	Suckling Swiss mouse	i.p.	−24 h	7 days	20 mg/kg/day	ZIKV	i.p.	40	Ferreira et al.⁷¹
	Suckling Swiss mouse	i.p.	2 dpi	7 days	20 mg/kg/day	ZIKV	i.p.	25	Ferreira et al.⁷¹
NITD008	AG129 mouse	p.o.	0 h	ND	25–50 mg/kg/day	DENV	i.v.	100	Yin et al.²⁶
	AG129 mouse	p.o.	1 dpi	ND	25 mg/kg/day	DENV	i.v.	70	Yin et al.²⁶
	A129 mouse	p.o.	ND	5 days	50 mg/kg/day	ZIKV	i.p.	50	Deng et al.⁷⁶
T-1106	Syrian golden hamster	i.p.	−4 h	8 days	100 mg/kg/2× day	YFV	i.p.	100	Julander et al.⁹³
			3 dpi					100
			5 dpi					20
BCX4430	Syrian golden hamster	i.p.	−4 h	7 days	12.5–125 mg/kg/day	YFV	i.p.	100	Julander et al.⁹²
	Syrian golden hamster	i.p.	4 dpi	7 days	200 mg/kg/day	YFV	i.p.	80	Julander et al.⁹²
	AG129 mouse	i.m.	−4 h	7–8 days	300 mg/kg/day	ZIKV	s.c.	90	Julander et al.⁹¹
			1 dpi					85
			3 dpi					10

DENV: dengue virus; i.c.: intracerebral administration; i.m.: intramuscular administration; i.p.: intraperitoneal administration; i.v.: intravenous administration; ND: not determined; p.o.: per os (oral administration); s.c.: subcutaneous administration; TBEV: tick-borne encephalitis virus; YFV: yellow fever virus; ZIKV: Zika virus.

Inhibitors of flaviviral NS5 RdRp

The flavivirus NS5 protein is approximately 900 amino acids in length and consists of the NH₂-terminal MTase domain required for the 5′-RNA capping process and the COOH-terminal RdRp domain responsible for the replication of the viral RNA genome.^10,36 Flaviviral RdRp is a right hand-shaped structure with fingers, palm, and thumb domains; the palm domain is the catalytic domain carrying the polymerase active site that coordinates two Mg²⁺ ions essential for catalyzing the polymerization reaction.³⁷ Nucleoside inhibitors of flaviviral RdRp are the most attractive targets for antiviral drug design; as human replication/transcription enzymes lack RdRp activity, such compounds are expected to show fewer deleterious side effects and favorable safety profiles.^12,15,38,39

The mode of action for nucleoside RdRp inhibitors is based on the premature termination of viral nucleic acid synthesis.⁴⁰ Following the intracellular phosphorylation, the 5′-triphosphate metabolites are competitively incorporated into the flaviviral RNA nascent chains (Figure 1). This prevents further extension of the incorporated analog by addition of the next nucleoside triphosphate resulting in formation of incomplete (nonfunctional) viral RNA chains.⁴¹ Nucleoside inhibitors of flaviviral RdRp act as “nonobligate chain terminators,” as their 3′-hydroxyl group is conformationally constrained or sterically/electronically hindered, thus decreasing the potency to form a phosphodiester linkage with the incoming nucleoside triphosphate.⁴⁰ The nonobligate terminators differ from “obligate chain terminators,” in which the 3′-hydroxy group is completely missing, as exemplified by numerous nucleoside reverse transcriptase inhibitors for the treatment of HIV infections.⁴² Chemical modifications of the heterobase moiety, different types of glycosidic bonds, and substitutions at different positions of the sugar ring, which are typical modifications for nucleoside inhibitors of flaviviral RdRps, are shown in Table 3.

Table 3.

Heterobase substitutions and ribose modifications of selected flaviviral RdRp nucleoside inhibitors.

Heterobase identity/modification	Type of the glycosidic bond	Ribose substitution	Ribose position	Example
4-Aza-7,9-dideazaadenie 4-Aza-7,9-dideazaadenie	C-glycosidic C-glycosidic	-CN (α) -CN (α)	C1′ C1′	GS-441524 GS-5734
Adenine 7-Deazaadenine Guanine Cytosine Uracil 6-O-methylguanine	N-glycosidic N-glycosidic N-glycosidic N-glycosidic N-glycosidic N-glycosidic	-CH₃ (β) -CH₃ (β) -CH₃ (β) -CH₃ (β) -CH₃ (β) -CH₃ (β)	C2′ C2′ C2′ C2′ C2′ C2′	2′-C-methyladenosine 7-Deaza-2′-C-methyladenosine 2′-C-methylguanosine 2′-C-methylcytidine 2′-C-methyluridine INX-08189
Uracil	N-glycosidic	-F (α), CH₃ (β)	C2′	Sofosbuvir
Adenine 7-Deazaadenine 7-Deaza-7-carbamoyladenine	N-glycosidic N-glycosidic N-glycosidic	-ethynyl (β) -ethynyl (β) -ethynyl (β)	C2′ C2′ C2′	2′-C-ethynyladenosine NITD008 NITD449
Cytosine Cytosine	N-glycosidic N-glycosidic	-H (α), OH (β) + N₃ (α) –N₃ (α)	C2′+ C4′ C4′	4′-Azido 4′-Azido-aracytidine
9-Deazaadenine	C-glycosidic	O exchanged for N	–	BCX4430
3-Oxopyrazine-2-carboxamide	N-glycosidic	No substitution	–	T-1106
6-Methyl-7-deazaadenine	N-glycosidic	No substitution	–	6-Methyl-7-deazadenosine
Uracil Uracil	N-glycosidic N-glycosidic	Trityl Trityl	C2′ and C5′ C3′ and C5′	2′,5′di-O-trityluridine 3′,5′di-O-trityluridine

1′-Cyano substituted nucleosides

GS-441524, a 1′-cyano substituted C-nucleoside derived from 4-aza-7,9-dideazaadenosine,⁴³ was developed by Gilead Sciences, Inc. as a treatment for filovirus infections also showing reasonable antiviral activity against paramyxo- and pneumoviruses.⁴⁴ Within the Flaviviridae family, this compound exerted micromolar inhibitory activity against YFV and DENV-2 (EC₅₀ values of 11 and 9.46 µM, respectively) in various cell-based screening systems.⁴³ Surprisingly, considerably less favorable in vitro activities (>30–51.2 µM) were reported for WNV and tick-borne flaviviruses, such as AHFV, KFDV, TBEV, and OHFV.^43,45

A phosphoramidate prodrug of GS-441524, referred to as GS-5734, recently entered Phase II clinical trials for treatment of Ebola infections, displayed 10- to 40-fold higher antiviral effect against members of the TBEV serocomplex when compared with its parental analog GS-441524. The increased antiviral potency could be related to an improved conversion of the prodrug to the biologically active form; however, the reported SI values (2.4–8.3) indicate a low therapeutic potential for this nucleoside to treat flaviviral infections.⁴⁵ Further substitutions of GS-441524 molecule at the C1′ position with methyl, vinyl, or methyl–ethynyl moieties yielded compounds with considerably reduced potency and a narrower spectrum of antiviral activity.⁴³

2′-C-methyl substituted nucleosides

2′-C-Methyl-nucleoside scaffolds represent the initial major class of therapeutic nucleosides developed by Merck Research Laboratories to demonstrate potent inhibition of HCV replication.^46–50 Antiviral activity of 2′-C-methylated nucleosides beyond the Flaviviridae family was reported for representatives of Picornaviridae and Caliciviridae families,^51–53 indicating the potential for broad-spectrum inhibitory activity for these compounds within the positive single-stranded RNA viruses.

The 2′-C-methyl substituent introduced at the nucleoside β-face appears to be an important structural element for highly selective micromolar inhibition of tick-borne flaviviruses, particularly for TBEV, AHFV, KDFV, OHFV, and POWV, when assayed in porcine stable kidney cells (PS), human neuroblastoma cells UKF-NB4, or adenocarcinomic human alveolar basal epithelial cells A549.^24,54,55 From mosquito-transmitted flaviviruses, 2′-C-methylated nucleosides inhibited WNV, DENV, and YFV in cell-based or cell-free reporter assay systems, showing low micromolar antiviral activities.^50,56,57 A phosphoramidate prodrug of 6-O-methyl-2′-C-methylguanosine, denoted as INX-08189, exerted nanomolar inhibitory activity against DENV-2, and the combination of INX-08189 with ribavirin resulted in significant synergistic anti-DENV activity in vitro.⁵⁸ 7-Deaza-2′-C-methyladenosine together with other 2′-C-methylated species was the first described nucleoside-based inhibitors of ZIKV, after its epidemiological outbreaks in Oceania and Latin America.^23,59 7-Deaza-2′-C-methyladenosine showed anti-ZIKV potency not only on immortalized cell lines, but also on induced pluripotent stem cell-derived neuronal cell types, such as cortical neurons, motor neurons, and astrocytes.⁶⁰ The triphosphate analogs of 2′-C-methylated nucleosides exhibited strong inhibitory activity in a polymerase-based in vitro assay using an active recombinant ZIKV RdRp.⁶¹

Strong antiflaviviral activity for several 2′-C-methyl modified nucleosides was also demonstrated using numerous in vivo efficacy models. For example, 7-deaza-2′-C-methyladenosine substantially improved disease outcome, increased survival, and reduced signs of neuroinfection and viral titers in the brains of BALB/c mice infected with a lethal dose of TBEV.⁶² This compound also reduced viremia in AG129 mice infected with the African strain of ZIKV.⁵⁹ Moreover, 2′-C-methylcytidine protected suckling mice challenged with DENV⁵⁶ and hamsters infected with a lethal dose of YFV, even when administered up to three days postinfection.⁶³

2′-Fluoro-2′-C-methyl substituted nucleosides

Similar to the 2′-C-methylated nucleosides, analogs possessing the 2′-α-fluoro-2′-β-C-methyl modification were initially identified as promising inhibitors of HCV polymerase activity.⁶⁴ Sofosbuvir, a phosphoramidate prodrug of 2′-fluoro-2′-C-methyluridine developed by Gilead Sciences, Inc., is one of the most potent and selective inhibitors in this series and was approved by the Food and Drug Administration (FDA) for the treatment of chronic HCV infection.⁶⁵ Sofosbuvir is nontoxic for most human cell lines and is a very poor substrate for human mitochondrial RdRp, resulting in an acceptable safety profile and negligible mitochondrial toxicity.^66,67

Sofosbuvir was demonstrated to inhibit the ZIKV RdRp in a recombinant polymerase assay⁶⁸ and to suppress ZIKV replication in different cell-based systems using U87 glioblastoma cells, baby hamster kidney fibroblasts (BHK-21), SH-sy5y neuroblasts, hepatocarcinoma Huh-7 cells, Jar human placental choriocarcinoma cells, neural stem cells, and brain organoids with nanomolar or low micromolar inhibitory activity.^25,69 Interestingly, no inhibition of ZIKV replication with sofosbuvir even at the 50 µM level was observed in Vero cells,²³ indicating that the sofosbuvir-mediated anti-ZIKV effect is strongly cell-type dependent.⁷⁰ Sofosbuvir protected inbred C57BL/6J mice, which were previously immunosuppressed with a single dose of anti-Ifnar1 blocking monoclonal antibody, against ZIKV-induced mortality.⁶⁹ Moreover, this compound reduced viral titer in blood plasma, spleen, kidney, and brain in suckling Swiss albino mice and prevented virus-induced neuromotor impairment in ZIKV-infected animals.⁷¹

Surprisingly however, sofosbuvir was inactive against TBEV, when screened on both PS and UKF-NB4 cells.⁵⁴ Another 2′-α-fluoro-2′-β-C-methyl modified nucleoside, PSI-6206, and its 3′,5′-diester prodrug mericitabine, also displayed no activity against TBEV replication. The lack of anti-TBEV activity for the 2′-α-fluoro-2′-β-C-methyl modified nucleosides could be ascribed to their inefficient intracellular conversion to their corresponding triphosphates and, moreover, to extensive deamination/demethylation in the tested cell cultures resulting in their conversion to uridine.^46,54

2′-C-ethynyl substituted nucleosides

2′-C-Ethynyl substituted nucleosides have been primarily identified as inhibitors of DENV.^22,72–75 2′-C-Ethynyladenosine represents the lead compound in this series, which inhibited DENV-2 replication with an EC₅₀ of 1.41 µM in cell-based assays and with a CC₅₀ value of 40 µM.²² The 7-deaza derivative of 2′-C-ethynyladenosine, denoted as NITD008, inhibited DENV of different serotypes at submicromolar or low micromolar concentrations when tested in BHK-21 cells, A549 cells, Huh-7 hepatocarcinoma cells, and human peripheral blood mononuclear cells (PBMCs), showing a significantly improved cytotoxicity profile (CC₅₀ of >100 µM) compared with that of 2′-C-ethynyladenosine.²⁶ NITD008 was also assayed against WNV, YFV, and ZIKV and exhibited excellent in vitro inhibitory parameters and a protective effect against WNV and ZIKV infections in mouse efficacy models.^26,76 NITD008 was also reported to effectively inhibit the in vitro replication of TBEV, AHFV, KDFV, OHFV, and POWV, with nanomolar or low micromolar antiviral levels observed in various cell-based screening systems.⁷⁷

Introduction of the C7 carbamoyl moiety to NITD008 molecule provided another 2′-ethynyl modified derivative, referred to as NITD449. Despite its low micromolar anti-DENV efficacy, this nucleoside disappointingly exhibited only low levels in plasma when dosed orally.⁷² To increase the oral bioavailability, the isobutyryl ester prodrug of NITD449, designed as NITD203, was synthesized. NITD203 successfully exhibited both nanomolar anti-DENV activity as well as improved pharmacokinetic parameters.⁷² Although NITD008 and NITD203 showed anti-DENV potency in rodent models, even when the treatment was delayed up to 48 h after infection, both nucleosides failed in preclinical toxicity studies in rats and dogs due to their insufficient safety profiles.^26,72 NITD008 failed to achieve no-observed-adverse-effect levels (NOAEL) when rats (10 mg/kg/day) and dogs (1 mg/kg/day) were dosed daily for two weeks.²⁶ Similarly, NOAEL was not achieved for NITD203 in the two-week toxicity test when rats were dosed at 30 and 75 mg/kg/day.⁷²

Further substitutions of the C7 position of NITD008 with fluoro or cyano moieties provided compounds with nano- or low micromolar anti-DENV activities and acceptable cytotoxicity profiles. In contrast, 2′-C-ethyl, -vinyl, or -methylethynyl substituted derivatives of 2′-C-ethynyladenosine were completely inactive when tested against DENV. Similarly, the exchange of the adenine base for cytosine or guanine also yielded inactive compounds.^22,72

2′-O-substituted nucleosides

2′-O-Methyl substituted adenosine, guanosine, cytidine, and uridine were evaluated for their potential anti-TBEV activity; however, no or negligible antiviral effects were observed when tested in both PS and UKF-NB4 cells.⁵⁴ Such abrogation of the nucleoside inhibitory activity could be related to the elimination of the 2′-α-hydroxy hydrogen bond donor/acceptor properties when the methyl moiety is introduced at the nucleoside O2′ position.^46,54 The ability of flaviviral RdRp to discriminate among nucleosides modified at the 2′-position on the nucleoside α-face is likely related to the need of the polymerase to avoid incorporation of 2′-α-deoxynucleoside monophosphates into the viral nascent RNA chain.⁴⁶

3′-C- and 3′-O-substituted nucleosides

Methylation of the C3′ or O3′ position to generate the corresponding 3′-C-methyl or 3′-O-methyl modified structures resulted in a complete loss of anti-TBEV activity, regardless of the purine/pyrimidine heterobase identity. These nucleosides exerted no cytotoxic effects and caused no morphological changes in PS or porcine embryo kidney (PEK) cell cultures.^35,54 Similarly, 3′-deoxynucleosides exhibited no detectable inhibitory effect on TBEV replication.⁵⁴ In contrast, several nucleosides with a trityl group at the C3′ position showed micromolar inhibitory activity against DENV and YFV (see below).⁷⁸ The observed inactivity of 3′-O-methylated and 3′-dehydroxylated nucleosides could be related to either a strict requirement of the TBEV RdRp active site for a 3′-hydroxyl group to form the appropriate hydrogen bonding interactions with the nucleoside triphosphate molecule, or, to inefficient cellular uptake and metabolism to convert the nucleoside molecule into the corresponding triphosphate form.^46,54

4′-Azido substituted nucleosides

Using high-throughput screening of large nucleoside libraries in combination with a rational drug design approach by investigators at Roche, several cytidine analogs with an azido group at the C4′ position were identified as potent inhibitors of HCV replication in subgenomic replicon assays.^79–81 It was later shown that these compounds were also highly active against henipaviruses and other paramyxoviruses.⁸² Two 4′-azido modified nucleoside analogs, 4′-azidocytidine (R-1479) and 4′-azido-aracytidine (RO-9187), showed nano- or micromolar in vitro antiviral activity against TBEV.⁵⁴ Moreover, both compounds were found to be active also against WNV (L. Eyer, manuscript in preparation).

The anti-TBEV activity of RO-9187 (the arabino-counterpart of 4′-azidocytidine) was unexpected, as this compound lacks the 2′-α-hydroxy moiety, a determinant which was generally considered to be crucial for specific hydrogen-bonding interactions with RdRps during the RNA replication process.^54,80 Some additional interactions of the polymerase active site with both the 2′-β-hydroxy substituent and the 4′-azido substituent are thought to compensate for the loss of the 2′-α-hydroxy interaction, resulting in the strong and selective anti-TBEV activity of RO-9187. Interestingly, the anti-TBEV efficacy of both 4′-azido modified nucleosides was cell-type dependent; the compounds were active only in PS cells, but not in UKF-NB4 cells.⁵⁴

An ester prodrug of 4′-azidocytidine, denoted as balapiravir, was completely inactive against TBEV in vitro, probably because of its poor intracellular uptake or insufficient kinase phosphorylation in the tested host cell lines.⁵⁴ In contrast, balapiravir was reported to show strong in vitro antiviral activity against DENV of various serotypes; it was the first direct antiviral agent tested clinically for DENV infection. Unfortunately, this compound failed to achieve antiviral efficacy in DENV patients, which was reflected in a negligible reduction of DENV viremia and persistence of clinical symptoms, even though the plasma concentration of the compound was higher than the 50% effective concentration.⁸³ One of the possible explanations is that DENV infection stimulates PBMCs to produce cytokines, which are responsible for the decreased efficiency of the conversion of balapiravir to its triphosphate form.⁸⁴

Interestingly, nucleoside analogs combining the 4′-azido moiety with the 2′-C-methyl group in one molecule (e.g. 2′-C-methyl-4′-azidocytidine) did not exhibit any antiviral activity when tested against HCV; however, the corresponding 5′-monophosphate prodrugs displayed considerably improved virus inhibitory effects (EC₅₀ values in the micromolar ranges) without apparent cytotoxicity.⁸⁵ Other interesting compounds, 4′-azido-2′-deoxy-2′-C-methylcytidine and its ester prodrugs, were found to show nano- or low micromolar antiviral efficacy in vitro.⁸⁶ Unfortunately, such nucleoside scaffolds have not been evaluated against arthropod-borne flaviviruses; the reported results originate from HCV replicon-based assays.^85,86

Imino-C-nucleoside analog BCX4430

BCX4430, developed by BioCryst Pharmaceuticals Inc., is an adenosine analog with the furanose oxygen on the ribose ring replaced by nitrogen and the heterobase nitrogen 9 replaced by carbon.⁸⁷ This interesting nucleoside is classified as imino-C-nucleoside.⁸⁸ BCX4430 was initially described as an inhibitor of filovirus infections, exerting antiviral activity against a broad spectrum of single-stranded RNA viruses, particularly against members of the Bunyaviridae, Arenaviridae, Picornaviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, and Flaviviridae families.⁸⁹ Currently, this compound has entered Phase I clinical trials for Ebola virus disease treatment focused on intramuscular administration of BCX4430 in healthy volunteers and to date has shown promising pharmacokinetics properties and good tolerability.⁸⁷

BCX4430 is active against numerous mosquito-transmitted flaviviruses, such as WNV (EC₅₀ of 2.33 µM)⁹⁰ and representatives of both the African and Asian lineages of ZIKV (3.8–11.7 µg/ml).⁹¹ For YFV, JEV, and DENV-2, micromolar EC₅₀ values were reported.⁸⁹ In vivo efficacy of BCX4430 was also demonstrated in a lethal hamster model of YFV infection and in a mouse model of ZIKV infection.^91,92 Low micromolar antiviral activity of BCX4430 was also described for some of the medically important tick-borne flaviviruses, such as TBEV, LIV, and KFDV.⁹⁰

Heterocyclic base-modified nucleosides

Heterocyclic base-modified nucleosides with demonstrated antiflaviviral activities include T-1106,^93–95 6-methyl-7-deazaadenosine,⁹⁶ and numerous N6-alkyl or aryl substituted nucleosides.^35,97 T-1106 is a ribosylated analog of the pyrazine derivative T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide, favipiravir),⁹⁸ which was described to inhibit the HCV RdRp in enzyme assays.⁹³ Nucleoside inhibitor T-1106 displayed a negligible in vitro activity against YFV in Vero cells, as well as in luciferase-based assays.⁹⁴ In contrast, this compound exerted favorable efficacy, bioavailability, and low toxicity in a hamster model of YFV infection, using a hamster-adapted Jimenez YFV strain. After intraperitoneal application of 100 mg/kg/day, T-1106 improved survival rates, serum parameters, weight gain, and mean day to death when administered up to four days after virus challenge.^93,94 In this model, the combination of T-1106 with ribavirin gave superior effects compared to monotherapy (with either T-1106 or ribavirin).⁹⁵

6-Methyl-7-deazaadenosine is a hydrophobic mimic of adenosine showing nanomolar antiviral activity against DENV-2 in a Vero cell-based screening system, as well as in luciferase-driven DENV-2 replicon assay, with no cytotoxic effects noted after 7 h of treatment.⁹⁶ Related compounds such as 6-methyl-1-deazaadenosine and 6-methyl-4-deazaadenosine were completely inactive when tested against DENV-2. Mechanistic studies of the 5′-triphosphate of 6-methyl-7-deazaadenosine revealed that this nucleotide is an efficient substrate for viral RdRp (screened against polio RdRp) and is incorporated into nascent viral RNA strains mimicking both ATP and GTP.⁹⁶

N6-Alkyl or aryl substituted nucleosides, originally identified as inhibitors of Lassa fever virus, Marburg virus, and enterovirus A71, showed interesting bioactivity profiles when tested against TBEV.^35,99,100 Whereas N6-methyladenosine and N6-benzyladenosine were completely inactive, nucleosides with bulky substituents, such as N6-(9-anthracenylmethyl)adenosine and N6-(1-pyrenylmethyl)adenosine, exerted a micromolar anti-TBEV effect. In contrast, N2- and N4-substituted analogs showed no antiviral activity. Moderate anti-YFV and anti-DENV activities were also observed in N6-subsitituted analogs of 5′,3′-O- and 5′,2′-O-tert-butyldiphenylsilyl-modified adenosine.⁹⁷ The mechanism of action of these nucleosides is poorly understood; however, they likely interact with the RdRp or MTase domain of the flaviviral NS5 protein, as demonstrated by docking studies.³⁵ Bulky aromatic substituents could also play a role in the interaction with the viral membrane resulting in cell entry inhibition.¹⁰¹

Tritylated nucleosides

In a large-scale cell-based screening campaign of alkylated, silylated, or acylated pyrimidine nucleosides, 2′,5′di-O-trityluridine and 3′,5′di-O-trytiluridine were identified as inhibitors of DENV-2 and YFV replication, showing high antiviral potency and favorable cytotoxicity profiles in Vero cells.^78,102,103 Substantial antiviral effect against YFV was observed also in 2′-deoxy-3′,5′-di-O-trityluridine and in several 5-halogenated bis-tritylated pyrimidine nucleosides; however, their anti-DENV activity was proven to be weaker.¹⁰⁴ Thymidine or 2′-deoxyuridine congeners of 2′,5′- and 3′,5′-tritylated nucleosides led to the loss of antiflavivirus activity or to increased compound cytotoxicity.

The mechanisms of action of tritylated nucleosides are not completely understood. Based on the observed inhibition of DENV replication in subgenomic replicon assays, it is assumed that these compounds may be acting as inhibitors of intracellular viral replication events rather than suppressing either early or late processes of viral infection, such as entry or assembly.¹⁰³ Although the presence of large, hydrophobic trityl moieties does not make these structures ideal candidates for further drug development, their chemical structures may provide valuable information for advanced mechanistic studies and for further development of related nucleoside scaffolds with improved biological parameters.¹⁰⁴

Nucleoside inhibitors of flaviviral MTase

The NH₂ domain of flaviviral NS5 protein is associated with the virus’s MTase activity, which is involved in methylation of the 5′-cap structure of genomic RNA.^8,105 The flaviviral cap structure is formed by the conserved dinucleotide sequence AG (m7GpppAm) and is crucial for mRNA stability and efficient translation.¹⁰⁶ Two topologically distinct methylation reactions are mediated by the flaviviral NS5 MTase: the N7 of guanine is methylated by the (guanine-N7)-MTase and the first nucleotide of RNA transcript is further methylated at the ribose 2′-hydroxyl by (nucleoside-2′-O)-MTase. The resulting product of the methyl donation for both methylation reactions by S-adenosyl-l-methionine is the nucleoside analog S-adenosyl-l-homocysteine (SAH).¹⁰⁷

SAH and the nucleoside antibiotic sinefungin are natural nonselective inhibitors of many eukaryotic and viral MTases, including those of DENV^108,109 or ZIKV.⁶¹ Chemical derivatization of SAH at the N6 position provided inhibitors with nano- or low micromolar activity against DENV MTase, which did not inhibit the corresponding human enzymes.¹¹⁰ Other rationally designed nucleosides with potent inhibitory activity against MTase contain a thymine base with a hydrophobic methyl tert-butyl substituent at the 5′ position of the sugar moiety.¹¹¹ Two of these nucleosides, GRL-002 and GRL-003, inhibited the N7 and 2′-O MTase activity of WNV in enzyme-based assays and the observed MTase inhibition was in agreement with the micromolar in vitro anti-WNV efficacy.¹¹¹ Another class of promising selective antiflavivirus compounds is represented by 5′-silylated 3′-1,2,3-triazole-substituted nucleoside scaffolds derived from 3′-azidothymidine. Similar structures were originally developed for HIV-1 inhibition.²⁷ Both the 5′-silyl protecting group and the 3′ bulky triazole substituent appeared to be crucial structural elements for low micromolar inhibition of flaviviral MTase in enzyme-based assays, as well as for inhibition of WNV and DENV replication in cell culture. These nucleosides inhibit the methylation reactions through competitive interactions with the substrate binding site and also with the GTP-binding pocket of the flaviviral NS5 MTase.²⁸

Recently, a novel series of flexible nucleoside analogs known as “fleximers” have exhibited activity against several hard to treat viruses, including filoviruses such as Ebola and Sudan,¹¹² coronaviruses including Severe Acute Respiratory Virus and Middle East Respiratory Virus,¹¹³ as well as most recently, flaviviruses including ZIKV and DENV. The fleximers feature a “split” purine nucleobase that has been shown to impart significant activity to the nucleoside scaffold as well as to allow it to overcome resistance related to point mutations.^114–116 The most recent series combined the fleximer approach with the acyclic nucleoside acyclovir, an FDA approved drug for herpes virus. While these analogs inhibited the aforementioned viruses, acyclovir shows no activity against any of those viruses, thereby underscoring the importance of the fleximer approach. Since those initial findings, these compounds have also demonstrated potent activity against DENV and ZIKV (K. Seley-Radtke, manuscript in preparation). Preliminary results indicate that these compounds target the DENV and ZIKV NS5 in at least its cap-MTase activity, with negligible effects on the cognate human N7-MTase. In that regard, initial screening revealed promising levels of MTase inhibition, particularly for the triphosphate of the compound (Flex 1-TP), with an IC₅₀ of 22 µM for both the DENV and ZIKV 2′-O-MTase (K. Seley-Radtke, manuscript in preparation). As a result, Flex 1-TP was further tested against DENV NS5, and while it was not incorporated, it successfully inhibited further incorporations of additional nucleotides, thereby halting replication, however not as a typical chain terminator. In addition, the acetate-protected dimethoxy analog (Flex 2) is a potent DENV inhibitor (3.2 µM, unpublished results, Smee laboratory, Utah); however, more work needs to be done to fully elucidate these novel compounds’ mechanism of action. It may well be that these nucleotide analogs target the NS5 protein at both the RdRp and MTase regions, which would make them highly effective viral inhibitors, with low probability of viral resistance developing.

Nucleoside inhibitors of flaviviral NTPase/helicase

Flaviviral NTPase and helicase activities are associated with the COOH-proximal domain of the NS3 protein.¹¹⁷ Flaviviral helicases are capable of unwinding duplex RNA structures during viral replication by disrupting the hydrogen bonds keeping the two strands together.^118,119 The helicase activity is strictly associated with NTP hydrolysis (NTPase activity); the released chemical energy is used for the translocation of the enzyme along the double-helix structures, capturing the exposed single strand regions or for a direct disruption of the hydrogen bonds between the two RNA strains.¹²⁰

Specific nucleoside inhibitors of flaviviral NTPase/helicase can interact with dsRNA or DNA resulting in the weakened stability of double-helix structures or by steric hindrance of translocation of the enzyme along the polynucleotide chain. Such a mechanism could modulate the efficacy of the unwinding reaction or NTPase activity of the enzyme and therefore, affect the viral replication process.^117–121 Weak inhibitory effects on flaviviral NTPase/helicases were described for ribavirin triphosphate,^124,125 5′-O-fluorosulfonylbenzoyl esters of purine nucleosides,^126,127 or halogenated benzotriazole-modified nucleosides.^30,122 These compounds were primarily evaluated in enzyme-based assays for their putative anti-HCV activity; however, some of them have been found to inhibit also NTPase/helicases of WNV, JEV, or DENV.^117–127

Ribavirin and other nucleoside synthesis inhibitors

Ribavirin, a nucleoside analog featuring a [1,2,4]triazole ring for a nucleobase, is a licensed drug against various RNA viruses. The predominant inhibitory mechanism for ribavirin against flaviviral replication is the suppression of de novo biosynthesis of guanine nucleotides through direct inhibition of inosine monophosphate dehydrogenase, an enzyme converting inosine monophosphate to xanthosine monophosphate, a precursor in GTP biosynthesis.³² Speculation over other modes of action for ribavirin includes specific inhibition of the viral RdRp,¹²⁸ accumulation of mutations in viral genomes resulting in error catastrophe,^129,130 interference with mRNA capping guanylation,¹³¹ and immuno-modulation promoting the Th1 antiviral response.^32,132,133

Ribavirin was shown to exert a moderate inhibitory effect for multiple mosquito-borne flaviviruses in various cell cultures,^31,134,135 often being used as a positive control in many in vitro^25,59,92 and in vivo antiviral studies.^91–94 Ribavirin administered to YVF-infected hamsters challenged intraperitoneally, resulted in significant improvement in survival rates, even if the therapy was started two days post-YFV infection.^93,94 In contrast however, in primates, only a weak prophylactic effect on viremia in rhesus monkeys challenged with DENV was observed.¹³⁶ Several ribavirin derivatives were recently synthesized and showed interesting bioactivity profiles: ETAR (1-β-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole) and IM18 (1-β-d-ribofuranosyl-4-ethynyl-[1,3]imidazole) inhibited DENV-2 replication in Vero cells by more than 10-fold compared with ribavirin and showed no detectable cytotoxic effects up to 1000 µM.¹³⁵ Another derivative, EICAR (1-β-d-ribofuranosyl-5-ethynyl-imidazole-4-carboxamide), was reported to possess a similar in vitro spectrum of antiviral activity, but lower selectivity compared with those of ribavirin.¹³⁷

Two nucleosides whose antiviral activity is based on the depletion of the intracellular nucleoside pool are 6-azauridine and 5-aza-7-deazaguanosine. 6-Azauridine and its derivatives are inhibitors of orotidine monophosphate decarboxylase blocking cellular de novo pyrimidine biosynthesis.^31,33 6-Azauridine proved to be active against numerous arthropod-borne flaviviruses,⁵⁵ however exhibited slight cytotoxicity with an inhibitory effect on the growth of host cells.²⁴ A triacetate prodrug of 6-azauridine showed low micromolar activity against AHFV and WNV in vitro⁵⁵ and very low toxicity in both animal and human studies.¹³⁸ Another derivative, 2-thio-6-azauridine, exerted a moderate inhibitory effect on WNV.³³ Similar results were achieved using 5-aza-7-deazaguanosine (ZX-2401)¹³⁹; this compound exhibited synergistic in vitro anti-YFV activity in combination with interferon. The mechanism of action for 5-aza-7-deazaguanosine is currently unknown; however, it is conceivable that it likely resembles that of ribavirin.³⁴

Rigid amphipathic nucleosides

Nucleoside derivatives containing bulky aromatic substituents (perylene or pyrene moieties) attached to the heterocyclic base were originally synthesized as fluorescent nucleoside probes^140–142; later these structures were identified as inhibitors of herpes simplex virus, type 1 and 2 (HSV-1 and HSV-2), vesicular stomatitis virus, and Sindbis virus replication.¹⁴³ Further studies demonstrated their broad-spectrum antiviral activity against other enveloped viruses, such as influenza virus, murine cytomegalovirus, and HCV.¹⁴⁴ The mechanism of action of these rigid amphipathic nucleosides is based on their incorporation into the viral or cellular membranes, preventing fusion.^143,144 Alternatively, these nucleosides may also function by photosensitization of lipid membranes, resulting in irreversible damage of enveloped virion particles.¹⁴⁵

5-(Perylen-3-yl)ethynyl-arabinouridine and 5-(perylen-3-yl)ethynyl-2′-deoxyuridine were shown to act as strong inhibitors of TBEV in PEK cell culture.¹⁰¹ The perylene moiety as well as the rigid ethynyl linker appeared as crucial structural elements for nanomolar anti-TBEV activity and low cytotoxicity (>50 µM). Interestingly, uracil nucleosides bearing the pyrene moiety, such as 5-[(pyren-3-yl)methoxypropyn-1-yl]-2′-deoxyuridine and 5-(pyren-1-yl)ethynyl-2′-deoxyuridine, showed almost a 10-fold lower anti-TBEV potency compared to their perylene-substituted counterparts. Such compounds, if not used as therapeutic agents, could still contribute to a better understanding of different modes of action of various nucleoside scaffolds.¹⁰¹

Challenges and complications of antiflavivirus nucleoside analog development

Introduction of various chemical substituents into different positions of the nucleoside scaffold can dramatically affect the physicochemical properties of the compound. Such modifications can also significantly influence the compound’s biological/pharmacokinetic parameters, such as cellular uptake,^15,146 the ability of the compound to be activated (phosphorylated) by cellular kinases,¹⁴⁷ degradation by nucleoside catabolic enzymes,¹⁴⁸ or cellular toxicity.^149,150 Use of nucleoside analogs can also result in the undesirable emergence of drug-resistant virus mutants.^151–153 This section highlights the most important challenges and complications toward the development of nucleoside inhibitors of arthropod-transmitted flaviviruses and suggests possible strategies to surmount these difficulties.

For most nucleoside analogs, the first kinase phosphorylation is the rate-limiting step for the conversion to the nucleoside triphosphates. This limitation has a major influence on nucleoside analog antiviral activity^147,154 but can be bypassed by the use of a monophosphate prodrug approach based on the introduction of the phosphorylated group into the 5′ nucleoside position. The phosphorylated group includes masking moieties on the charged phosphate leading to a neutral and eventually hydrophobic entity able to deliver the nucleoside 5′-monophosphate into the cells (Figure 1).¹⁵ The monophosphate prodrug approach has been shown to convert some inactive nucleosides into strong inhibitors, or, has improved the kinetics parameters of intracellular nucleoside triphosphate formation.⁸⁵ This strategy, together with enantioselective purification, led to the development of the phosphoramidate prodrug sofosbuvir, which exhibited considerably increased phosphorylation efficacy compared to the parent nucleoside, 2′-C-fluoro-2′-C-methyluridine.^155,156

Rapid degradation of nucleoside analogs by nucleoside catabolic pathways (Figure 1) is another undesirable phenomenon, which can adversely affect the antiviral potency of some nucleoside analogs.¹⁴⁸ To address this problem, appropriate structural changes can be introduced into the nucleoside scaffolds to protect the nucleosides from metabolic deactivation.¹⁵⁷ Such a strategy was successful for 2′-C-methyladenosine, which was found to be rapidly deaminated by cellular adenosine deaminase to the inactive inosine derivative and/or degraded by purine nucleoside phosphorylase, resulting in poor bioavailability and rapid clearance of the nucleoside in plasma.^49,50 Substitution of the adenine N7 nitrogen for a carbon provided metabolically stable 7-deaza-2′-C-methyladenosine, which is a poor substrate for both nucleoside catabolic enzymes. This compound was characterized by excellent bioavailability and half-life in beagle dogs and rhesus monkeys.⁵⁰ Another possible strategy to increase the metabolic stability of therapeutic nucleosides is based on the introduction of a C-glycosidic bond into the nucleoside scaffold to generate C-nucleoside analogs, such as BCX4430⁸⁹ or GS-5734.⁴³ The major advantage of C-nucleosides over the canonical N-nucleosides lies in their resistance to unwanted phosphorolysis by intracellular phosphorylases, which otherwise would cleave the N-glycosidic linkage.⁸⁸

Individual host cellular types can display differences in expression levels of nucleoside kinases and other enzymes/proteins involved in nucleoside metabolism and transport. This can then result in cell-type dependent antiviral activity as manifested by different EC₅₀ values for the same inhibitor when assayed on different cell lines.^158,159 Strong anti-TBEV activity for 2′-C-methylguanosine, 4′-azidocytidine, and 4′-azido-aracytidine in PS cells was associated with rapid and efficient nucleoside conversion to the corresponding triphosphates. On the other hand, no anti-TBEV effect or phosphorylation products were observed when both compounds were tested in UKF-NB4.⁵⁴ Similarly, the loss of anti-ZIKV activity in Vero cells for sofosbuvir is likely related to the increased expression of the multidrug resistance ABC transporter in this cell line, resulting in the efflux of the compound from the cells.^23,25 Clearly, cell-type dependent antiviral activity of some nucleosides can considerably affect the results of antiviral screens and therefore, using multiple clinically relevant cell lines for evaluation of compounds for antiviral activity is important.⁷⁰

Another possible complication in nucleoside drug development is an undesirable toxicity profile for the nucleoside inhibitor, which can be related to poor selectivity between viral and human enzymes.^39,149,150 A typical example of a nonselective nucleoside analog is 7-deazaadenosine (tubercidin), which exhibits high in vitro cytotoxicity. This has been attributed to the incorporation of tubercidin monophosphate into cellular DNA/RNA by human polymerases.⁵⁰ In contrast, two derivatives of tubercidin, 7-deaza-2′-C-methyladenosine and 6-methyl-7-deazaadenosine, are selectively recognized by flaviviral RdRp and are nontoxic in most mammalian cell lines.^50,96 Some nucleoside analogs inhibit the mitochondrial DNA or RNA polymerase γ, resulting in mitochondrial toxicity.^149,160 This has been the primary reason for the failure of several promising nucleosides/prodrugs in clinical trials, as has been shown for some 2′-C-methyl- and 4′-azido modified nucleosides.⁶⁷ Newly developed compounds should also be evaluated for their genotoxicity and mutagenicity, as well as for renal, cardiovascular, or liver toxicity using various biochemical in vitro assays.^161–163 Nevertheless, even if a compound successfully passes through numerous in vitro tests, it can still exhibit harmful side effects when tested in animals, as observed with the adenosine analog NITD008.²⁶

In that regard, the availability of suitable animal models is crucial for the successful evaluation of a nucleoside’s therapeutic in vivo antiviral efficacy. Some flaviviruses, particularly DEVN and ZIKV, do not readily replicate or cause pathology in immunocompetent mice and, therefore, the use of these rodents as animal infection models is substantially limited.^164–166 To overcome this problem, suckling or young mice,¹⁶⁷ AG129 mice lacking INF-α/β and INF-γ receptors,¹⁶⁸ IFNAR^−/− mice lacking only the IFN-α/β receptor,¹⁶⁹ or immunosuppressed mice¹⁷⁰ can be used as appropriate models to evaluate nucleosides in vivo. However, as these animals are defective in an immune response, this model may also underestimate the real efficacy of the test compounds. Rodent-adapted flavivirus strains, such as the hamster-adapted YFV strain Jimenez,¹⁷¹ mice-adapted DENV strain D2S10,²⁶ or ZIKV African strain Dakar 41519,¹⁷² represent other possible options for in vivo antiviral studies; the biological properties of such viruses can be, however, considerably different compared with those of the parent human-adapted strains.¹⁶⁹

Another issue is related to the length of therapeutic treatment. Most tick- and mosquito-borne flaviviruses cause acute infections, in which short-time treatment duration is expected, ranging between several days to weeks.¹⁷³ This is in contrast to chronic diseases, such as HCV, HBV, and HIV infections, which require long-lasting, and sometimes lifelong, treatment regimens.^72,173 The differences between the acute and chronic diseases should be considered during preclinical development of antiflaviviral inhibitors. Thus, some compounds that show insufficient safety profiles when tested for treatment of chronic infections can be still suitable and safe for short-term therapy of acute flaviviral diseases.⁷²

Antiviral therapies based on chemical inhibitors of viral replication can be accompanied with a rapid emergence of drug-resistant mutants which substantially complicates the course of infection treatment, as seen in HIV, HBV, or HCV infections.^152,153 In flaviviruses, a rapid evolution of resistance to 2′-C-methylated nucleoside inhibitors was observed; this resistance was associated with a signature mutation S603T (in AHVF and TBEV)^55,62 or S604T (in ZIKV)⁶⁸ within the active site of the viral RdRp. Interestingly, the biological properties of the TBEV mutant viruses were dramatically affected, which was manifested by resistance-associated loss of viral replication efficacy in cell culture and a highly attenuated virulence phenotype in mice. This resulted in an unusually low mortality rate when mice were infected with the mutant strain.⁶² As TBEV mutants resistant to 2′-C-methylated nucleosides are highly sensitive to 4′-azido substituted nucleosides,⁶² a combination treatment based on two or more inhibitors could be a possible strategy in order to minimize the risks for the emergence of viral drug resistance.^174–176

Conclusions

More than 200 million clinical cases caused by arthropod-borne flaviviruses, including numerous deaths, are reported worldwide annually. So many cases of infection indicate the importance for the pursuit of new small molecule-based therapeutics to combat emerging viral pathogens. Among these, inhibitors of flaviviruses represent a critical unmet medical need. In that regard, nucleoside inhibitors of flaviviral RdRps are the most attractive targets for antiviral drug design. Nucleosides with the methyl- or ethynyl- modification at the C2′ position and their 2′-fluoro derivatives are the best understood antiflavivirus nucleoside analogs, many of which were initially developed for treatment of HCV infections and later reemployed to suppress replication of other non-HCV flaviviruses. Other important antiflavivirus nucleosides are represented by inhibitors of nucleoside biosynthesis, whose mode of action is predominantly based on depletion of the intracellular nucleoside pool. Nucleoside inhibitors of flaviviral MTase and NTPase/helicase, as well as some newly discovered flavivirus inhibitors, such as tritylated nucleosides, rigid amphipathic nucleosides, or N6-aryl-substituted adenosine derivatives, whose mechanisms of action are still poorly understood, can be used as initial structures or starting points for further developments of new generations of nucleoside scaffolds with improved biological parameters. Taken together, specific nucleoside analog-based antiviral therapy in combination with effective vaccination strategies could provide potent prophylactic and curative tools to treat human infections caused by flaviviruses.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant from the Ministry of Health of the Czech Republic (grant no. 16–34238A), Czech Science Foundation (grant no. 16–20054S) (to DR and RN), and by Project “FIT” (Pharmacology, Immunotherapy, nanotoxicology; CZ.02.1.01/0.0/0.0/15_003/0000495), which was funded by the European Regional Development Fund (to DR).

References

Baier

Flaviviral infections and potential targets for antiviral therapy. In: Ruzek

(ed) Flavivirus encephalitis. 1st ed. Rijeka: InTech, 2011, pp.89–104.

Lazear

Stringer

and de Silva

AM.

The emerging zika virus epidemic in the Americas research priorities. J Am Med Assoc 2016; 315: 1945–1946.

Jeffries

Mansfield

Phipps

et al . Louping ill virus: an endemic tick-borne disease of Great Britain. J Gen Virol 2014; 95: 1005–1014.

Ashraf

Ruan

et al . Usutu virus: an emerging flavivirus in Europe. Viruses 2015; 7: 219–238.

Rumyantsev

Murphy

and Pletnev

AG.

A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice. J Virol 2006; 80: 1427–1439.

Blackburn

and Swanepoel

An investigation of flavivirus infections of cattle in Zimbabwe-Rhodesia with particular reference to Wesselsbron virus. J Hyg 1980; 85: 1–33.

Chambers

Hahn

Galler

et al . Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 1990; 44: 649–688.

Koonin

EV.

Computer-assisted identification of a putative methyltransferase domain in Ns5 protein of flaviviruses and lambda-2 protein of reovirus. J Gen Virol 1993; 74: 733–740.

Zhou

Ray

Zhao

et al . Structure and function of flavivirus NS5 methyltransferase. J Virol 2007; 81: 3891–3903.

10.

Liu

and Gong

A structural overview of RNA-dependent RNA polymerases from the Flaviviridae family. Int J Mol Sci 2015; 16: 12943–12957.

11.

Zou

Chen

Dong

et al . Functional analysis of two cavities in flavivirus NS5 polymerase. J Biol Chem 2011; 286: 14362–14372.

12.

Deval

Symons

and Beigelman

Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus. Curr Opin Virol 2014; 9: 1–7.

13.

Puig-Basagoiti

Tilgner

Forshey

et al . Triaryl pyrazoline compound inhibits flavivirus RNA replication. Antimicrob Agents Chemother 2006; 50: 1320–1329.

14.

De Clercq

A 40-year journey in search of selective antiviral chemotherapy. Annu Rev Pharmacol Toxicol 2011; 51: 1–24.

15.

Jordheim

Durantel

Zoulim

et al

Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat Rev Drug Discov 2013; 12: 447–464.

16.

Benhamou

Tubiana

and Thibault

Tenofovir disoproxil fumarate in patients with HIV and lamivudine-resistant hepatitis B virus. N Engl J Med 2003; 348: 177–178.

17.

Ray

Fordyce

and Hitchcock

MJM.

Tenofovir alafenamide: a novel prodrug of tenofovir for the treatment of human immunodeficiency virus. Antiviral Res 2016; 125: 63–70.

18.

Huang

Chang

Sheng

et al . Virological response to tenofovir disoproxil fumarate in HIV-positive patients with lamivudine-resistant hepatitis B virus coinfection in an area hyperendemic for hepatitis B virus infection. PLoS One 2016; 11: e0169228.

19.

Lam

Seto

Wong

et al . Seven-year treatment outcome of entecavir in a real-world cohort: effects on clinical parameters, HBsAg and HBcrAg levels. Clin Trans Gastroenterol 2017; 8: e125.

20.

Stedman

Sofosbuvir, a NS5B polymerase inhibitor in the treatment of hepatitis C: a review of its clinical potential. Ther Adv Gastroenterol 2014; 7: 131–140.

21.

De Clercq

and Holý

Acyclic nucleoside phosphonates: a key class of antiviral drugs. Nat Rev Drug Discov 2005; 4: 928–940.

22.

Chen

Yin

Duraiswamy

et al . Inhibition of dengue virus RNA synthesis by an adenosine nucleoside. Antimicrob Agents Chemother 2010; 54: 2932–2939.

23.

Eyer

Nencka

Huvarova

et al . Nucleoside inhibitors of zika virus. J Infect Dis 2016; 214: 707–711.

24.

Eyer

Valdes

Gil

et al . Nucleoside inhibitors of tick-borne encephalitis virus. Antimicrob Agents Chemother 2015; 59: 5483–5493.

25.

Sacramento

de Melo

de Freitas

et al . The clinically approved antiviral drug sofosbuvir inhibits zika virus replication. Sci Rep 2017; 7: 40920.

26.

Yin

Chen

Schul

et al . An adenosine nucleoside inhibitor of dengue virus. Proc Natl Acad Sci USA 2009; 106: 20435–20439.

27.

Vernekar

Qiu

and Zacharias

Synthesis and antiviral evaluation of 4′-(1,2,3-triazol-1-yl)thymidines. MedChemComm 2014; 5: 603–608.

28.

Vernekar

Qiu

Zhang

et al . 5′-Silylated 3′-1,2,3-triazolyl thymidine analogues as inhibitors of West Nile Virus and Dengue Virus. J Med Chem 2015; 58: 4016–4028.

29.

Borowski

Lang

Haag

et al . Characterization of imidazo[4,5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus nucleoside triphosphatase/helicase: evidence for activity on the level of substrate and/or enzyme. Antimicrob Agents Chemother 2002; 46: 1231–1239.

30.

Borowski

Deinert

Schalinski

et al . Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses. Eur J Biochem 2003; 270: 1645–1653.

31.

Crance

Scaramozzino

Jouan

et al . Interferon, ribavirin, 6-azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses. Antiviral Res 2003; 58: 73–79.

32.

Leyssen

Balzarini

De Clercq

et al . The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of inosine monophosphate dehydrogenase. J Virol 2005; 79: 1943–1947.

33.

Morrey

Smee

Sidwell

et al . Identification of active antiviral compounds against a New York isolate of West Nile virus. Antiviral Res 2002; 55: 107–116.

34.

Ojwang

Ali

Smee

et al . Broad-spectrum inhibitor of viruses in the Flaviviridae family. Antiviral Res 2005; 68: 49–55.

35.

Orlov

Drenichev

Oslovsky

et al . New tools in nucleoside toolbox of tick-borne encephalitis virus reproduction inhibitors. Bioorg Med Chem Lett 2017; 27: 1267–1273.

36.

Lescar

and Canard

RNA-dependent RNA polymerases from flaviviruses and Picornaviridae. Curr Opin Struct Biol 2009; 19: 759–767.

37.

Choi

and Rossmann

MG.

RNA-dependent RNA polymerases from Flaviviridae. Curr Opin Struct Biol 2009; 19: 746–751.

38.

Behnam

MAM

Nitsche

Boldescu

et al . The medicinal chemistry of Dengue virus. J Med Chem 2016; 59: 5622–5649.

39.

Boldescu

Behnam

MAM

Vasilakis

et al . Broad-spectrum agents for flaviviral infections: Dengue, Zika and beyond. Nat Rev Drug Discov 2017; 16: 565–586.

40.

De Clercq

and Neyts

Antiviral agents acting as DNA or RNA chain terminators. Handb Exp Pharmacol 2009; 189: 53–84.

41.

De Clercq

Antivirals and antiviral strategies. Nat Rev Microbiol 2004; 2: 704–720.

42.

Cihlar

and Ray

AS.

Nucleoside and nucleotide HIV reverse transcriptase inhibitors: 25 years after zidovudine. Antiviral Res 2010; 85: 39–58.

43.

Cho

Saunders

Butler

et al . Synthesis and antiviral activity of a series of 1′-substituted 4-aza-7,9-dideazaadenosine C-nucleosides. Bioorg Med Chem Lett 2012; 22: 2705–2707.

44.

Warren

Jordan

et al . Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016; 531: 381–385.

45.

Jordan

Arvey

et al . GS-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. Sci Rep 2017; 7: 43395.

46.

Eldrup

Allerson

Bennett

et al . Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase. J Med Chem 2004; 47: 2283–2295.

47.

Carroll

Tomassini

Bosserman

et al . Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs. J Biol Chem 2003; 278: 11979–11984.

48.

Carroll

Koeplinger

Vavrek

et al . Antiviral efficacy upon administration of a HepDirect prodrug of 2′-C-methylcytidine to hepatitis C virus-infected chimpanzees. Antimicrob Agents Chemother 2011; 55: 3854–3860.

49.

Migliaccio

Tomassini

Carroll

et al . Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J Biol Chem 2003; 278: 49164–49170.

50.

Olsen

Eldrup

Bartholomew

et al . A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. Antimicrob Agents Chemother 2004; 48: 3944–3953.

51.

Lefebvre

De Vleeschauwer

Goris

et al . Proof of concept for the inhibition of foot-and-mouth disease virus replication by the anti-viral drug 2′-C-methylcytidine in severe combined immunodeficient mice. Transbound Emerg Dis 2014; 61: E89–E91.

52.

Rocha-Pereira

Jochmans

Dallmeier

et al . Inhibition of norovirus replication by the nucleoside analogue 2′-C-methylcytidine. Biochem Biophys Res Commun 2012; 427: 796–800.

53.

Rocha-Pereira

Jochmans

Debing

et al . The viral polymerase inhibitor 2′-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model. J Virol 2013; 87: 11798–11805.

54.

Eyer

Smidkova

Nencka

et al . Structure-activity relationships of nucleoside analogues for inhibition of tick-borne encephalitis virus. Antiviral Res 2016; 133: 119–129.

55.

Flint

McMullan

Dodd

et al . Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses. Antimicrob Agents Chemother 2014; 58: 3206–3216.

56.

Lee

Tseng

et al . Characterization of the activity of 2′-C-methylcytidine against dengue virus replication. Antiviral Res 2015; 116: 1–9.

57.

Mateo

Nagamine

and Kirkegaard

Suppression of drug resistance in Dengue virus. MBio 2015; 6: e01960–15.

58.

Yeo

Chen

et al . Synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors. Antimicrob Agents Chemother 2015; 59: 2086–2093.

59.

Lanko

Eggermont

Patel

et al . Replication of the Zika virus in different iPSC-derived neuronal cells and implications to assess efficacy of antivirals. Antiviral Res 2017; 145: 82–86.

60.

Hercik

Brynda

Nencka

et al . Structural basis of Zika virus methyltransferase inhibition by sinefungin. Arch Virol 2017; 162: 2091–2096.

61.

Eyer

Kondo

Zouharova

et al. Escape of tick-borne flavivirus from 2′ -C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness. J Virol 2017; e01028–17.

62.

Zmurko

Marques

Schols

et al . The viral polymerase inhibitor 7-deaza-2′-C-methyladenosine is a potent inhibitor of in vitro Zika virus replication and delays disease progression in a robust mouse infection model. PLoS Negl Trop Dis 2016; 10: e0004695.

63.

Julander

Jha

Choi

et al . Efficacy of 2′-C-methylcytidine against yellow fever virus in cell culture and in a hamster model. Antiviral Res 2010; 86: 261–267.

64.

Sofia

Chang

Furman

et al . Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase. J Med Chem 2012; 55: 2481–2531.

65.

Keating

and Vaidya

Sofosbuvir: first global approval. Drugs 2014; 74: 273–282.

66.

Arnold

Sharma

Feng

et al . Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides. PLoS Pathog 2012; 8: e1003030.

67.

Feng

Barauskas

et al . Role of mitochondrial RNA polymerase in the toxicity of nucleotide inhibitors of hepatitis C virus. Antimicrob Agents Chemother 2016; 60: 806–817.

68.

Hassounah

Colby-Germinario

et al . Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors. J Antimicrob Chemoth 2017; 72: 727–734.

69.

Bullard-Feibelman

Govero

Zhu

et al . The FDA-approved drug sofosbuvir inhibits Zika virus infection. Antiviral Res 2017; 137: 134–140.

70.

Mumtaz

Jimmerson

Bushman

et al . Cell-line dependent antiviral activity of sofosbuvir against Zika virus. Antiviral Res 2017; 146: 161–163.

71.

Ferreira

Zaverucha-do-Valle

Reis

et al . Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequelae. Sci Rep 2017; 7: 9409.

72.

Chen

Yin

Lakshminarayana

et al . Inhibition of Dengue Virus by an ester prodrug of an adenosine analog. Antimicrob Agents Chemother 2010; 54: 3255–3261.

73.

Chen

Yokokawa

and Shi

PY.

The search for nucleoside/nucleotide analog inhibitors of dengue virus. Antiviral Res 2015; 122: 12–19.

74.

Latour

Jekle

Javanbakht

et al . Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-D-2′-ethynyl-7-deaza-adenosine triphosphate. Antiviral Res 2010; 87: 213–222.

75.

Yin

and Schneller

SW1.

Deaza-5′-noraisteromycin. Nucleosides Nucleotides Nucleic Acids 2004; 23: 67–76.

76.

Deng

Zhang

et al . Adenosine analog NITD008 is a potent inhibitor of Zika virus. Open Forum Infect Dis 2016; 3: ofw175.

77.

Shi

Chen

et al . In vitro antiviral activity of adenosine analog NITD008 against tick borne flaviviruses. Antiviral Res 2016; 130: 46–49.

78.

McGuigan

Serpi

Slusarczyk

et al . Anti-flavivirus activity of different tritylated pyrimidine and purine nucleoside analogues. Chem Open 2016; 5: 227–235.

79.

Klumpp

Leveque

Le Pogam

et al . The novel nucleoside analog R1479 (4′-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem 2006; 281: 3793–3799.

80.

Klumpp

Kalayanov

et al . Deoxy-4′-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus replication despite the lack of 2′-alpha-hydroxyl groups. J Biol Chem 2008; 283: 2167–2175.

81.

Smith

Kalayanov

Sund

et al . The design, synthesis, and antiviral activity of 4′-azidocytidine analogues against hepatitis C virus replication: the discovery of 4′-azidoarabinocytidine. J Med Chem 2009; 52: 219–223.

82.

Hotard

Nichol

et al . 4′-Azidocytidine (R1479) inhibits henipaviruses and other paramyxoviruses with high potency. Antiviral Res 2017; 144: 147–152.

83.

Nguyen

Tran

CNB

Phung

et al . A randomized, double-blind placebo controlled trial of balapiravir, a polymerase inhibitor, in adult dengue patients. J Infect Dis 2013; 207: 1442–1450.

84.

Chen

Ghafar

Karuna

et al . Activation of peripheral blood mononuclear cells by Dengue virus infection depotentiates balapiravir. J Virol 2014; 88: 1740–1747.

85.

Rondla

Coats

McBrayer

et al . Anti-hepatitis C virus activity of novel beta-d-2′-C-methyl-4′ -azido pyrimidine nucleoside phosphoramidate prodrugs. Antivir Chem Chemother 2009; 20: 99–106.

86.

Nilsson

Kalayanov

Winqvist

et al . Discovery of 4′-azido-2′-deoxy-2′-C-methyl cytidine and prodrugs thereof: a potent inhibitor of hepatitis C virus replication. Bioorg Med Chem Lett 2012; 22: 3265–3268.

87.

Taylor

Kotian

Warren

et al . BCX4430-A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease. J Infect Public Health 2016; 9: 220–226.

88.

De Clercq

C-nucleosides to be revisited. J Med Chem 2016; 59: 2301–2311.

89.

Warren

Wells

Panchal

et al . Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature 2014; 508: 402–405.

90.

Eyer

Zouharova

Sirmarova

et al . Antiviral activity of the adenosine analogue BCX4430 against West Nile virus and tick-borne flaviviruses. Antiviral Res 2017; 142: 63–67.

91.

Julander

Siddharthan

Evans

et al . Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model. Antiviral Res 2017; 137: 14–22.

92.

Julander

Bantia

Taubenheim

et al . BCX4430, a novel nucleoside analog, effectively treats yellow fever in a hamster model. Antimicrob Agents Chemother 2014; 58: 6607–6614.

93.

Julander

Furuta

Shafer

et al . Activity of T-1106 in a hamster model of yellow fever virus infection. Antimicrob Agents Chemother 2007; 51: 1962–1966.

94.

Julander

Shafer

Smee

et al . Activity of T-705 in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, T-1106. Antimicrob Agents Chemother 2009; 53: 202–209.

95.

Julander

Shafer

Smee

et al . Efficacy of T-1106 or T-705, alone or in combination with ribavirin, in the treatment of hamsters infected with yellow fever virus. Antiviral Res 2008; 78: A34.

96.

Smidansky

et al . Synthesis of a 6-methyl-7-deaza analogue of adenosine that potently inhibits replication of polio and Dengue viruses. J Med Chem 2010; 53: 7958–7966.

97.

Angusti

Manfredini

Durini

et al . Design, synthesis and anti Flaviviridae activity of N-6-, 5′,3′-O- and 5′,2′-O-substituted adenine nucleoside analogs. Chem Pharm Bull 2008; 56: 423–432.

98.

Morrey

Taro

Siddharthan

et al . Efficacy of orally administered T-705 pyrazine analog on lethal West Nile virus infection in rodents. Antiviral Res 2008; 80: 377–379.

99.

Drenichev

Oslovsky

Sun

et al . Modification of the length and structure of the linker of N-6-benzyladenosine modulates its selective antiviral activity against enterovirus 71. Eur J Med Chem 2016; 111: 84–94.

100.

Tararov

Tijsma

Kolyachkina

et al . Chemical modification of the plant isoprenoid cytokinin N-6-isopentenyladenosine yields a selective inhibitor of human enterovirus 71 replication. Eur J Med Chem 2015; 90: 406–413.

101.

Orlov

Chistov

Kozlovskaya

et al . Rigid amphipathic nucleosides suppress reproduction of the tick-borne encephalitis virus. Med Chem Commun 2016; 7: 495–499.

102.

Chatelain

Debing

De Burghgraeve

et al . In search of flavivirus inhibitors: evaluation of different tritylated nucleoside analogues. Eur J Med Chem 2013; 65: 249–255.

103.

De Burghgraeve

Selisko

Kaptein

et al . 3′,5′ Di-O-trityluridine inhibits in vitro flavivirus replication. Antiviral Res 2013; 98: 242–247.

104.

Saudi

Zmurko

Kaptein

et al . In search of flavivirus inhibitors part 2: tritylated, diphenylmethylated and other alkylated nucleoside analogues. Eur J Med Chem 2014; 76: 98–109.

105.

Koonin

and Dolja

VV.

Evolution taxonomy of positive-strand RNA viruses – implications of comparative-analysis of amino-acid-sequences. Crit Rev Biochem Mol Biol 1993; 28: 375–430.

106.

Cleaves

and Dubin

DT.

Methylation status of intracellular dengue type-2 40-S RNA. Virology 1979; 96: 159–165.

107.

Dong

Fink

Zuest

et al . Flavivirus RNA methylation. J Gen Virol 2014; 95: 763–778.

108.

Chung

Dong

Chao

et al . Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2′-O methyltransferase activity in dengue virus. Virology 2010; 402: 52–60.

109.

Lim

Wen

Yap

et al . A scintillation proximity assay for dengue virus NS5 2′-O-methyltransferase-kinetic and inhibition analyses. Antiviral Res 2008; 80: 360–369.

110.

Lim

Sonntag

Noble

et al . Small molecule inhibitors that selectively block dengue virus methyltransferase. J Biol Chem 2011; 286: 6233–6240.

111.

Chen

Liu

Jones

et al . Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs. Antiviral Res 2013; 97: 232–239.

112.

Yates

Raje

Chatterjee

et al . Flex-nucleoside analogues – novel therapeutics against filoviruses. Bioorg Med Chem Lett 2017; 27: 2800–2802.

113.

Peters

Jochmans

de Wilde

et al . Design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity. Bioorg Med Chem Lett 2015; 25: 2923–2926.

114.

Seley

Zhang

Hagos

et al . “ Fleximers”. Design and synthesis of a new class of novel shape-modified nucleosides. J Org Chem 2002; 67: 3365–3373.

115.

Seley

Quirk

Salim

et al . Unexpected inhibition of S-adenosyl-L-homocysteine hydrolase by a guanosine nucleoside. Bioorg Med Chem Lett 2003; 13: 1985–1988.

116.

Quirk

and Seley

KL.

Substrate discrimination by the human GTP fucose pyrophosphorylase. Biochemistry 2005; 44: 10854–10863.

117.

Gallinari

Brennan

Nardi

et al . Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus. J Virol 1998; 72: 6758–6769.

118.

Hodgman

TC.

A new superfamily of replicative proteins. Nature 1988; 333: 22–23.

119.

Kim

Morgenstern

Griffith

et al . Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 1998; 6: 89–100.

120.

Yao

Hesson

Cable

et al . Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol 1997; 4: 463–467.

121.

Tai

Chi

Chen

et al . The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol 1996; 70: 8477–8484.

122.

Bretner

Baier

Kopańska

et al . Synthesis and biological activity of 1H-benzotriazole and 1H-benzimidazole analogues – inhibitors of the NTpase/helicase of HCV and of some related Flaviviridae. Antivir Chem Chemother 2005; 16: 315–326.

123.

Borowski

Kuehl

Mueller

et al . Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus. Eur J Biochem 1999; 266: 715–723.

124.

Borowski

Lang

Niebuhr

et al . Inhibition of the helicase activity of HCV NTPase/helicase by 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5′-triphosphate (ribavirin-TP). Acta Biochim Pol 2001; 48: 739–744.

125.

Borowski

Mueller

Niebuhr

et al . ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy. Acta Biochim Pol 2000; 47: 173–180.

126.

Bretner

Schalinski

Borowski

et al . 5′-O-fluorosulfonylbenzoyl esters of purine nucleosides as potential inhibitors of NTPase/helicase and polymerase of Flaviviridae viruses. Nucleosides Nucleotides Nucleic Acids 2003; 22: 1531–1533.

127.

Bretner

Schalinski

Haag

et al . Synthesis and evaluation of ATP-binding site directed potential inhibitors of nucleoside triphosphatases/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses. Antivir Chem Chemother 2004; 15: 35–42.

128.

Eriksson

Helgstrand

Johansson

et al . Inhibition of influenza-virus ribonucleic-acid polymerase by ribavirin triphosphate. Antimicrob Agents Chemother 1977; 11: 946–951.

129.

Crotty

Cameron

and Andino

RNA virus error catastrophe: direct molecular test by using ribavirin.

Proc Natl Acad Sci USA 2001; 98: 6895–6900.

130.

Graci

and Cameron

CE.

Quasispecies, error catastrophe, and the antiviral activity of ribavirin. Virology 2002; 298: 175–180.

131.

Guillen

Rabah

et al . mRNA capping by Venezuelan Equine Encephalitis virus nsP1: functional characterization and implications for antiviral research. J Virol 2015; 89: 8292–8303.

132.

Graci

and Cameron

CE.

Mechanisms of action of ribavirin against distinct viruses. Rev Med Virol 2006; 16: 37–48.

133.

Hultgren

Milich

Weiland

et al . The antiviral compound ribavirin modulates the T helper (Th)1/Th2 subset balance in hepatitis B and C virus-specific immune responses. J Gen Virol 1998; 79: 2381–2391.

134.

Jordan

Briese

Fischer

et al . Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells. J Infect Dis 2000; 182: 1214–1217.

135.

McDowell

Gonzales

Kumarapperuma

et al . A novel nucleoside analog, 1-beta-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), exhibits efficacy against a broad range of flaviviruses in vitro. Antiviral Res 2010; 87: 78–80.

136.

Malinoski

Hasty

Ussery

et al . Prophylactic ribavirin treatment of dengue type-1 infection in rhesus-monkeys. Antiviral Res 1990; 13: 139–150.

137.

Gabrielsen

Phelan

Barthelrosa

et al . Synthesis and antiviral evaluation of N-carboxamidine-substituted analogs of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride. J Med Chem 1992; 35: 3231–3238.

138.

Crutcher

and Moschella

SL.

Double-blind controlled crossover high-dose study of azaribine in psoriasis. Br J Dermatol 1975; 92: 199–205.

139.

Dukhan

Leroy

Peyronnet

et al . Synthesis of 5-aza-7-deazaguanine nucleoside derivatives as potential anti-flavivirus agents. Nucleosides Nucleotides Nucleic Acids 2005; 24: 671–674.

140.

Korshun

Prokhorenko

Gontarev

et al . New pyrene derivatives for fluorescent labeling of oligonucleotides. Nucleosides Nucleotides 1997; 16: 1461–1464.

141.

Korshun

Manasova

Balakin

et al . New fluorescent nucleoside derivatives – 5-alkynylated 2′-deoxyuridines. Nucleosides Nucleotides 1998; 17: 1809–1812.

142.

Skorobogatyi

Malakhov

Pchelintseva

et al . Fluorescent 5-alkynyl-2′-deoxyuridines: high emission efficiency of a conjugated perylene nucleoside in a DNA duplex. Chembiochem 2006; 7: 810–816.

143.

St Vincent

Colpitts

Ustinov

et al . Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses. Proc Natl Acad Sci USA 2010; 107: 17339–17344.

144.

Colpitts

Ustinov

Epand

et al . 5-(Perylen-3-yl) ethynyl-arabino-uridine (aUY11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses. J Virol 2013; 87: 3640–3654.

145.

Vigant

Hollmann

Lee

et al . The rigid amphipathic fusion inhibitor dUY11 acts through photosensitization of viruses. J Virol 2014; 88: 1849–1853.

146.

Maag

and Alfredson

Prodrugs of nucleoside analogues for improved oral absorption and tissue targeting. J Pharm Sci 2008; 97: 1109–1134.

147.

Hecker

and Erion

MD.

Prodrugs of phosphates and phosphonates. J Med Chem 2008; 51: 2328–2345.

148.

Lane

and Fan

TWM.

Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res 2015; 43: 2466–2485.

149.

Johnson

Ray

Hanes

et al . Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase. J Biol Chem 2001; 276: 40847–40857.

150.

Lee

Hanes

and Johnson

KA.

Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry 2003; 42: 14711–14719.

151.

Locarnini

and Warner

Major causes of antiviral drug resistance and implications for treatment of hepatitis B virus monoinfection and coinfection with HIV. Antiviral Ther 2007; 12: H15–H23.

152.

Menendez-Arias

Alvarez

and Pacheco

Nucleoside/nucleotide analog inhibitors of hepatitis B virus polymerase: mechanism, of action and resistance. Curr Opin Virol 2014; 8: 1–9.

153.

Poveda

Wyles

Mena

et al . Update on hepatitis C virus resistance to direct-acting antiviral agents. Antiviral Res 2014; 108: 181–191.

154.

McGuigan

Cahard

Sheeka

et al . Aryl phosphoramidate derivatives of d4T have improved anti-HIV efficacy in tissue culture and may act by the generation of a novel intracellular metabolite. J Med Chem 1996; 39: 1748–1753.

155.

Lam

Espiritu

Bansal

et al . Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob Agents Chemother 2012; 56: 3359–3368.

156.

Ross

Reddy

Zhang

et al . Synthesis of diastereomerically pure nucleotide phosphoramidates. J Org Chem 2011; 76: 8311–8319.

157.

Peterson

LW and

McKenna

CE.

Prodrug approaches to improving the oral absorption of antiviral nucleotide analogues. Expert Opin Drug Deliv 2009; 6: 405–420.

158.

Becher

Landman

Mboup

et al . Monitoring of didanosine and stavudine intracellular trisphosphorylated anabolite concentrations in HIV-infected patients. AIDS 2004; 18: 181–187.

159.

Gao

Shirasaka

Johns

et al . Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral-blood mononuclear-cells. J Clin Invest 1993; 91: 2326–2333.

160.

Kohler

and Lewis

A brief overview of mechanisms of mitochondrial toxicity from NRTIs. Environ Mol Mutagen 2007; 48: 166–172.

161.

Maurer

HH.

Screening procedures for simultaneous detection of several drug classes used for high throughput toxicological analyses and doping control. A review. Comb Chem High Throughput Screen 2000; 3: 467–480.

162.

Mckim

JM.

Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance. Comb Chem High Throughput Screen 2010; 13: 188–206.

163.

Szymanski

Markowicz

and Mikiciuk-Olasik

Adaptation of high-throughput screening in drug discovery-toxicological screening tests. Int J Mol Sci 2012; 13: 427–452.

164.

Adachi

and Miura

Animal model studies on viral infections. Front Microbiol 2014; 5: 672.

165.

Dowall

Graham

Rayner

et al . A susceptible mouse model for Zika virus infection. PLoS Negl Trop Dis 2016; 10: e0004658.

166.

Zompi

and Harris

Animal models of Dengue virus infection. Viruses 2012; 4: 62–82.

167.

Lee

Huang

Lei

et al . Suckling mice were used to detect infectious dengue-2 viruses by intracerebral injection of the full-length RNA transcript. Intervirology 2005; 48: 161–166.

168.

Sarathy

White

et al . A lethal murine infection model for Dengue Virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease. J Virol 2015; 89: 1254–1266.

169.

Zellweger

and Shresta

Mouse models to study dengue virus immunology and pathogenesis. Front Immunol 2014; 5: 151.

170.

Chan

JFW

Zhang

Chan

CCS

et al . Zika virus infection in dexamethasone-immunosuppressed mice demonstrating disseminated infection with multi-organ involvement including orchitis effectively treated by recombinant type I interferons. EBioMedicine 2016; 14: 112–122.

171.

McArthur

Suderman

Mutebi

et al . Molecular characterization of a hamster viscerotropic strain of yellow fever virus. J Virol 2003; 77: 1462–1468.

172.

Govero

Esakky

Scheaffer

et al . Zika virus infection damages the testes in mice. Nature 2016; 540: 438–442.

173.

Gubler

Kuno

and Markoff

Flaviviruses In: Knipe

and Howley

(eds) Fields virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2007, pp.1153–1253.

174.

Carey

and Harrison

PM.

Monotherapy versus combination therapy for the treatment of chronic hepatitis B. Expert Opin Investig Drug 2009; 18: 1655–1666.

175.

Wong

GLH

Wong

VWS

and Chan

HLY.

Combination therapy of interferon and nucleotide/nucleoside analogues for chronic hepatitis B. J Viral Hepat 2014; 21: 825–834.

176.

and Liu

CJ.

Combination therapy for chronic hepatitis B: current updates and perspectives. Gut Liver 2017; 11: 590–603.